ORCID Profile
0000-0003-0685-2952
Current Organisation
University of Queensland
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Animal Physiology - Systems | Physiology | Cell Physiology | Animal Physiology - Biophysics | Animal Physiology - Cell | Medical Physiology | Neurobiology | Animal Physiology—Systems | Zoology | Genetic Technologies: Transformation, Site-Directed Mutagenesis, Etc. | Cardiology (incl. Cardiovascular Diseases) | Biochemistry and Cell Biology not elsewhere classified |
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Digestive System Disorders | Health Related to Ageing | Nervous system and disorders | Cardiovascular system and diseases | Expanding Knowledge in the Medical and Health Sciences
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Publications
THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: G protein-coupled receptors.
Publisher: Wiley
Date: 16-09-2021
DOI: 10.1111/BPH.15538
Abstract: The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at oi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is ided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
Angiotensin Type 1A Receptors in C1 Neurons of the Rostral Ventrolateral Medulla Modulate the Pressor Response to Aversive Stress
Publisher: Society for Neuroscience
Date: 08-02-2012
DOI: 10.1523/JNEUROSCI.5360-11.2012
Abstract: The rise in blood pressure during an acute aversive stress has been suggested to involve activation of angiotensin type 1A receptors (AT 1A Rs) at various sites within the brain, including the rostral ventrolateral medulla. In this study we examine the involvement of AT 1A Rs associated with a subclass of sympathetic premotor neurons of the rostral ventrolateral medulla, the C1 neurons. The distribution of putative AT 1A R-expressing cells was mapped throughout the brains of three transgenic mice with a bacterial artificial chromosome-expressing green fluorescent protein under the control of the AT 1A R promoter. The overall distribution correlated with that of the AT 1A Rs mapped by other methods and demonstrated that the majority of C1 neurons express the AT 1A R. Cre-recombinase expression in C1 neurons of AT 1A R-floxed mice enabled demonstration that the pressor response to microinjection of angiotensin II into the rostral ventrolateral medulla is dependent upon expression of the AT 1A R in these neurons. Lentiviral-induced expression of wild-type AT 1A Rs in C1 neurons of global AT 1A R knock-out mice, implanted with radiotelemeter devices for recording blood pressure, modulated the pressor response to aversive stress. During prolonged cage-switch stress, expression of AT 1A Rs in C1 neurons induced a greater sustained pressor response when compared to the control viral-injected group (22 ± 4 mmHg for AT 1A R vs 10 ± 1 mmHg for GFP p 0.001), which was restored toward that of the wild-type group (28 ± 2 mmHg). This study demonstrates that AT 1A R expression by C1 neurons is essential for the pressor response to angiotensin II and that this pathway plays an important role in the pressor response to aversive stress.
Angiotensin II Receptor Endocytosis Involves Two Distinct Regions of the Cytoplasmic Tail
Publisher: Elsevier BV
Date: 09-1995
Helix I of β-Arrestin Is Involved in Postendocytic Trafficking but Is Not Required for Membrane Translocation, Receptor Binding, and Internalization
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 02-11-2004
Abstract: beta-Arrestins bind to phosphorylated, seven-transmembrane-spanning, G protein-coupled receptors (GPCRs), including the type 1 angiotensin II receptor (AT(1)R), to promote receptor desensitization and internalization. The AT(1) R is a class B GPCR that recruits both beta-arrestin1 and beta-arrestin2, forming stable complexes that cotraffic to deep-core endocytic vesicles. beta-Arrestins contain one hipathic and potentially hitropic (membrane-targeting) alpha-helix (helix I) that may promote translocation to the membrane or influence receptor internalization or trafficking. Here, we investigated the trafficking and function of beta-arrestin1 and beta-arrestin2 mutants bearing substitutions in both the hydrophobic and positively charged faces of helix I. The level of expression of these mutants and their cytoplasmic localization (in the absence of receptor activation) was similar to wild-type beta-arrestins. After angiotensin II stimulation, both wild-type and beta-arrestin mutants translocated to the cell membrane, although recruitment was weaker for mutants of the hydrophobic face of helix I. For all beta-arrestin mutants, the formation of deep-core vesicles was less observed compared with wild-type beta-arrestins. Furthermore, helix I conjugated to green fluorescent protein is not membrane-localized, suggesting that helix I, in isolation, is not hitropic. Bioluminescence resonance energy transfer analysis revealed that both wild-type and beta-arrestin mutants retained a capacity to interact with the AT(1)R, although the interaction with the mutants was less stable. Finally, wild-type and mutant beta-arrestins fully supported receptor internalization in human embryonic kidney cells and mouse embryonic fibroblasts deficient in beta-arrestin1 and -2. Thus, helix I is implicated in postmembrane trafficking but is not strongly hitropic.
A single beta-amino acid substitution to angiotensin II confers AT2 receptor selectivity and vascular function
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-2011
DOI: 10.1161/HYPERTENSIONAHA.110.164301
Abstract: Novel AT 2 R ligands were designed by substituting in idual β-amino acid in the sequence of the native ligand angiotensin II (Ang II). Relative ATR selectivity and functional vascular assays (in vitro AT 2 R-mediated vasorelaxation and in vivo vasodepressor action) were determined. In competition binding experiments using either AT 1 R- or AT 2 R- transfected HEK-293 cells, only β-Asp 1 -Ang II and Ang II fully displaced [ 125 I]-Ang II from AT 1 R. In contrast, β-substitutions at each position of Ang II exhibited AT 2 R affinity, with β-Tyr 4 -Ang II and β-Ile 5 -Ang II exhibiting ≈1000-fold AT 2 R selectivity. In mouse aortic rings, β-Tyr 4 -Ang II and β-Ile 5 -Ang II evoked vasorelaxation that was sensitive to blockade by the AT 2 R antagonist PD123319 and the nitric oxide synthase inhibitor L-NAME. When tested with a low level of AT 1 R blockade, β-Ile 5 -Ang II (15 pmol/kg per minute IV for 4 hours) reduced blood pressure (BP) in conscious spontaneously hypertensive rats (β-Ile 5 -Ang II plus candesartan, −24±4 mm Hg) to a greater extent than candesartan alone (−11±3 mm Hg, n=7, P .05), an effect that was abolished by concomitant PD123319 infusion. However, in an identical experimental protocol, β-Tyr 4 -Ang II had no influence on BP (n=10), and it was less stable than β-Ile 5 -Ang II in plasma stability assays. Thus, this study demonstrated that a single β-amino acid substitution resulted in a compound that demonstrated both in vitro vasorelaxation and in vivo depressor activity via AT 2 R. This approach to the design and synthesis of novel AT 2 R-selective peptidomimetics shows great potential to provide insight into AT 2 R function.
Variability in human bitter taste sensitivity to chemically diverse compounds can be accounted for by differential TAS2R activation
Publisher: Oxford University Press (OUP)
Date: 21-05-2015
Abstract: The human population displays high variation in taste perception. Differences in in idual taste sensitivity may also impact on nutrient intake and overall appetite. A well-characterized ex le is the variable perception of bitter compounds such as 6-n-propylthiouracil (PROP) and phenylthiocarbamide (PTC), which can be accounted for at the molecular level by polymorphic variants in the specific type 2 taste receptor (TAS2R38). This phenotypic variation has been associated with influencing dietary preference and other behaviors, although the generalization of PROP/PTC taster status as a predictor of sensitivity to other tastes is controversial. Here, we proposed that the taste sensitivities of different bitter compounds would be correlated only when they activate the same bitter taste receptor. Thirty-four volunteers were exposed to 8 bitter compounds that were selected based on their potential to activate overlapping and distinct repertoires of TAS2Rs. Taste intensity ratings were evaluated using the general Labeled Magnitude Scale. Our data demonstrate a strong interaction between the intensity for bitter substances when they activate common TAS2Rs. Consequently, PROP/PTC sensitivity was not a reliable predictor of general bitter sensitivity. In addition, our findings provide a novel framework to predict taste sensitivity based on their specific T2R activation profile.
Evaluation of the membrane-binding properties of the proximal region of the angiotensin II receptor (AT1A) carboxyl terminus by surface plasmon resonance
Publisher: Springer Science and Business Media LLC
Date: 02-2005
Abstract: The proximal region of the angiotensin II receptor (AT1A) carboxyl-terminus (known as helix VIII) is important for receptor function. In this study, we used surface plasmon resonance (SPR) to examine the interaction of helix VIII-derived peptides with three model lipid membranes. The membrane-binding properties of these synthetic peptides, as well as a series of peptide analogues with modified amino acid sequences, could be explained by both amino acid sequence and kinetic binding data by SPR. The helix VIII peptides showed a higher affinity for lipid membranes that contained negatively charged phospholipid, rather than zwitterionic phospholipid. The findings of an SPR study may be useful for estimating the cooperative binding of intracellular receptor domains with G proteins and the components of the lipid bilayer.
Direct actions of Urotensin II on the heart: Implications for cardiac fibrosis and hypertrophy
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 08-08-2003
DOI: 10.1161/01.RES.0000084382.64418.BC
Abstract: Urotensin II (UII) is a somatostatin-like peptide recently identified as a potent vasoconstrictor. In this study, we examined whether UII promotes cardiac remodeling through nonhemodynamic effects on the myocardium. In a rat model of heart failure after myocardial infarction (MI), increased UII peptide and UII receptor protein expression was observed in both infarct and noninfarct regions of the left ventricle compared with sham. Moreover, post-MI remodeling was associated with a significant 75% increase in UII receptor gene expression in the heart ( P .05 versus sham controls), with this increase noted in both regions of the left ventricle. In vitro, UII (10 −7 mol/L) stimulation of neonatal cardiac fibroblasts increased the level of mRNA transcripts for procollagens α 1 (I), α 1 (III), and fibronectin by 139±15% ( P .01), 59±5% ( P .05), and 141±14% ( P .01), respectively, with a concomitant 23±2% increase in collagen peptide synthesis as determined by 3 H-proline incorporation ( P .01). UII had no effect on cellular hypertrophy, as determined by changes in total protein content in isolated neonatal cardiomyocytes. However, expression of recombinant rat UII receptor in neonatal cardiomyocytes resulted in significant UII-dependent activation of hypertrophic signaling as demonstrated by increased total protein content (unstimulated, 122.4±4.0 μg/well rat UII, 147.6±7.0 μg/well P .01) and activation of the hypertrophic phenotype through Gαq- and Ras-dependent pathways. These results indicate that, in addition to potent hemodynamic effects, UII may be implicated in myocardial fibrogenesis through increased collagen synthesis by cardiac fibroblasts and may also be an important determinant of pathological cardiac hypertrophy in conditions characterized by UII receptor upregulation.
High-density lipoprotein modulates glucose metabolism in patients with type 2 diabetes mellitus
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 21-04-2009
DOI: 10.1161/CIRCULATIONAHA.108.843219
Abstract: Background— Low plasma high-density lipoprotein (HDL) is associated with elevated cardiovascular risk and aspects of the metabolic syndrome. We hypothesized that HDL modulates glucose metabolism via elevation of plasma insulin and through activation of the key metabolic regulatory enzyme, AMP-activated protein kinase, in skeletal muscle. Methods and Results— Thirteen patients with type 2 diabetes mellitus received both intravenous reconstituted HDL (rHDL: 80 mg/kg over 4 hours) and placebo on separate days in a double-blind, placebo-controlled crossover study. A greater fall in plasma glucose from baseline occurred during rHDL than during placebo (at 4 hours rHDL=−2.6±0.4 placebo=−2.1±0.3mmol/L P =0.018). rHDL increased plasma insulin (at 4 hours rHDL=3.4±10.0 placebo= −19.2±7.4 pmol/L P =0.034) and also the homeostasis model assessment β-cell function index (at 4 hours rHDL=18.9±5.9 placebo=8.6±4.4% P =0.025). Acetyl-CoA carboxylase β phosphorylation in skeletal muscle biopsies was increased by 1.7±0.3-fold after rHDL, indicating activation of the AMP-activated protein kinase pathway. Both HDL and apolipoprotein AI increased glucose uptake (by 177±12% and 144±18%, respectively P .05 for both) in primary human skeletal muscle cell cultures established from patients with type 2 diabetes mellitus (n=5). The mechanism is demonstrated to include stimulation of the ATP-binding cassette transporter A1 with subsequent activation of the calcium/calmodulin-dependent protein kinase kinase and the AMP-activated protein kinase pathway. Conclusions— rHDL reduced plasma glucose in patients with type 2 diabetes mellitus by increasing plasma insulin and activating AMP-activated protein kinase in skeletal muscle. These findings suggest a role for HDL-raising therapies beyond atherosclerosis to address type 2 diabetes mellitus.
Phospholipase C/Protein Kinase C Pathway Mediates Angiotensin II-Dependent Apoptosis in Neonatal Rat Cardiac Fibroblasts Expressing AT1 Receptor
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 08-2008
Hijacking epidermal growth factor receptors by angiotensin II: New possibilities for understanding and treating cardiac hypertrophy
Publisher: Springer Science and Business Media LLC
Date: 11-2004
DOI: 10.1007/S00018-004-4244-3
Abstract: Activation of the type 1 angiotensin II receptor (AT(1)R) is associated with the aetiology of left ventricular hypertrophy, although the exact intracellular signalling mechanism(s) remain unclear. Transactivation of the epidermal growth factor receptor (EGFR) has emerged as a central mechanism by which the G protein-coupled AT(1)R, which lacks intrinsic tyrosine kinase activity, can stimulate the mitogen-activated protein kinase signalling pathways thought to mediate cardiac hypertrophy. Current studies support a model whereby AT(1)R-dependent transactivation of EGFRs on cardiomyocytes involves stimulation of membrane-bound metalloproteases, which in turn cleave EGFR ligands such as heparin-binding EGF from a plasma membrane-associated precursor. Numerous aspects of the 'triple membrane-passing signalling' paradigm of AT(1)R-induced EGFR transactivation remain to be characterised, including the identity of the specific metalloproteases involved, the intracellular mechanism for their activation and the exact EGFR subtypes required. Here we examine how 'hijacking' of the EGFR might explain the ability of the AT(1)R to elicit the temporally and qualitatively erse responses characteristic of the hypertrophic phenotype, and discuss the ramifications of delineating these pathways for the development of new therapeutic strategies to combat cardiac hypertrophy.
A Novel Inhibitory Role for Glucocorticoids in the Secretion of Angiotensinogen by C6 Glioma Cells
Publisher: Wiley
Date: 04-1994
DOI: 10.1046/J.1471-4159.1994.62041296.X
Abstract: Astrocytes have been identified as the primary source of brain angiotensinogen (Ao), but the regulation of the secretion of this protein from astrocytes is poorly defined. In this study, the rat C6 glioma cell line was used as an astrocyte model to investigate the regulation of Ao secretion. C6 cultures secreted Ao at a rate of 4.05 +/- 1.52 (mean +/- SD) ng of Ao/10(6) cells/24 h as determined by a direct radioimmunoassay. This rate was not significantly altered by the hormones thyroxine, estradiol, angiotensin II, growth hormone, and prostaglandins or by increased levels of intracellular cyclic AMP. Treatment with the synthetic glucocorticoid dexamethasone (DEX 10(-6) M) reduced the rate of Ao secretion to 1.82 +/- 0.28 ng of Ao/10(6) cells/24 h. By comparison, the basal secretion rate for rat H4 hepatoma cells was 142.4 +/- 10.0 ng of Ao/10(6) cells/24 h, and this increased fourfold (572.4 +/- 173.1 ng/10(6) cells/24 h) in the presence of 10(-6) M DEX. Both these inhibitory (C6) and stimulatory (H4) actions of DEX were dose related. The inhibition observed in C6 cells was mimicked by RU28362, a pure glucocorticoid agonist, and reversed by the antagonist RU486, demonstrating that DEX was functioning as a true glucocorticoid. The action of DEX was also antagonized by the cyclic AMP analogue N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dBcAMP) (control, DEX, and DEX + dBcAMP, 3.58 +/- 0.73, 1.69 +/- 0.82, and 4.93 +/- 1.88 ng of Ao/10(6) cells/24 h, respectively, and by the beta-adrenergic agonist isoprenaline, which stimulates cyclic AMP production.(ABSTRACT TRUNCATED AT 250 WORDS)
Electrostatic and hydrophobic forces tether the proximal region of the angiotensin II receptor (AT1a ) carboxyl terminus to anionic lipids
Publisher: American Chemical Society (ACS)
Date: 22-05-2002
DOI: 10.1021/BI0121813
Abstract: The carboxyl terminus of the type-1 angiotensin II receptor (AT(1A)) is a focal point for receptor activation and deactivation. Synthetic peptides corresponding to the membrane-proximal, first 20 amino acids of the carboxyl terminus adopt an alpha-helical conformation in organic solvents, suggesting that the secondary structure of this region may be sensitive to hydrophobic environments. Using surface plasmon resonance, immobilized lipid chromatography, and circular dichroism, we examined whether this positively charged, hipathic alpha-helical region of the AT(1A) receptor can interact with lipid components in the cell membrane and thereby modulate local receptor attachment and structure. A synthetic peptide corresponding to the proximal region of the AT(1A) receptor carboxyl terminus (Leu(305) to Lys(325)) was shown by surface plasmon resonance to bind with high affinity to the negatively charged lipid, dimyristoyl L-alpha-phosphatidyl-DL-glycerol (DMPG), but poorly to the zwitterionic lipid, dimyristoyl L-alpha-phosphatidylcholine (DMPC). In contrast, a peptide analogue possessing substitutions at four lysine residues (corresponding to Lys(307,308,310,311)) displayed poor association with either lipid, indicating a crucial anionic component to the interaction. Circular dichroism analysis revealed that both the wild-type and substituted peptides possessed alpha-helical propensity in methanol and trifluoroethanol, while the wild-type peptide also adopted partially inserted helical structure in DMPG and DMPC liposomes. In contrast, the substituted peptide exhibited spectra that suggested the presence of beta-sheet and alpha-helical structure in both liposomes. Immobilized lipid chromatography was used to characterize the hydrophobic component of the membrane interaction, and the results demonstrated that hydrophobic and electrostatic interactions mediated the binding of the wild-type peptide but that the substituted peptide bound to the model membranes mainly via hydrophobic forces. We propose that, in intact AT(1A) receptors, the proximal carboxyl terminus associates with the cytoplasmic face of the cell membrane via a high-affinity, anionic phospholipid-specific tethering that serves to increase the hipathic helicity of this region. Such associations may be important for receptor function and common for G protein-coupled receptors.
Association of β-arrestin 1 with the type 1A angiotensin II receptor involves phosphorylation of the receptor carboxyl terminus and correlates with receptor internalization
Publisher: The Endocrine Society
Date: 10-2001
UBF levels determine the number of active ribosomal RNA genes in mammals
Publisher: Rockefeller University Press
Date: 22-12-2008
Abstract: In mammals, the mechanisms regulating the number of active copies of the ∼200 ribosomal RNA (rRNA) genes transcribed by RNA polymerase I are unclear. We demonstrate that depletion of the transcription factor upstream binding factor (UBF) leads to the stable and reversible methylation-independent silencing of rRNA genes by promoting histone H1–induced assembly of transcriptionally inactive chromatin. Chromatin remodeling is abrogated by the mutation of an extracellular signal-regulated kinase site within the high mobility group box 1 domain of UBF1, which is required for its ability to bend and loop DNA in vitro. Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased. We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression. Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation.
Adrenomedullin inhibits angiotensin AT1A receptor expression and function in cardiac fibroblasts
Publisher: Elsevier BV
Date: 04-2003
DOI: 10.1016/S0167-0115(03)00031-4
Abstract: Adrenomedullin (AM) is a multifunctional peptide hormone with wide-ranging actions related to cardiovascular homeostasis. AM receptors are highly expressed in the heart and AM has antihypertrophic and antiproliferative effects on cardiac myocytes and fibroblasts, respectively. We have investigated the interaction between AM and angiotensin II (Ang II) signalling in neonatal cardiac fibroblast cultures to determine whether the antagonistic effects of AM are mediated via the modulation of Ang II receptors. Cardiac fibroblasts exclusively expressed the Ang II type 1 receptor (AT(1)R) and binding to this site was downregulated by 35% following an 18-h incubation with 100 nM AM. Levels of AT(1A)R mRNA were dose-dependently lowered by AM, with a maximal 40-50% inhibition by 6 h. The decreases in both AT(1)R binding and AT(1A)R mRNA levels were mimicked by 8-Br-cAMP or forskolin, suggesting that the effects of AM were mediated via an elevation of cAMP. In cardiac fibroblasts pretreated with AM, the Ang II induction of collagen biosynthesis was attenuated, although basal collagen synthesis was unaffected. These data suggest that AM mediates the heterologous downregulation of AT(1)R expression via a relatively rapid decrease in AT(1A)R mRNA pools. This interaction may represent a relevant pathophysiological mechanism for modulating Ang II responsiveness in the diseased heart.
Stimulation of the four isoforms of receptor tyrosine kinase ErbB4, but not ErbB1, confers cardiomyocyte hypertrophy
Publisher: Wiley
Date: 29-09-2525
DOI: 10.1002/JCP.30487
Abstract: Epidermal growth factor (EGF) receptors (ErbB1–ErbB4) promote cardiac development and growth, although the specific EGF ligands and receptor isoforms involved in growth/repair versus pathology remain undefined. We challenged ventricular cardiomyocytes with EGF‐like ligands and observed that selective activation of ErbB4 (the receptor for neuregulin 1 [NRG1]), but not ErbB1 (the receptor for EGF, EGFR), stimulated hypertrophy. This lack of direct ErbB1‐mediated hypertrophy occurred despite robust activation of extracellular‐regulated kinase 1/2 (ERK) and protein kinase B. Hypertrophic responses to NRG1 were unaffected by the tyrosine kinase inhibitor (AG1478) at concentrations that are selective for ErbB1 over ErbB4. NRG1‐induced cardiomyocyte enlargement was suppressed by small interfering RNA (siRNA) knockdown of ErbB4 and ErbB2, whereas ERK phosphorylation was only suppressed by ErbB4 siRNA. Four ErbB4 isoforms exist (JM‐a/JM‐b and CYT‐1/CYT‐2), generated by alternative splicing, and their expression declines postnatally and following cardiac hypertrophy. Silencing of all four isoforms in cardiomyocytes, using an ErbB4 siRNA, abrogated NRG1‐induced hypertrophic promoter/reporter activity, which was rescued by coexpression of knockdown‐resistant versions of the ErbB4 isoforms. Thus, ErbB4 confers cardiomyocyte hypertrophy to NRG1, and all four ErbB4 isoforms possess the capacity to mediate this effect.
Phosphorylation of the angiotensin II (AT(1A)) receptor carboxyl terminus: A role in receptor endocytosis
Publisher: The Endocrine Society
Date: 10-1998
Abstract: The molecular mechanism of angiotensin II type I receptor (AT1) endocytosis is obscure, although the identification of an important serine/threonine rich region (Thr332Lys333Met334Ser335Thr336Leu337Ser338) within the carboxyl terminus of the AT1A receptor subtype suggests that phosphorylation may be involved. In this study, we examined the phosphorylation and internalization of full-length AT1A receptors and compared this to receptors with truncations and mutations of the carboxyl terminus. Epitope-tagged full-length AT1A receptors, when transiently transfected in Chinese hamster ovary (CHO)-K1 cells, displayed a basal level of phosphorylation that was significantly enhanced by angiotensin II (Ang II) stimulation. Phosphorylation of AT1A receptors was progressively reduced by serial truncation of the carboxyl terminus, and truncation to Lys325, which removed the last 34 amino acids, almost completely inhibited Ang II-stimulated 32P incorporation into the AT1A receptor. To investigate the correlation between receptor phosphorylation and endocytosis, an epitope-tagged mutant receptor was produced, in which the carboxyl-terminal residues, Thr332, Ser335, Thr336, and Ser338, previously identified as important for receptor internalization, were substituted with alanine. Compared with the wild-type receptor, this mutant displayed a clear reduction in Ang II-stimulated phosphorylation. Such a correlation was further strengthened by the novel observation that the Ang II peptide antagonist, Sar1Ile8-Ang II, which paradoxically causes internalization of wild-type AT1A receptors, also promoted their phosphorylation. In an attempt to directly relate phosphorylation of the carboxyl terminus to endocytosis, the internalization kinetics of wild-type AT1A receptors and receptors mutated within the Thr332-Ser338 region were compared. The four putative phosphorylation sites (Thr332, Ser335, Thr336, and Ser338) were substituted with either neutral [alanine (A)] or acidic amino acids [glutamic acid (E) and aspartic acid (D)], the former to prevent phosphorylation and the latter to reproduce the acidic charge created by phosphorylation. Wild-type AT1A receptors, expressed in Chinese hamster ovary cells, rapidly internalized after Ang II stimulation [t1/2 2.3 min maximal level of internalization (Ymax) 78.2%], as did mutant receptors carrying single acidic substitutions (T332E, t1/2 2.7 min, Ymax 76.3% S335D, t1/2 2.4 min, Ymax 76.7% T336E, t1/2 2.5 min, Ymax 78.2% S338D, t1/2 2.6 min, Ymax 78.4%). While acidic amino acid substitutions may simply be not as structurally disruptive as alanine mutations, we interpret the tolerance of a negative charge in this region as suggestive that phosphorylation may permit maximal internalization. Substitution of all four residues to alanine produced a receptor with markedly reduced internalization kinetics (T332A/S335A/T336A/S338A, t1/2 10.1 min, Ymax 47.9%), while endocytosis was significantly rescued in the corresponding quadruple acidic mutant (T332E/S335D/T336E/S338D, t1/2 6.4 min, Ymax 53.4%). Double mutation of S335 and T336 to alanine also diminished the rate and extent of endocytosis (S335A/T336A, 3.9 min, Ymax 69.3%), while the analogous double acidic mutant displayed wild type-like endocytotic parameters (S335D/T336E, t1/2 2.6 min, Ymax 77.5%). Based on the apparent rescue of internalization by acidic amino acid substitutions in a region that we have identified as a site of Ang II-induced phosphorylation, we conclude that maximal endocytosis of the AT1A receptor requires phosphorylation within this serine/threonine-rich segment of the carboxyl terminus.
Silencing relaxin-3 in nucleus incertus of adult rodents: A viral vector-based approach to investigate neuropeptide function
Publisher: Public Library of Science (PLoS)
Date: 02-08-2012
Liver angiotensin II receptors in the rat: Binding properties and regulation by dietary Na+ and angiotensin II
Publisher: Bioscientifica
Date: 07-1985
Abstract: Recent evidence suggests that angiotensin II (AII) acts on the liver via specific receptors. The aims of this study were to examine the general binding properties of these receptors in the rat and to determine the role of dietary Na+ and AII in the regulation of AII receptors. Binding of 125 I-labelled Ile 5 -AII to liver membranes was saturable and behaved as a single class of sites with an affinity ( K a ) of 2·9 l/nmol and a Hill coefficient of 0·99. Kinetic analysis of AII binding gave estimates for the rates of association and dissociation of 42 l/μmol per min and 1·5 × 10 −2 /min respectively. The binding of analogues exhibited the following order of potency: Val 5 -AII Ile 5 -AII AIII Sar 1 -Ala 8 -AII Sar 1 -Gly 8 -AII AI des-Asp-AI C 4 −C 8 pentapeptide Phe 1 -Tyr 8 -AII neurotensin luteinizing hormone-releasing hormone. Binding was enhanced by Mg 2+ and Ca 2+ and inhibited by EDTA and the reducing agent dithiothreitol. Low dietary Na + affected in a biphasic manner both the K a and concentration ( R o ) of liver AII receptors. Initially, R o decreased from 25·9±3·8 (control) to 16±1·9 ( s.e.m. ) pmol/g tissue by 1·5 days but thereafter it rapidly increased to 47·3±8·7 pmol/g tissue (3·5 days) and remained elevated to the end of the experiment, 8·5 days later. The K a initially increased from 2·7±0·3 (control) to 4·3±0·5 l/nmol (1·5 days) and then decreased steadily to 1·2±0·1 l/mol (8·5 days). The effect of AII was examined by infusing 24 pmol AII/kg per min for 5 days into the jugular vein by subcutaneous osmotic minipumps. The response was qualitatively similar to that for low dietary Na + . It was concluded that the rat liver has a high content of specific AII receptors that are responsive to changes in Na + and AII. J. Endocr. (1985) 106 , 103–111
Immunoprecipitation and phosphorylation of G protein-coupled receptors.
Publisher: Humana Press
Date: 2009
DOI: 10.1007/978-1-60327-317-6_26
Abstract: G protein-coupled receptors (GPCRs) are integral membrane proteins with seven transmembrane-spanning alpha-helices. Following ligand activation, many GPCRs are rapidly phosphorylated on serine/threonine residues in their cytoplasmic domains, principally the carboxyl-terminus. GPCR phosphorylation recruits arrestin proteins to the activated receptor leading to receptor internalization and desensitization. Arrestins also act as scaffolds to recruit other regulatory and signaling molecules to the receptor. The low level of expression of GPCRs in tissues, the difficulty in developing antibodies that can specifically detect and harvest receptor protein, and the hydrophobic and heterogeneric nature of GPCRs makes examination of their structure, function, and biology an interesting challenge. Receptor phosphorylation is typically performed in cells transfected with wild and mutated receptors usually bearing an epitope tag and equilibrated with [(32)Pi] to radiolabel cellular ATP pools. Following ligand stimulation, receptor protein is extracted using a detergent lysis buffer and immunoprecipitated with antibodies raised against the epitope tag following separation on SDS-polyacrylamide gel electrophoresis, phosphorylated receptors are quantified using phosphorimaging.
Real-Time Measurement of F-Actin Remodelling during Exocytosis Using Lifeact-EGFP Transgenic Animals
Publisher: Public Library of Science (PLoS)
Date: 02-07-2012
The angiotensin II type 2 (AT2) receptor: an enigmatic seven transmembrane receptor
Publisher: IMR Press
Date: 2009
DOI: 10.2741/3289
Abstract: Angiotensin II (AngII) interacts with two receptor subtypes, AT1 and AT2, belonging to the seven transmembrane receptor superfamily. Pharmacological investigations initially suggested that AT2 receptors antagonize AT1 effects. Data from AT2 receptor transgenic and knock-out mice have not been entirely consistent with this interpretation. At the cellular level, a clear mechanistic model of AT2 transduction and signalling has yet to emerge. The AT2 receptor displays the hallmark motifs and signature residues of a G protein-coupled receptor (GPCR), but fails to demonstrate most of the classic features of GPCR signalling. In recent years, unbiased screens for AT2-interacting proteins have identified novel partner proteins involved in AT2 signalling, providing new insight into the mechanisms of AT2 action. A growing body of evidence suggests that the AT2 receptor is constitutively active (i.e. signals without AngII). This review critically evaluates controversies surrounding physiological functions and signalling mechanisms of the AT2 receptor, primarily in a cardiovascular context. Recent advances in the field are highlighted and findings challenging the concept that the AT2 receptor is a conventional angiotensin receptor are considered.
Emerging role of the urotensin II system in cardiovascular disease
Publisher: S. Karger AG
Date: 2003
DOI: 10.1159/000072742
Abstract: Urotensin II is a potent 11-amino-acid vasoconstrictor peptide. The discovery of its endogenous receptor has led to renewed interest in its role in human cardiovascular physiology and pathophysiology. The cardiovascular actions of urotensin II are complex and include direct effects on the vasculature, systemic hemodynamic effects and effects on the myocardium, including actions on contractility, myocyte hypertrophy and extracellular matrix deposition. Plasma levels of urotensin have been found to be elevated in patients with chronic heart failure, diabetes mellitus and chronic renal disease. These elevations raise the possibility that urotensin II might be involved in the pathogenesis and/or progression of these disease states. Highly selective urotensin II antagonists have been developed, which should help to definitively clarify the role of urotensin II in these and other conditions.
Bitter taste receptor agonists elicit G-protein-dependent negative inotropy in the murine heart
Publisher: Wiley
Date: 07-2014
DOI: 10.1096/FJ.14-256305
Abstract: G-protein-coupled receptors (GPCRs) are key mediators in cardiovascular physiology, yet frontline therapies for heart disease target only a small fraction of the cardiac GPCR repertoire. Moreover, there is emerging evidence that GPCRs implicated in taste (Tas1r and Tas2rs) have specific functions beyond the oral cavity. Our recent description of these receptors in heart tissue foreshadows a potential novel role in cardiac cells. In this study, we identified novel agonist ligands for cardiac-Tas2rs to enable the functional investigation of these receptors in heart tissue. Five Tas2rs cloned from heart tissue were screened against a panel of 102 natural or synthetic bitter compounds in a heterologous expression system. We identified agonists for Tas2r108, Tas2r137, and Tas2r143 that were then tested in Langendorff-perfused mouse hearts (from 8-wk-old male C57BL/6 mice). All Tas2r agonists tested exhibited concentration-dependent effects, with agonists for Tas2r108 and Tas2r137, leading to a ∼40% decrease in left ventricular developed pressure and an increase in aortic pressure, respectively. These responses were abrogated in the presence of Gαi and Gβγ inhibitors (pertussis toxin and gallein). This study represents the first demonstration of profound Tas2r agonist-induced, G protein-dependent effects on mouse heart function.
G protein-coupled receptors in cardiac biology: old and new receptors
Publisher: Springer Science and Business Media LLC
Date: 13-01-2015
Functional role for the angiotensin II receptor (AT1A) 3′-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins
Publisher: Portland Press Ltd.
Date: 15-01-1998
DOI: 10.1042/BJ3290255
Abstract: We demonstrate a functional role for the 3ʹ-untranslated region (3ʹ-UTR) of the angiotensin II (Ang II) receptor subtype AT1A mRNA in Chinese hamster ovary (CHO-K1) cells by stably transfecting the coding region of the receptor gene with or without the 845 bp 3ʹ-UTR. Two cell lines expressing similar levels of cell-surface receptors (with 3ʹ-UTR, Bmax = 571 fmol/mg protein without 3ʹ-UTR, Bmax = 663 fmol/mg protein) were used in the present study. Both cell lines expressed high-affinity receptors (with 3ʹ-UTR, Kd = 0.83 nM without 3ʹ-UTR, Kd = 0.82 nM), and binding studies with 125I-labelled Ang II in the presence of GTP[S] demonstrated that both coupled to heterotrimeric G-proteins. Despite these similarities, significant differences were observed for receptor-mediated cell signalling pathways. In cells without the 3ʹ-UTR, Ang II stimulated an increase in cAMP accumulation (11-fold above control) and in cells with the 3ʹ-UTR no stimulation was observed, which was consistent with previous observations in most endogenous Ang II receptor (AT1)-expressing cells. Activation of cAMP by Ang II in cells without the 3ʹ-UTR correlated with an inhibition of DNA synthesis, determined by [3H]thymidine incorporation. Ang II-mediated responses were blocked by EXP3174, a selective non-peptide receptor antagonist. We also observed differences in the transient profiles of intracellular calcium between cells with and without the 3ʹ-UTR in response to Ang II. In cells with the 3ʹ-UTR, a sustained level of intracellular calcium was observed after Ang II stimulation, whereas cells without the 3ʹ-UTR displayed a full return to basal level within 50 s of Ang II treatment. Even though the expressed exogenous gene is under the control of a constitutively expressing promoter (cytomegalovirus promoter), Northern-blot analysis revealed a considerably greater accumulation of AT1A mRNA in cells without the 3ʹ-UTR compared with cells with the 3ʹ-UTR. Analysis of the decay rate of the AT1A mRNA in cells with and without the 3ʹ-UTR revealed that the normally unstable AT1A receptor mRNA became highly stable by removing its 3ʹ-UTR, identifying a role for the 3ʹ-UTR in mRNA destabilization. Interestingly, both cells express similar levels of receptors at the cell surface, suggesting that the 3ʹ-UTR is also involved in the efficient translation and/or translocation of the receptor protein to the plasma membrane. We hypothesized that these 3ʹ-UTR-mediated functions of the receptor are regulated by RNA-binding proteins. To identify possible RNA-binding proteins for the AT1A 3ʹ-UTR, cellular extracts were prepared from parental CHO-K1 cells and 3ʹ-UTR-binding assays, electrophoretic mobility-shift assays and UV crosslinking studies were performed. A major cellular protein of 55 kDa was identified, which specifically interacted with the 3ʹ-UTR. Our data suggest that the 3ʹ-UTR of the AT1A can control specific receptor functions, perhaps via selective recognition of the 3ʹ-UTR by RNA-binding proteins.
CRIM1 is necessary for coronary vascular endothelial cell development and homeostasis
Publisher: Springer Science and Business Media LLC
Date: 11-2017
DOI: 10.1007/S10735-016-9702-3
Abstract: Endothelial cells form a critical component of the coronary vasculature, yet the factors regulating their development remain poorly defined. Here we reveal a novel role for the transmembrane protein CRIM1 in mediating cardiac endothelial cell development. In the absence of Crim1 in vivo, the coronary vasculature is malformed, the number of endothelial cells reduced, and the canonical BMP pathway dysregulated. Moreover, we reveal that CRIM1 can bind IGFs, and regulate IGF signalling within endothelial cells. Finally, loss of CRIM1 from human cardiac endothelial cells results in misregulation of endothelial genes, predicted by pathway analysis to be involved in an increased inflammatory response and cytolysis, reminiscent of endothelial cell dysfunction in cardiovascular disease pathogenesis. Collectively, these findings implicate CRIM1 in endothelial cell development and homeostasis in the coronary vasculature.
Extrasensory perception: Odorant and taste receptors beyond the nose and mouth
Publisher: Elsevier BV
Date: 04-2014
DOI: 10.1016/J.PHARMTHERA.2013.11.004
Abstract: G protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptors and are prime therapeutic targets. The odorant and taste receptors account for over half of the GPCR repertoire, yet they are generally excluded from large-scale, drug candidate analyses. Accumulating molecular evidence indicates that the odorant and taste receptors are widely expressed throughout the body and functional beyond the oronasal cavity - with roles including nutrient sensing, autophagy, muscle regeneration, regulation of gut motility, protective airway reflexes, bronchodilation, and respiratory disease. Given this expanding array of actions, the restricted perception of these GPCRs as mere mediators of smell and taste is outdated. Moreover, delineation of the precise actions of odorant and taste GPCRs continues to be h ered by the relative paucity of selective and specific experimental tools, as well as the lack of defined receptor pharmacology. In this review, we summarize the evidence for expression and function of odorant and taste receptors in tissues beyond the nose and mouth, and we highlight their broad potential in physiology and pathophysiology.
CNTF reverses obesity-induced insulin resistance by activating skeletal muscle AMPK
Publisher: Springer Science and Business Media LLC
Date: 09-04-2006
DOI: 10.1038/NM1383
Abstract: Ciliary neurotrophic factor (CNTF) induces weight loss and improves glucose tolerance in humans and rodents. CNTF is thought to act centrally by inducing hypothalamic neurogenesis to modulate food intake and peripherally by altering hepatic gene expression, in a manner similar to that of leptin. Here, we show that CNTF signals through the CNTFRalpha-IL-6R-gp130beta receptor complex to increase fatty-acid oxidation and reduce insulin resistance in skeletal muscle by activating AMP-activated protein kinase (AMPK), independent of signaling through the brain. Thus, our findings further show that the antiobesogenic effects of CNTF in the periphery result from direct effects on skeletal muscle, and that these peripheral effects are not suppressed by diet-induced or genetic models of obesity, an essential requirement for the therapeutic treatment of obesity-related diseases.
Side-chain substitutions within angiotensin II reveal different requirements for signaling, internalization, and phosphorylation of type 1A angiotensin receptors.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 04-2002
DOI: 10.1124/MOL.61.4.768
Abstract: Binding of the peptide hormone angiotensin II (AngII) to the type 1 (AT(1A)) receptor and the subsequent activation of phospholipase C-mediated signaling, involves specific determinants within the AngII peptide sequence. In contrast, the contribution of such determinants to AT(1A) receptor internalization, phosphorylation and activation of mitogen-activated protein kinase (MAPK) signaling is not known. In this study, the internalization of an enhanced green fluorescent protein-tagged AT(1A) receptor (AT(1A)-EGFP), in response to AngII and a series of substituted analogs, was visualized and quantified using confocal microscopy. AngII-stimulation resulted in a rapid, concentration-dependent internalization of the chimeric receptor, which was prevented by pretreatment with the nonpeptide AT(1) receptor antagonist EXP3174. Remarkably, AT(1A) receptor internalization was unaffected by substitution of AngII side chains, including single and double substitutions of Tyr(4) and Phe(8) that abolish phospholipase C signaling through the receptor. AngII-induced receptor phosphorylation was significantly inhibited by several substitutions at Phe(8) as well as alanine replacement of Asp(1). The activation of MAPK was only significantly inhibited by substitutions at position eight in the peptide and specific substitutions did not equally inhibit inositol phosphate production, receptor phosphorylation and MAPK activation. These results indicate that separate, yet overlapping, contacts made between the AngII peptide and the AT(1A) receptor select/induce distinct receptor conformations that preferentially affect particular receptor outcomes. The requirements for AT(1A) receptor internalization seem to be less stringent than receptor activation and signaling, suggesting an inherent bias toward receptor deactivation.
Adenoviral-directed expression of the type 1A angiotensin receptor promotes cardiomyocyte hypertrophy via transactivation of the epidermal growth factor receptor
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 08-02-2002
Abstract: Angiotensin II (Ang II) may cause cardiac hypertrophy via type 1 Ang II receptors (AT 1 ) on cardiomyocytes and through growth factors released from cardiac fibroblasts. Whereas cardiomyocyte-specific AT 1 receptor expression produces cardiac hypertrophy and remodeling in vivo, delineation of the signals that mediate growth to Ang II is challenging because the prevailing in vitro model (cultured neonatal cardiomyocytes) expresses low levels of AT 1 receptor and responds inconsistently to Ang II. In this study, when AT 1A receptors were expressed using adenovirus in cultured neonatal cardiomyocytes, Ang II stimulated a robust hypertrophy that was not secondary to the release of cardiac fibroblast-derived factors, specifically endothelin-1. Hypertrophy was accompanied by the induction of the immediate-early response genes, c-fos and c-jun , and reexpression of atrial natriuretic peptide (ANP). Ang II–induced activation of an ANP promoter-reporter was inhibited by the dominant/negative mutants, GαqI and N17Ras, indicating that hypertrophic signaling by the AT 1A receptor is via heterotrimeric G protein coupling and downstream Ras pathways. AT 1A -mediated cardiomyocyte hypertrophy and mitogen-activated protein kinase (MAPK) activation were inhibited by the MAPK kinase inhibitor, PD98059, and the epidermal growth factor (EGF) receptor kinase antagonist, AG1478, but not by PKC inhibitor, bisindolylmaleimide-1. Moreover, Ang II–induced MAPK activation was prevented by treatment with a matrix metalloproteinase inhibitor, consistent with the tyrosine phosphorylation of the EGF receptor in response to AT 1A receptor activation. These data unequivocally demonstrate that Ang II can directly promote cardiac myocyte growth via AT 1A receptors expressed on these cells and reveal for the first time the important contribution of EGF receptor–transactivated MAPK signaling to this process.
Endothelin-1 activates ETA receptors on human vascular smooth muscle cells to yield proteoglycans with increased binding to LDL
Publisher: Elsevier BV
Date: 08-2009
DOI: 10.1016/J.ATHEROSCLEROSIS.2009.01.024
Abstract: Lipid retention in the vessel wall by glycosaminoglycan (GAG) chains on chondroitin/dermatan sulfate proteoglycans synthesized by vascular smooth muscle cells (VSMC) have recently been established as an early event in human coronary artery atherosclerosis. GAG structure can be altered by growth factors resulting in enhanced binding to low density lipoprotein (LDL). The aim of this study was to determine if proteoglycans produced by endothelin-1 treated VSMCs had increased binding to human LDL, to examine the effect of endothelin-1 on the synthesis and structure of proteoglycans and to elucidate the signalling pathway. Endothelin-1 stimulated an increase in [(35)S]sulfate and [(3)H]glucosamine incorporation into proteoglycans produced by human VSMC. The increase was due to an increase in GAG chain size assessed by SDS-PAGE and size exclusion chromatography. Increased radiolabel incorporation was inhibited by an ET(A) but not an ET(B) receptor antagonist. Endothelin-1 stimulated an increase in the 6:4 position sulfation ratio on the disaccharides of the GAG chains, an effect that was blocked by bosentan. The EGF receptor antagonist AG1478 did not affect the increase in GAG size mediated by endothelin-1. Inhibition of protein kinase C (PKC) with GF109203X or down regulation by PMA pre-treatment attenuated the effect of endothelin-1 on GAG synthesis. These data demonstrate that endothelin-1 stimulates changes in GAG chain structure that increase binding to LDL. This action of endothelin-1 may represent a new target for the prevention of lipid binding within the vascular wall and the associated complications resulting from this interaction.
A Bitter Taste in Your Heart
Publisher: Frontiers Media SA
Date: 08-05-2020
Cavin-1 deficiency modifies myocardial and coronary function, stretch responses and ischaemic tolerance: roles of NOS over-activity
Publisher: Springer Science and Business Media LLC
Date: 25-03-2017
DOI: 10.1007/S00395-017-0613-6
Abstract: Caveolae and associated cavin and caveolins may govern myocardial function, together with responses to mechanical and ischaemic stresses. Abnormalities in these proteins are also implicated in different cardiovascular disorders. However, specific roles of the cavin-1 protein in cardiac and coronary responses to mechanical/metabolic perturbation remain unclear. We characterised cardiovascular impacts of cavin-1 deficiency, comparing myocardial and coronary phenotypes and responses to stretch and ischaemia-reperfusion in hearts from cavin-1
Oxytocin Receptors in the Mammary Gland and Reproductive Tract of a Marsupial, the Brushtail Possum (Trichosurus Vulpecula)1
Publisher: Oxford University Press (OUP)
Date: 11-1991
DOI: 10.1095/BIOLREPROD45.5.673
Abstract: Previous studies of marsupial lactation have shown that the milk-ejection reflex changes in sensitivity, being greater in small mammary glands sucked by small pouch young and lesser in larger glands supplying milk to larger young. The involvement of oxytocin receptors in these changes was examined in the brushtail possum Trichosurus vulpecula. Oxytocin receptors were measured in the mammary glands, uterus, and medial vaginal sacs by radioreceptor assay, using [3H]oxytocin as radioligand. In the mammary gland, a single oxytocin binding site was found with an affinity and receptor concentration of 0.81 +/- 0.41 l/nmol and 10.2 +/- 4.8 pmol/g tissue respectively (SD, 10 possums). Competitive displacement curves with related peptides and analogs showed the following order of specificity: d(CH2)5[Tyr(Me)2,Thr4,Tyr9-NH2]-vasotocin much greater than vasotocin greater than oxytocin = Arg-vasopressin greater than mesotocin greater than [Thr4,Gly7]-oxytocin = Lys-vasopressin greater than [deamino-Pen1, O-methyl-Tyr2, Arg8]-vasopressin greater than isotocin much greater than [d(CH2)5, D-Phe2, Ile4, Ala9-NH2]-AVP. [3H]Oxytocin did not bind to vasopressin receptors in the thoracic aorta. The concentration of oxytocin receptors was very high in small mammary glands (18.6 pmol/g tissue in a 2-g gland) and decreased logarithmically as the size of the mammary gland increased. It is suggested that the changes in the sensitivity of milk ejection to oxytocin is related to the concentration of mammary oxytocin receptors. The presence of oxytocin receptors in both uterus and median vaginal sacs extends previous observations and supports the hypothesis that in marsupial parturition, the uterus and medial vaginal sacs respond as a single functional unit to oxytocin.
Expression of constitutively active angiotensin receptors in the rostral ventrolateral medulla increases blood pressure
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 06-2006
DOI: 10.1161/01.HYP.0000218576.36574.54
Abstract: Angiotensin type 1A (AT 1A ) receptors are expressed within the rostral ventrolateral medulla, and microinjections of angiotensin II into this region increase sympathetic vasomotor tone. To determine the effect of sustained increases in AT 1A receptor density or activity in rostral ventrolateral medulla, we used radiotelemetry to monitor blood pressure in conscious rats before and after bilateral microinjection into the rostral ventrolateral medulla of adenoviruses encoding the wild-type AT 1A receptor or a constitutively active version of the receptor (Asn 111 Gly, [N111G]AT 1A ). The constitutively active receptor signals in the absence of angiotensin II. Adenovirus-directed receptor expression was extensively characterized both in vitro and in vivo. We established that adenoviral infection was limited to the rostral ventrolateral medulla and that receptor expression was sustained for ≥10 days we also observed that adenoviral transgene expression occurs in glia, with no transgene expression observed in neurons of the rostral ventrolateral medulla. Rats receiving the wild-type AT 1A receptor showed no change in blood pressure, whereas animals receiving the [N111G]AT 1A receptor displayed an increase in blood pressure that persisted for 3 to 4 days before returning to basal levels. These data indicate that increased AT 1A receptor activity (not just overexpression) is a primary determinant of efferent drive from rostral ventrolateral medulla and reveal counterregulatory processes that moderate AT 1A receptor actions at this crucial relay point. More importantly, they imply that constitutive receptor signaling in glia of the rostral ventrolateral medulla can modulate the activity of adjacent neurons to change blood pressure.
Australian Policy Online
Publisher: Regional Development Australia
Date: 2002
Activation of the STAT Pathway by Angiotensin II in T3CHO/AT1A Cells
Publisher: Elsevier BV
Date: 08-1995
Development and optimization of MicroRNA against relaxin-3
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2008.03816.X
Abstract: Recent in vivo studies suggest a role for relaxin-3 in feeding and stress. To further elucidate the function of relaxin-3 in the central nervous system, we have employed a complementary approach, based on RNA interference, to modulate relaxin-3 expression. We have designed, constructed, and characterized three microRNAs (miRNAs) targeting different regions of the relaxin-3 transcript. These were tested to determine the amount of miRNA required to achieve the greatest knockdown and for their ability to reduce the expression of relaxin-3 in transfected HEK293Tcells. All miRNA constructs significantly reduced relaxin-3-induced cAMP responses however, miR499 was most effective. This should be a useful tool for in vitro studies and central targeting of relaxin-3 in vivo using viral delivery systems.
THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: G protein‐coupled receptors
Publisher: Wiley
Date: 11-11-2019
DOI: 10.1111/BPH.14748
Abstract: The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands ( www.guidetopharmacology.org ), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at oi/10.1111/bph.14748 . G protein‐coupled receptors are one of the six major pharmacological targets into which the Guide is ided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest
Publisher: Proceedings of the National Academy of Sciences
Date: 15-09-2017
Abstract: Engineered cardiac muscle can be used to promote the structural and functional maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). However, previous studies have not yet produced cardiac tissues with metabolic and proliferative maturation. Here, we develop a 96-well screening platform and screen for cardiac maturation conditions in engineered cardiac muscle. We found that simulating the postnatal switch in metabolic substrates from carbohydrates to fatty acids promoted a switch in metabolism, DNA damage response, and cell cycle arrest in hPSC-CM. Our study shows that this mechanism can be harnessed to enhance the maturation of human hPSC-CM and cardiac tissues, which has major implications for stem cell sciences, drug discovery, and regenerative medicine.
Immunocytochemical localization of angiotensinogen in the rat brain
Publisher: Elsevier BV
Date: 04-1988
DOI: 10.1016/0306-4522(88)90029-2
Abstract: The distribution of angiotensinogen-like immunoreactivity in the rat brain was investigated using specific antisera against pure rat plasma angiotensinogen in conjunction with the sensitive streptavidin-biotin peroxidase method. Angiotensinogen antisera were shown by radioimmunoassay and Western blotting to recognize angiotensinogen from both rat plasma and cerebrospinal fluid, and to cross-react with des-AI-angiotensinogen (100%) but not with angiotensin I and II, tetradecapeptide, luteinizing hormone-releasing hormone, rat albumin and angiotensinogen from eight other species. Angiotensinogen-like immunoreactivity was detected throughout the rat brain in both neuroglia and neurons. The highest concentration of neuroglial angiotensinogen-like immunoreactivity was in the hypothalamus and preoptic areas, with moderate to heavy concentrations in the mesencephalon and myelencephalon. The cerebellum demonstrated neuroglial staining in the granular layer and fibre tracts. Very little neuroglial staining was noted in the cerebral cortex or olfactory bulbs. Neuronal immunostaining was observed throughout the globus pallidus and the caudate putamen, in various parts of the thalamus and the supraoptic nucleus of the hypothalamus. In the midbrain moderate immunostaining was observed in periaquaductal central gray, the deep mesencephalic nucleus, the inferior colliculus and in scattered cells in the anterior mesencephalon. In the medulla, neuronal staining was localized to the vestibular nuclei and to other cell bodies mainly in the dorsolateral regions. In the cerebellum, staining was noted mainly in the deeper cerebellar nuclei and in the Purkinje cells. Immunostaining in the cerebral cortex was localized to the cingulate cortex and the primary olfactory cortex. Light staining was present in the endopiriform cortex and in scattered neurons adjacent to the external capsule. In the olfactory bulbs light neuronal staining was mainly associated with the mitral cell layer. The widespread distribution of angiotensinogen-like immunoreactivity supports the view that it is synthesized in the central nervous system and forms part of a brain renin-angiotensin system. In addition, its presence at sites other than those normally associated with the control of blood pressure and fluid and electrolyte homeostasis suggests that its involvement may not be limited to these regulatory functions.
Complex interactions between the angiotensin II type 1 receptor, the epidermal growth factor receptor and TRIO-dependent signaling partners
Publisher: Elsevier BV
Date: 06-2021
Extracellular Surface Residues of theα1B-Adrenoceptor Critical for G Protein–Coupled Receptor Function
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 28-10-2014
Abstract: Ligand binding and conformational changes that accompany signaling from G protein-coupled receptors (GPCRs) have mostly focused on the role of transmembrane helices and intracellular loop regions. However, recent studies, including several GPCRs cocrystallized with bound ligands, suggest that the extracellular surface (ECS) of GPCRs plays an important role in ligand recognition, selectivity, and binding, as well as potentially contributing to receptor activation and signaling. This study applied alanine-scanning mutagenesis to investigate the role of the complete ECS of the α1B-adrenoreceptor on norepinephrine (NE) potency, affinity, and efficacy. Half (24 of 48) of the ECS mutations significantly decreased NE potency in an inositol 1-phosphate assay. Most mutations reduced NE affinity (17) determined from [(3)H]prazosin displacement studies, whereas four mutations at the entrance to the NE binding pocket enhanced NE affinity. Removing the influence of NE affinity and receptor expression levels on NE potency gave a measure of NE efficacy, which was significantly decreased for 11 of 48 ECS mutants. These different effects tended to cluster to different regions of the ECS, which is consistent with different regions of the ECS playing discrete functional roles. Exposed ECS residues at the entrance to the NE binding pocket mostly affected NE affinity, whereas buried or structurally significant residues mostly affected NE efficacy. The broad potential for ECS mutations to affect GPCR function has relevance for the increasing number of nonsynonymous single nucleotide polymorphisms now being identified in GPCRs.
Interleukin-6 increases insulin-stimulated glucose disposal in humans and glucose uptake and fatty acid oxidation in vitro via AMP-activated protein kinase
Publisher: American Diabetes Association
Date: 10-2006
DOI: 10.2337/DB05-1404
Abstract: Although interleukin-6 (IL-6) has been associated with insulin resistance, little is known regarding the effects of IL-6 on insulin sensitivity in humans in vivo. Here, we show that IL-6 infusion increases glucose disposal without affecting the complete suppression of endogenous glucose production during a hyperinsulinemic-euglycemic cl in healthy humans. Because skeletal muscle accounts for most of the insulin-stimulated glucose disposal in vivo, we examined the mechanism(s) by which IL-6 may affect muscle metabolism using L6 myotubes. IL-6 treatment increased fatty acid oxidation, basal and insulin-stimulated glucose uptake, and translocation of GLUT4 to the plasma membrane. Furthermore, IL-6 rapidly and markedly increased AMP-activated protein kinase (AMPK). To determine whether the activation of AMPK mediated cellular metabolic events, we conducted experiments using L6 myotubes infected with dominant-negative AMPK α-subunit. The effects described above were abrogated in AMPK dominant-negative–infected cells. Our results demonstrate that acute IL-6 treatment enhances insulin-stimulated glucose disposal in humans in vivo, while the effects of IL-6 on glucose and fatty acid metabolism in vitro appear to be mediated by AMPK.
Tackling the EGFR in pathological tissue remodelling
Publisher: Elsevier BV
Date: 02-2006
DOI: 10.1016/J.PUPT.2005.04.005
Abstract: Tissue remodelling is an adaptive physiological event initiated by physical and/or hormonal stimuli and characterised by extracellular matrix modifications, inflammation, cellular hypertrophy, proliferation and/or apoptosis. Although its initial effects may be beneficial for the maintenance of organ function, it is evident that sustained remodelling processes can lead to pathological outcomes, such as fibrosis in asthma, and cardiac hypertrophy in heart failure. Our research is focussed upon cardiac hypertrophy and the significant contribution of the molecular pathway, termed 'the triple membrane-passing signalling' paradigm (TMPS), to this phenomenon. Cardiac hypertrophy describes the enlargement, but not proliferation, of cardiomyocytes in response to mechanical or hormonal factors to normalise cardiac output and accompanies other features of cardiac remodelling. As a major independent risk factor for heart failure, it is imperative that the molecular mechanisms that govern this phenotype are determined to identify possible therapeutic targets. This review will focus on the importance of matrix metalloproteases and epidermal growth factor receptors in the TMPS pathway and their potential as pharmacological targets for heart failure therapy. The evidence provided may have implications for pathological tissue remodelling in other organs.
The immunocytochemical localization of angiotensinogen in the rat ovary
Publisher: Springer Science and Business Media LLC
Date: 08-1990
DOI: 10.1007/BF00318679
Stable Expression of a Truncated AT1A Receptor in CHO-K1 Cells
Publisher: Elsevier BV
Date: 1995
The renin-angiotensin system and cancer: Old dog, new tricks
Publisher: Springer Science and Business Media LLC
Date: 22-10-2010
DOI: 10.1038/NRC2945
Abstract: For cancers to develop, sustain and spread, the appropriation of key homeostatic physiological systems that influence cell growth, migration and death, as well as inflammation and the expansion of vascular networks are required. There is accumulating molecular and in vivo evidence to indicate that the expression and actions of the renin-angiotensin system (RAS) influence malignancy and also predict that RAS inhibitors, which are currently used to treat hypertension and cardiovascular disease, might augment cancer therapies. To appreciate this potential hegemony of the RAS in cancer, an expanded comprehension of the cellular actions of this system is needed, as well as a greater focus on translational and in vivo research.
PAQR10 and PAQR11 mediate Ras signaling in the Golgi apparatus
Publisher: Springer Science and Business Media LLC
Date: 04-10-2011
DOI: 10.1038/CR.2011.161
Determination of the Exact Molecular Requirements for Type 1 Angiotensin Receptor Epidermal Growth Factor Receptor Transactivation and Cardiomyocyte Hypertrophy
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 05-2011
DOI: 10.1161/HYPERTENSIONAHA.110.166710
Abstract: Major interest surrounds how angiotensin II triggers cardiac hypertrophy via epidermal growth factor receptor transactivation. G protein–mediated transduction, angiotensin type 1 receptor phosphorylation at tyrosine 319, and β-arrestin–dependent scaffolding have been suggested, yet the mechanism remains controversial. We examined these pathways in the most reductionist model of cardiomyocyte growth, neonatal ventricular cardiomyocytes. Analysis with [ 32 P]-labeled cardiomyocytes, wild-type and [Y319A] angiotensin type 1 receptor immunoprecipitation and phosphorimaging, phosphopeptide analysis, and antiphosphotyrosine blotting provided no evidence for tyrosine phosphorylation at Y319 or indeed of the receptor, and mutation of Y319 (to A/F) did not prevent either epidermal growth factor receptor transactivation in COS-7 cells or cardiomyocyte hypertrophy. Instead, we demonstrate that transactivation and cardiomyocyte hypertrophy are completely abrogated by loss of G-protein coupling, whereas a constitutively active angiotensin type 1 receptor mutant was sufficient to trigger transactivation and growth in the absence of ligand. These results were supported by the failure of the β-arrestin–biased ligand SII angiotensin II to transactivate epidermal growth factor receptor or promote hypertrophy, whereas a β-arrestin–uncoupled receptor retained these properties. We also found angiotensin II–mediated cardiomyocyte hypertrophy to be attenuated by a disintegrin and metalloprotease inhibition. Thus, G-protein coupling, and not Y319 phosphorylation or β-arrestin scaffolding, is required for epidermal growth factor receptor transactivation and cardiomyocyte hypertrophy via the angiotensin type 1 receptor.
Type 1 angiotensin receptor pharmacology: Signaling beyond G proteins
Publisher: Elsevier BV
Date: 2007
Angiotensin receptors in an Australian marsupial, the brushtail possum Trichosurus vulpecula
Publisher: Elsevier BV
Date: 1990
DOI: 10.1016/0016-6480(90)90212-5
Abstract: In this study, the binding properties of angiotensin receptors were examined in the liver, adrenal, brain, and vascular tissue of the brushtail possum, Trichosurus vulpecula. With 125I-Ile5-angiotensin II as the radioligand, the binding affinity (Ka) and receptor number (R0) were estimated for the liver (Ka = 3.60 +/- 0.31 liters/nmol R0 = 23.8 +/- 1.30 pmol/g tissue n = 8) and adrenal (Ka = 1.68 +/- 0.29 liters/nmol R0 = 1.67 +/- 0.23 pmol/g tissue n = 8). Specific binding was not found in any of seven areas of the possum brain (n = 6), whereas the expected binding was present in similar areas of the rat brain. Using angiotensin III or the antagonist Sar1-Ala8-angiotensin II as radioligands or changing the composition of the incubation buffer did not alter the outcome. Moreover, the intracerebroventricular injection of 1 and 5 nmol of angiotensin II did not elicit an increase in blood pressure which could be attributed to brain angiotensin II (AII) receptors. Ligand affinities of the adrenal and liver receptors were found to be in the following decreasing order: Val5-AII greater than Ile5-AII = Ile5-AIII greater than Sar1-Ala8-AII greater than Sar1-Gly8-AII greater than Sar1-Leu8-AII greater than Ile5-AI greater than hexapeptide greater than Phe3-Tyr8-AII. The cardiovascular AII receptor was investigated by generating dose-response curves of the pressor activity of Ile5-AII and six AII analogs infused intravenously. It was concluded that liver, adrenal, and vascular AII receptors in the marsupial possum have characteristics similar to those in eutherian mammals. However, the failure to find brain AII receptors raises the possibility that those functions mediated by such receptors in the eutherian brain are absent in the possum and perhaps other marsupials.
Crim1 has cell-autonomous and paracrine roles during embryonic heart development
Publisher: Springer Science and Business Media LLC
Date: 29-01-2016
DOI: 10.1038/SREP19832
Abstract: The epicardium has a critical role during embryonic development, contributing epicardium-derived lineages to the heart, as well as providing regulatory and trophic signals necessary for myocardial development. Crim1 is a unique trans-membrane protein expressed by epicardial and epicardially-derived cells but its role in cardiogenesis is unknown. Using knockout mouse models, we observe that loss of Crim1 leads to congenital heart defects including epicardial defects and hypoplastic ventricular compact myocardium. Epicardium-restricted deletion of Crim1 results in increased epithelial-to-mesenchymal transition and invasion of the myocardium in vivo, and an increased migration of primary epicardial cells. Furthermore, Crim1 appears to be necessary for the proliferation of epicardium-derived cells (EPDCs) and for their subsequent differentiation into cardiac fibroblasts. It is also required for normal levels of cardiomyocyte proliferation and apoptosis, consistent with a role in regulating epicardium-derived trophic factors that act on the myocardium. Mechanistically, Crim1 may also modulate key developmentally expressed growth factors such as TGFβs, as changes in the downstream effectors phospho-SMAD2 and phospho-ERK1/2 are observed in the absence of Crim1 . Collectively, our data demonstrates that Crim1 is essential for cell-autonomous and paracrine aspects of heart development.
Transactivation of the epidermal growth factor receptor in responses to myocardial stress and cardioprotection
Publisher: Elsevier BV
Date: 02-2017
DOI: 10.1016/J.BIOCEL.2016.12.014
Abstract: The epidermal growth factor receptor (EGFR) family comprises the ErbB1 (EGFR) and ErbB4 receptors as well as the 'co-receptors' ErbB2 (which does not bind EGF ligands) and ErbB3 (which lack tyrosine kinase activity). This family of receptors is essential for cardiac development, myocardial, renal and vascular function, and cardiac responses to physiological and pathological perturbations. The EGFR appears critical in protecting cardiac cells from injury, while considerable attention has focussed on neuregulin/ErbB4 signalling in potentially ameliorating cardiomyopathy/heart failure. Indeed, the EGFRs provide a signalling nexus, upon which multiple cardioprotective stimuli appear to converge, including ischaemic preconditioning and various G protein-coupled receptors (opioid, muscarinic, adenosine, adrenergic, bradykinin, sphingosine 1-phosphate). These stimuli engage the EGFR axis (in a process referred to as transactivation) in differing ways, involving both G protein-dependent and -independent mechanisms, to promote myocardial cell survival during and following ischaemia/infarction. Elucidating the molecular processes that underpin EGFR transactivation and mediate cardiac protection will advance our understanding of the intrinsic capacity of the heart to withstand pathological insult. It should also reveal new approaches to facilitate cardioprotective therapy to limit damage during and following myocardial ischaemia/infarction, which despite intense investigation remains an unrealised, yet highly desirable, clinical goal. This review focuses on the cardiovascular functions of the EGFR, its role in cardioprotection, and the potential influences of common disease states on this signalling.
P38 mitogen-activated protein kinase inhibition improves cardiac function and attenuates left ventricular remodeling following myocardial infarction in the rat
Publisher: Elsevier BV
Date: 10-2004
DOI: 10.1016/J.JACC.2004.07.038
Abstract: The aim of this study was to examine the effect of the p38 mitogen-activated protein kinase (MAPK) inhibitor, RWJ-67657 (RWJ), on left ventricular (LV) dysfunction and remodeling post-myocardial infarction (MI) in rats. p38 MAPK signaling has been implicated in the progression of chronic heart failure. From day 7 post-MI (coronary artery ligation), rats received either RWJ (50 mg/day, by gavage, n = 8, MI+RWJ) or vehicle (by gavage, n = 8, MI+V) for 21 days. Echocardiography was performed on day 6, before the commencement of treatment, and on day 27. In vivo hemodynamic measurements were made on day 28. Sham-operated rats served as controls. The LV end-diastolic pressure and lung/body weight ratio were reduced, whereas the maximum rate of rise of LV pressure was increased towards sham levels in MI+RWJ compared with MI+V. Baseline echocardiographic studies demonstrated uniform LV remodeling and dysfunction in MI rats. Fractional shortening (FS) further deteriorated in MI+V, whereas FS was preserved in MI+RWJ. Progressive LV dilation and infarct expansion observed in MI+V were inhibited in MI+RWJ. MI+RWJ also demonstrated increased myocyte hypertrophy in the peri-infarct and non-infarct zones, and reduced myocardial collagen and alpha-smooth muscle actin (SMA) immunoreactivity compared with MI+V. The antifibrotic effects of RWJ in vivo may reflect direct effects on cardiac fibroblasts, because RWJ attenuated transforming growth factor beta-1-stimulated collagen synthesis and alpha-SMA expression in isolated cardiac fibroblasts. RWJ also protected cultured myocytes from hydrogen peroxide-induced apoptosis. RWJ-67657 treatment post-MI had beneficial effects on LV remodeling and dysfunction, supporting a key role for p38 MAPK in pathologic cell signaling in these processes and its inhibition as a novel therapy.
Cardiovascular role of urotensin II: Effect of chronic infusion in the rat
Publisher: Elsevier BV
Date: 10-2004
DOI: 10.1016/J.PEPTIDES.2004.03.029
Abstract: Urotensin II (UII) is a potent vaso-active peptide thought to have multiple roles in the regulation of cardiovascular physiology and pathophysiology. The actions of UII are complex and difficult to interpret given its systemic hemodynamic effects and variable action on different vascular beds and isolated vessels. Direct effects of UII on the myocardium, include myocyte hypertrophy, extracellular matrix deposition and contractility. These observations, together with elevated plasma levels found in disease, are common traits reported in other pathophysiologically implicated neurohormonal systems. In this review, we include original data obtained from chronic infusion of UII in rats. We report a reduction in first derivative of left ventricular pressure (+dP/dt), as well as an increase in the ratio of left ventricular collagen I:III, that may contribute to the reduced myocardial contractility observed in these animals.
Corrigendum to ‘BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes’. Biochemical Pharmacology 158(2018) 232-242
Publisher: Elsevier BV
Date: 10-2021
Role of helix 8 in G protein-coupled receptors based on structure-function studies on the type 1 angiotensin receptor
Publisher: Elsevier BV
Date: 04-2009
DOI: 10.1016/J.MCE.2009.01.002
Abstract: G protein-coupled receptors (GPCRs) are transmembrane receptors that convert extracellular stimuli to intracellular signals. The type 1 angiotensin II receptor is a widely studied GPCR with roles in blood pressure regulation,water and salt balance and cell growth. The complex molecular and structural changes that underpin receptor activation and signaling are the focus of intense research. Increasingly, there is an appreciation that the plasma membrane participates in receptor function via direct, physical interactions that reciprocally modulate both lipid and receptor and provide microdomains for specialized activities. Reversible protein:lipid interactions are commonly mediated by hipathic -helices in proteins and one such motif - a short helix, referred to as helix VIII/8 (H8), located at the start of the carboxyl (C)-terminus of GPCRs - is gaining recognition for its importance to GPCR function. Here, we review the identification of H8 in GPCRs and examine its capacity to sense and interact with erse proteins and lipid environment, most notably with acidic lipids that include phosphatidylinositol phosphates.
Uterine oxytocin receptors in an australian marsupial, the brushtail possum, Trichosurus vulpecula
Publisher: Elsevier BV
Date: 1990
DOI: 10.1016/0300-9629(90)90021-J
Abstract: 1. Oxytocin receptors in the uterus of the brushtail possum (T. vulpecula) were characterized by radioreceptor assay and compared with those of the sheep and rat uterus. 2. A single oxytocin binding site was found with an affinity (Kd) and receptor concentration (Ro) of 3.0 +/- 0.8 nmol/l and 200 +/- 60 fmol/mg protein, respectively (SEM n = 5). The receptor was stable at -20 degrees C alent ions were required for optimum binding. 3. Competitive displacement curves with related peptides showed the following order of specificity: vasotocin greater than oxytocin greater than mesotocin = arginine-vasopressin = [Thr4, Gly7]-oxytocin greater than lysine-vasopressin = isotocin much greater than [d(CH2)5, D-Phe2, Ile4, Ala9-NH2]-AVP. 4. It was concluded that oxytocin receptors in the possum have similar characteristics to those of placental mammals.
Arresting angiotensin type 1 receptors
Publisher: Elsevier BV
Date: 04-2003
DOI: 10.1016/S1043-2760(03)00023-7
Abstract: The type 1 angiotensin (AT(1)) receptor mediates the homeostatic and pathological actions of the peptide hormone, angiotensin II. With regard to the processes that activate and deactivate seven-transmembrane-spanning, G-protein-coupled receptors (GPCRs), AT(1) receptors are among the most widely studied, serving as prototypes for GPCRs that bind and respond to peptide hormones. Arrestins are proteins that bind to activated and phosphorylated GPCRs, terminating initial signals emanating from these receptors, in addition to mediating receptor internalization. New aspects of arrestin function continue to emerge, such as their capacity to act as scaffolds to recruit regulatory and signaling molecules to increase the repertoire of receptor responses. Here, we examine the evidence that arrestins contribute to the signaling, deactivation and trafficking of AT(1) receptors.
Unravelling the molecular complexity of GPCR-mediated EGFR transactivation using functional genomics approaches
Publisher: Wiley
Date: 23-09-2013
DOI: 10.1111/FEBS.12509
Abstract: To influence physiology and pathophysiology, G protein-coupled receptors (GPCRs) have evolved to appropriate additional signalling modalities, such as activation of adjacent membrane receptors. Epidermal growth factor receptors (EGFRs) mediate growth and remodelling actions of GPCRs, although the precise network of gene products and molecular cascades linking GPCRs to EGFRs (termed EGFR transactivation) remains incomplete. In this review, we describe the current view of GPCR-EGFR transactivation, identifying the established models of receptor cross-talk. We consider the limitations in our current knowledge, and propose that recent advances in molecular and cell biology technology, including functional genomics approaches, will allow a renewed focus of efforts to understand the mechanism underlying EGFR transactivation. Using an unbiased approach for identification of the molecules required for GPCR-mediated EGFR transactivation will provide a contemporary and more complete representation from which to extrapolate therapeutic control in diseases from cardiovascular remodelling to cancer.
Prolonged RXFP1 and RXFP2 signaling can be explained by poor internalization and a lack of β-arrestin recruitment
Publisher: American Physiological Society
Date: 05-2009
DOI: 10.1152/AJPCELL.00581.2008
Abstract: Relaxin induces sustained physiological responses, which brings into question the deactivation processes typical of most G protein-coupled receptors (GPCR) for its receptor, relaxin family peptide receptor 1 (RXFP1). Here, we examined relaxin-dependent phosphorylation of RXFP1 and the related insulin-like peptide 3 (INSL3) receptor, RXFP2, as well as the capacity of these receptors to recruit β-arrestins and internalize in response to ligand stimulation. We confirmed in human embryonic kidney (HEK)-293T cells, expressing RXFP1 or RXFP2, that both receptors elicit prolonged cAMP responses up to 6 h after stimulation. Receptors immunoprecipitated from 32 P metabolically labeled cells were used to investigate the agonist-specific phosphorylation. Rapid and robust receptor phosphorylation was not observed for either RXFP1 or RXFP2, although some 32 P-incorporation was observed at 30 min however, this was not statistically significant. In accord with this result, RXFP1 and RXFP2 demonstrated poor internalization in response to relaxin or INSL3, as compared with the angiotensin II type 1 receptor (AT 1 R), which undergoes rapid and robust phosphorylation and internalization in response to angiotensin II. Additionally, coexpression of GPCR kinases has no effect on the rate of internalization for either RXFP1 or RXFP2. Confocal microscopy was used to follow the trafficking of green fluorescent protein-labeled β-arrestins after receptor activation. Neither RXFP1 nor RXFP2 activation results in recruitment of β-arrestins to the cell surface, whereas AT 1 R rapidly recruits both β-arrestins-1 and -2. The apparent lack of classical regulation for RXFP1 and RXFP2 provides the molecular basis for the prolonged signaling and physiological actions of relaxin and related peptides.
Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein–protein interactions in live cells
Publisher: Elsevier BV
Date: 10-2006
DOI: 10.1016/J.CELLSIG.2006.01.004
Abstract: Bioluminescence resonance energy transfer (BRET) is an increasingly popular technique for studying protein-protein interactions in live cells. It is particularly suitable for real-time monitoring of such interactions, however, the timescale over which assays can be carried out is currently relatively short (minutes) due to substrate instability. We present a new derivation of the BRET technology, termed 'extended BRET' (eBRET), which now enables protein-protein interactions to be monitored in real-time for many hours. This capability has significant benefits for investigating cellular function over extended timescales, as we have illustrated using the agonist-induced G-protein coupled receptor/beta-arrestin interaction. The potential for studying the modulation of such interactions by agonists, antagonists, inhibitors, dominant negative mutants and co-expressed accessory proteins is substantial. Furthermore, the advantages of eBRET have important implications for the development of high-throughput BRET screening systems, an ever-expanding area of interest for the pharmaceutical industry.
The power of observation: Making research visible, accessible, and usable for the Asian digital economy
Publisher: Digital Asia Hub
Date: 2015
Molecular forms of rat angiotensinogen in plasma and brain: identification by isoelectric focusing and immunoblot analysis
Publisher: Elsevier BV
Date: 09-1995
DOI: 10.1016/0167-0115(95)00071-I
Abstract: Angiotensinogen (Ao) is the glycoprotein precursor of the vasoactive peptide angiotensin II. While Ao is synthesized as multiple molecular forms, the biochemical characteristics of this protein in blood and other tissues have not been defined. In this study, the charge heterogeneity of Ao in rat plasma, cerebrospinal fluid and that secreted by astrocyte and neuronal cultures was examined using analytical isoelectric focusing in combination with immunoblotting and quantitative image analysis. Normal rat male plasma Ao separated into 9 isoforms in the pI range 4.34-4.92 (1, 4.34 2, 4.41 3, 4.48 4, 4.58 5, 4.61 6, 4.66 7, 4.68 8, 4.81 9, 4.92) the percentage contribution of each to total plasma Ao was 13, 20, 23, 18, 2, 7, 10, 5, and < 1, respectively. A similar isoelectric focusing pattern was observed in female rat plasma with the exception that the relative contribution of isoform 6 was reduced to 2% of total Ao. Cerebrospinal fluid Ao displayed a more erse charge heterogeneity than plasma Ao, focusing over a broader pI range of 4.42-5.24. Astrocytes and neurons secreted Ao isoforms in the pI range 4.44-5.29 and 4.42-4.95, respectively, with the astrocyte cultures showing additional bands towards the cathode. It was concluded that rat Ao is secreted as multiple charged forms that are regulated in a sex- and cell-specific manner. These differences between plasma and brain Ao suggest a functional ersity, a view which is supported by recent evidence linking Ao variants to hypertension.
Fine mapping of Lvm1: A quantitative trait locus controlling heart size independently of blood pressure
Publisher: Elsevier BV
Date: 02-2006
DOI: 10.1016/J.PUPT.2005.02.010
Abstract: We have previously reported a quantitative trait locus (QTL) on rat chromosome 2 that influences heart size independently of blood pressure (Left Ventricular Mass Locus 1 Lvm1). The recent release of the rat genome sequence allowed us to retest and refine this relatively broad QTL with a view to identifying within it candidate genes worthy of structural investigation. We sought to achieve this 'fine mapping' by increasing the marker density within the interval and undertaking a linkage analysis in a previously defined population of F2 hybrids generated from inbred spontaneously hypertensive rats (SHR) of the Okamoto strain and Fischer rat (F344) progenitors. We were able to reconfirm and resolve Lvm1 from its original width of approximately 45 to 15 cM. By reference to the ENSEBL rat genome data bank, we identified within Lvm1 27 known genes, 109 predicted genes and 7 pseudogenes. Of the known genes, candidates include potential regulators of cardiac growth, a sodium channel and calcium channel as well as the fibroblast growth factor 2 gene. Located nearby the Lvm1 locus was the gene for the angiotensin Type 1B receptor. Given the evidence that the ligand for the angiotensin Type 1B receptor-angiotensin II-is a potent cardiotroph, we also consider this gene a potential candidate. The identification of the precise allelic variant(s) within Lvm1 involved in the control of pressure-independent cardiac growth awaits further molecular studies.
Agonist-induced phosphorylation of the angiotensin II (AT(1A)) receptor requires generation of a conformation that is distinct from the inositol phosphate-signaling state
Publisher: Elsevier BV
Date: 2000
Relative affinity of angiotensin peptides and novel ligands at AT1 and AT2 receptors
Publisher: Portland Press Ltd.
Date: 07-06-2011
DOI: 10.1042/CS20110036
Abstract: AT1R (angiotensin type 1 receptor) and AT2R (angiotensin type 2 receptor) are well known to be involved in the complex cardiovascular actions of AngII (angiotensin II). However, shorter peptide fragments of AngII are thought to have biological activity in their own right and elicit effects that oppose those mediated by AngII. In the present study, we have used HEK (human embryonic kidney)-293 cells stably transfected with either AT1R or AT2R to perform a systematic analysis of binding affinities of all the major angiotensin peptides. Additionally, we tested the novel AT2R agonist Compound 21, as well as the MasR (Mas receptor) agonist and antagonist AVE0991 and A-779 respectively, for their ability to bind to AT1R or AT2R. Candesartan, CGP42214 and PD123319 were used as reference compounds. Binding studies using 125I-[Sar1Ile8]AngII on the AT1R-transfected HEK-293 cells revealed only AngII, AngIII [angiotensin III angiotensin-(2–8)] and candesartan to have high affinity for AT1R. In the AT2R-transfected HEK-293 cells, competition for 125I-[Sar1Ile8]AngII binding was observed for all ligands except candesartan, AVE0991 and A-779, the latter two compounds having negligible affinity at either AT1R or AT2R. The rank order of affinity of ligands at AT2R was CGP42112& AngII≥AngIII& Compound 21≥PD123319≫AngIV [angiotensin IV angiotensin-(3–8)]& Ang-(1–7) [angiotensin-(1–7)]. Of note, although AngIV and Ang-(1–7) exhibited only modest affinity at AT2R compared with AngII, these two angiotensin peptides, together with AngIII, had substantial AT2R selectivity over AT1R. Collectively, our results suggest that shorter angiotensin peptides can act as endogenous ligands at AT2R.
BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes
Publisher: Elsevier BV
Date: 12-2018
Dual Pathways for Nuclear Factor κB Activation by Angiotensin II in Vascular Smooth Muscle
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 11-11-2005
DOI: 10.1161/01.RES.0000190589.52286.41
Abstract: Activation of nuclear factor (NF)-κB by angiotensin II (Ang II) plays an essential role in stimulating expression of vascular adhesion molecules, which are essential for vascular inflammation. We report that Ang II activates NF-κB by phosphorylating its p65 subunit via a pathway mediated partially by ribosomal S6 kinase (RSK). In investigating other pathway(s) that may be involved, we found that the ability of Ang II to activate NF-κB in mouse embryonic fibroblast is suppressed (& %) either by deletion of IκB Kinase (IKK) or by inhibiting or knocking down IKK in vascular smooth muscle cells using a dominant-negative IKK adenovirus or small interference RNA to IKKβ. Thus, Ang II also stimulates NF-κB via IKK. In vitro, we found that Ang II stimulates IKK to phosphorylate myelin basic protein and the p65 subunit of NF-κB. The mechanism by which Ang II activates IKK is to increase phosphorylation of IKKβ in its activation loop (Ser181) rather than IκB phosphorylation. Inhibiting both the RSK and IKK pathways completely blocks the Ang II–induced p65 phosphorylation and NF-κB activation. These 2 pathways are independent: inhibiting IKK does not block Ang II–induced phosphorylation of RSK, whereas inhibiting mitogen-activated protein kinase 1 does not affect phosphorylation of IKK. Finally, we found that Ang II can induce expression of vascular adhesion molecules by 2 pathways both IKK and RSK lead to phosphorylation of the p65 subunit of NF-κB to increase vascular cell adhesion molecule-1 transcription. The 2 pathways are functionally important because inhibiting IKK and RSK in vascular smooth muscle cells blocks Ang II–induced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 to limit vascular inflammation.
Where is the evidence: realising the value of grey literature for public policy and practice
Publisher: Swinburne Institute for Social Research
Date: 2014
Effect of intra-ovarian infusion of oxytocin on plasma progesterone concentrations in pregnant ewes
Publisher: Bioscientifica
Date: 07-1991
Abstract: The function of oxytocin receptors in the corpus luteum of pregnant ewes was investigated by infusing saline or oxytocin (100 ng/min) into the utero-ovarian artery of pregnant ewes (62 +/- 5 days, n = 12). During a 4-h infusion, plasma oxytocin (OT) concentration increased to 268 +/- 80 pg OT/ml in the OT-infused group and remained unchanged at 2.5 +/- 1.5 pg OT/ml in the saline-infused group. Progesterone concentration in jugular venous plasma (17 +/- 9 ng/ml) rapidly decreased during oxytocin infusion to 59 +/- 10% and 26 +/- 9% of control at 1.5 and 2 h, respectively the utero-ovarian venous concentration of 64 +/- 38 ng/ml decreased by a similar magnitude during oxytocin infusion. Electron microscopy of corpora lutea, removed at the end of the experiments, showed no indication of luteolytic changes following oxytocin infusion. It was concluded that oxytocin markedly and rapidly reduces progesterone secretion in pregnant ewes.
Evidence against a role for protein kinase C in the regulation of the angiotensin II (AT1A) receptor
Publisher: Elsevier BV
Date: 1996
DOI: 10.1016/0014-2999(95)00732-6
Abstract: Three putative protein kinase C phosphorylation sites in the carboxyl-terminal region of the angiotensin II AT1A receptor suggest that protein kinase C is involved in the regulation and desensitisation of this receptor. We investigated this possibility by measuring angiotensin II induced Ca2+ transients in cultures of neonatal rat cardiac fibroblasts which express predominantly the angiotensin AT1A receptor. Stimulating or inhibiting protein kinase C activity had no effect on angiotensin II stimulated Ca2+ transients. In addition, in situ and in vitro kinase assays revealed that a peptide, corresponding to the region of the angiotensin AT1A receptor containing the protein kinase C sites, was a poor substrate for protein kinase C. Thus, a heterologous desensitising role for this kinase on angiotensin AT1A receptors in these fibroblasts appears unlikely.
Type I Diabetes Mellitus Increases the Cardiovascular Complications of Influenza Virus Infection
Publisher: Frontiers Media SA
Date: 14-09-2021
DOI: 10.3389/FCIMB.2021.714440
Abstract: People with diabetes mellitus are susceptible to both cardiovascular disease and severe influenza A virus infection. We hypothesized that diabetes also increases risks of influenza-associated cardiac complications. A murine type 1 (streptozotocin-induced) diabetes model was employed to investigate influenza-induced cardiac distress. Lung histopathology and viral titres revealed no difference in respiratory severity between infected control and diabetic mice. However, compared with infected control mice, infected diabetic mice had increased serum cardiac troponin I and creatine-kinase MB, left ventricular structural changes and right ventricular functional alterations, providing the first experimental evidence of type I diabetes increasing risks of influenza-induced cardiovascular complications.
Information and research for policy and practice: survey of collection and information services dataset
Publisher: Swinburne Institute for Social Research
Date: 2015
Helix 8 of the angiotensin- II type 1A receptor interacts with phosphatidylinositol phosphates and modulates membrane insertion
Publisher: Springer Science and Business Media LLC
Date: 30-06-2015
DOI: 10.1038/SREP09972
Abstract: The carboxyl-terminus of the type 1 angiotensin II receptor (AT 1A ) regulates receptor activation/deactivation and the hipathic Helix 8 within the carboxyl-terminus is a high affinity interaction motif for plasma membrane lipids. We have used dual polarisation interferometry (DPI) to examine the role of phosphatidylinositdes in the specific recognition of Helix 8 in the AT 1A receptor. A synthetic peptide corresponding to Leu 305 to Lys 325 (Helix 8 AT 1A ) discriminated between PIPs and different charges on lipid membranes. Peptide binding to PtdIns(4)P-containing bilayers caused a dramatic change in the birefringence (a measure of membrane order) of the bilayer. Kinetic modelling showed that PtdIns(4)P is held above the bilayer until the mass of bound peptide reaches a threshold, after which the peptides insert further into the bilayer. This suggests that Helix 8 can respond to the presence of PI(4)P by withdrawing from the bilayer, resulting in a functional conformational change in the receptor.
PAQR3 modulates insulin signaling by shunting phosphoinositide 3-kinase p110α to the Golgi apparatus
Publisher: American Diabetes Association
Date: 17-01-2013
DOI: 10.2337/DB12-0244
Abstract: Phosphoinositide 3-kinase (PI3K) mediates insulin actions by relaying signals from insulin receptors (IRs) to downstream targets. The p110α catalytic subunit of class IA PI3K is the primary insulin-responsive PI3K implicated in insulin signaling. We demonstrate here a new mode of spatial regulation for the p110α subunit of PI3K by PAQR3 that is exclusively localized in the Golgi apparatus. PAQR3 interacts with p110α, and the intracellular targeting of p110α to the Golgi apparatus is reduced by PAQR3 downregulation and increased by PAQR3 overexpression. Insulin-stimulated PI3K activity and phosphoinositide (3,4,5)-triphosphate production are enhanced by Paqr3 deletion and reduced by PAQR3 overexpression in hepatocytes. Deletion of Paqr3 enhances insulin-stimulated phosphorylation of AKT and glycogen synthase kinase 3β, but not phosphorylation of IR and IR substrate-1 (IRS-1), in hepatocytes, mouse liver, and skeletal muscle. Insulin-stimulated GLUT4 translocation to the plasma membrane and glucose uptake are enhanced by Paqr3 ablation. Furthermore, PAQR3 interacts with the domain of p110α involved in its binding with p85, the regulatory subunit of PI3K. Overexpression of PAQR3 dose-dependently reduces the interaction of p85α with p110α. Thus, PAQR3 negatively regulates insulin signaling by shunting cytosolic p110α to the Golgi apparatus while competing with p85 subunit in forming a PI3K complex with p110α.
Glucocorticoids suppress growth in neonatal cardiomyocytes co-expressing AT(2) and AT(1) angiotensin receptors
Publisher: S. Karger AG
Date: 30-10-2010
DOI: 10.1159/000253757
Abstract: i Background: /i Perinatal glucocorticoid treatment is associated with hypertrophic cardiomyopathy, but the cellular mechanism is controversial. An underlying interaction between glucocorticoids and the renin-angiotensin system may be important, but whether glucocorticoids modulate angiotensin II (AngII)-dependent cardiomyocyte growth responses in the neonate has not been investigated. i Objectives: /i The major aim of this investigation was to determine whether glucocorticoids modulate the neonatal cardiomyocyte growth response to AngII. In particular we sought evidence to determine whether angiotensin II type 2 (AT sub /sub ) receptor co-expression with angiotensin II type 1 (AT sub /sub ) receptor is of specific importance in this modulatory function. i Methods: /i In this study, we used AT sub /sub and AT sub /sub receptor-expressing adenoviruses (Ad-AT sub /sub and Ad-AT sub /sub ) in a well-defined in vitro neonatal cardiomyocyte culture model to assess whether glucocorticoids affect cardiomyocyte growth responses (i.e. total protein content). i Results: /i Following addition of AngII (0.1 µmol/l) to neonatal cardiomyocytes infected with Ad-AT sub /sub alone, a significant growth response was measured (133.2 ± 4.8%). Expression of Ad-AT sub /sub alone induced a ∼20% increase in total cellular protein content, which was unaffected by addition of AngII. Neither corticosterone (1 µmol/l) nor dexamethasone (1 µmol/l) had any significant effect on the AT sub /sub - or AT sub /sub -mediated growth responses. In contrast, the growth response to AngII was augmented following co-expression of AT sub /sub and AT sub /sub receptors (149.2 ± 4.2%), which was reduced by ∼20% in the presence of either corticosterone or dexamethasone (p 0.05). i Conclusions: /i The present study provides novel evidence that glucocorticoids suppress neonatal cardiomyocyte growth responsiveness when AT sub /sub and AT sub /sub receptor subtypes are co-expressed.
Cardiac gene expression data and in silico analysis provide novel insights into human and mouse taste receptor gene regulation
Publisher: Springer Science and Business Media LLC
Date: 20-05-2015
DOI: 10.1007/S00210-015-1118-1
Abstract: G protein-coupled receptors are the principal mediators of the sweet, umami, bitter, and fat taste qualities in mammals. Intriguingly, the taste receptors are also expressed outside of the oral cavity, including in the gut, airways, brain, and heart, where they have additional functions and contribute to disease. However, there is little known about the mechanisms governing the transcriptional regulation of taste receptor genes. Following our recent delineation of taste receptors in the heart, we investigated the genomic loci encoding for taste receptors to gain insight into the regulatory mechanisms that drive their expression in the heart. Gene expression analyses of healthy and diseased human and mouse hearts showed coordinated expression for a subset of chromosomally clustered taste receptors. This chromosomal clustering mirrored the cardiac expression profile, suggesting that a common gene regulatory block may control the taste receptor locus. We identified unique domains with strong regulatory potential in the vicinity of taste receptor genes. We also performed de novo motif enrichment in the proximal promoter regions and found several overrepresented DNA motifs in cardiac taste receptor gene promoters corresponding to ubiquitous and cardiac-specific transcription factor binding sites. Thus, combining cardiac gene expression data with bioinformatic analyses, this study has provided insights into the noncoding regulatory landscape for taste GPCRs. These findings also have broader relevance for the study of taste GPCRs outside of the classical gustatory system, where understanding the mechanisms controlling the expression of these receptors may have implications for future therapeutic development.
Angiotensin 1A receptors transfected into caudal ventrolateral medulla inhibit baroreflex gain and stress responses
Publisher: Oxford University Press (OUP)
Date: 06-08-2012
DOI: 10.1093/CVR/CVS252
Abstract: The caudal ventrolateral medulla (CVLM) is important for autonomic regulation and is rich in angiotensin II type 1A receptors (AT(1A)R). To determine their function, we examined whether the expression of AT(1A)R in the CVLM of mice lacking AT(1A)R (AT(1A)(-/-)) alters baroreflex sensitivity and cardiovascular responses to stress. Bilateral microinjections into the CVLM of AT(1A)(-/-) mice of lentivirus with the phox-2 selective promoter (PRSx8) were made to express either AT(1A)R (Lv-PRSx8-AT(1A)) or green fluorescent protein (Lv-PRSx8-GFP) as a control. Radiotelemetry was used to record mean arterial pressure (MAP), heart rate (HR), and locomotor activity. Following injection of Lv-PRSx8-GFP, robust neuronal expression of GFP was observed with ∼60% of the GFP-positive cells also expressing the catecholamine-synthetic enzyme tyrosine hydroxylase. After 5 weeks, there were no differences in MAP or HR between groups, but the Lv-PRSx8-AT(1A)- injected mice showed reduced baroreflex sensitivity (-25%, P = 0.003) and attenuated pressor responses to cage-switch and restraint stress compared with the Lv-PRSx8-GFP-injected mice. Reduced MAP mid-frequency power during cage-switch stress reflected attenuated sympathetic activation (Pgroup × stress = 0.04). Fos-immunohistochemistry indicated greater activation of forebrain and hypothalamic neurons in the Lv-PRSx8-AT(1A) mice compared with the control. The expression of AT(1A)R in CVLM neurons, including A1 neurons, while having little influence on the basal blood pressure or HR, may play a tonic role in inhibiting cardiac vagal baroreflex sensitivity. However, they strongly facilitate the forebrain response to aversive stress, yet reduce the pressor response presumably through greater sympatho-inhibition. These findings outline novel and specific roles for angiotensin II in the CVLM in autonomic regulation.
Baroreceptor reflex stimulation does not induce cytomegalovirus promoter-driven transgene expression in the ventrolateral medulla in vivo
Publisher: Elsevier BV
Date: 06-2006
DOI: 10.1016/J.AUTNEU.2006.02.024
Abstract: Adenoviruses are being employed to induce transgene expression in the central nervous system in vivo. In these studies, the cytomegalovirus (CMV) promoter is commonly employed to drive expression of the transgene because of its strong, constitutive activity in a wide range of cell types. However, using this promoter, expression in neurons is variable, with strongest expression being observed in non-neuronal cells. Indeed, even in vitro, CMV driven expression in neurons is variable. In cultured sympathetic ganglion cells it has been demonstrated that CMV-driven expression requires activation of cAMP-response element-binding protein (CREB) and that this can be induced by depolarization. In this study we tested whether depolarization might induce CMV-driven transgene expression, delivered by microinjection of an adenovirus, in the rostral ventrolateral medulla (RVLM) of rats. Prior to stimulation, transgene expression occurs in non-neuronal cells in the RVLM. Some neuronal expression was observed in neighbouring regions, in the nucleus ambiguus and in facial motor neurons. Within the RVLM, depolarization, induced by intraperitoneal administration of the ganglion blocking drug, pentolinium, did not lead to induction of transgene expression. This stimulus is known to induce expression of the immediate early gene c-fos. We conclude that either this experimental paradigm was not sufficient for activation of the CREB pathway or that possibly the virus does not gain access to the neurons of the RVLM. The adoption of specific promoters or viruses with higher neuronal transduction efficiency appears to be essential for the genetic modification of RVLM presympathetic neurons in vivo.
Casein Kinase II Sites in the Intracellular C-terminal Domain of the Thyrotropin-releasing Hormone Receptor and Chimeric Gonadotropin-releasing Hormone Receptors Contribute to β-Arrestin-dependent Internalization
Publisher: Elsevier BV
Date: 05-2001
Acid sensing ion channel 1a is a key mediator of cardiac ischemia-reperfusion injury
Publisher: Cold Spring Harbor Laboratory
Date: 10-12-2019
DOI: 10.1101/869826
Abstract: The proton-gated acid-sensing ion channel 1a (ASIC1a) is implicated in the injury response to cerebral ischemia but little is known about its role in cardiac ischemia. We provide genetic evidence that ASIC1a is involved in myocardial ischemia-reperfusion injury (IRI) and show that pharmacological inhibition of ASIC1a yields robust cardioprotection in rodent and human models of cardiac ischemia, resulting in improved post-IRI cardiac viability and function. Consistent with a key role for ASIC1a in cardiac ischemia, we show that polymorphisms in the ASIC1 genetic locus are strongly associated with myocardial infarction. Collectively, our data provide compelling evidence that ASIC1a is a key target for cardioprotective drugs to reduce the burden of disease associated with myocardial ischemia.
Expression of angiotensin type 1A receptors in C1 neurons restores the sympathoexcitation to angiotensin in the rostral ventrolateral medulla of angiotensin type 1A knockout mice
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-2010
DOI: 10.1161/HYPERTENSIONAHA.110.151704
Abstract: In adult mice we determined whether expression of angiotensin II (Ang II) type 1A receptors (AT 1A Rs) in C1 neurons mediates the excitation of the rostral ventrolateral medulla (RVLM) by Ang II. Blood pressure, heart rate, and sympathetic nerve activity were measured in anesthetized, artificially ventilated wild-type (n=15) and AT 1A R knockout (AT 1A −/− n=9) mice. Microinjection of Ang II (50 nL of 0.1 to 1.0 mmol/L) into the RVLM induced a dose-related, sympathetically mediated pressor response (maximum of 17±2 mm Hg) in wild-type mice. These microinjections had no effect in AT 1A −/− mice. Endogenous AT 1 Rs occur on catecholaminergic C1 neurons in the RVLM. We induced AT 1A R or green fluorescent protein expression in C1 neurons of AT 1A −/− mice through bilateral microinjection of replication-deficient lentiviruses, with transgene expression under the control of a phox2 transcription factor binding promoter (PRSx8) (Lv-PRSx8-AT 1A , n=10, and Lv-PRSx8-GFP, n=5). Transgene expression was observed in a significant proportion of RVLM C1 neurons. In anesthetized Lv-PRSx8-AT 1A injected mice, unilateral RVLM microinjection of Ang II (50 nL of 1 mmol/L) increased blood pressure (17±4 mm Hg) and sympathetic nerve activity (155±32%). No response to Ang II occurred in Lv-PRSx8-GFP microinjected mice. These results show that Ang II–mediated excitation of RVLM neurons in adult mice depends on the AT 1A R with little or no effect of type 1B or 2 receptors. Expression of the AT 1A R predominantly in C1 catecholamine neurons restores the response to Ang II in the AT 1A −/− mouse and demonstrates that these neurons are sympathoexcitatory in the mouse.
Agonist-dependent internalization of the angiotensin II type one receptor (AT1): role of C-terminus phosphorylation in recruitment of β-arrestins
Publisher: Elsevier BV
Date: 08-2004
Angiotensin II type 2 receptor antagonizes angiotensin II type 1 receptor-mediated cardiomyocyte autophagy
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 06-2009
DOI: 10.1161/HYPERTENSIONAHA.108.128488
Abstract: Autophagy has emerged as an important process in the pathogenesis of cardiovascular diseases, but the proximal triggers for autophagy are unknown. Angiotensin II plays a central role in the pathogenesis of cardiac hypertrophy and heart failure. In this study, we used angiotensin II type 1 (AT 1 ) and type 2 (AT 2 ) receptor–expressing adenoviruses in cultured neonatal cardiomyocytes to provide the first demonstration that neonatal cardiomyocyte autophagic activity is differentially modulated by AT 1 and AT 2 receptor subtypes. Angiotensin II stimulation (48 hours) of neonatal cardiomyocytes expressing the AT 1 receptor alone (Ad-AT 1 10 multiplicities of infection) induced a significant increase in the number of HcRed-LC3 autophagosomes per cell (17.3±1.6 versus 33.3±4.1 autophagosomes per cell P .05). Coexpression of a high ratio of AT 2 :AT 1 (Ad-AT 2 :Ad-AT 1 multiplicity of infection ratio: 20:5) receptors completely abrogated the AT 1 -mediated increase in autophagy (9.3±1.4 versus 33.3±4.1 autophagosomes per cell P .05). Treatment with the AT 2 receptor antagonist PD123319 did not reverse the AT 2 -mediated antiautophagic effect. AT 1 - and AT 2 -mediated autophagic responses were also assessed in cardiomyocytes from a genetic model that exhibits neonatal myocardial growth suppression. In these neonate myocyte cultures, AT 1 receptor activation induced a marked increase in the number of myocytes containing cytoplasmic vacuoles compared with the control (22.7±4.1% versus 1.1±0.6% P .001) and was characterized by a nonapoptotic autophagic phenotype. The incidence of cardiomyocyte autophagic vacuolization in this myocyte population decreased dramatically to only 0.4±0.2% in myocytes infected with a high ratio of Ad-AT 2 :Ad-AT 1 . This study provides the first description of reciprocal regulation of cardiomyocyte autophagic induction by the AT 1 and AT 2 receptor subtypes.
Effect of dominant-negative epidermal growth factor receptors on cardiomyocyte hypertrophy
Publisher: Informa UK Limited
Date: 2006
DOI: 10.1080/10799890600923187
Abstract: Angiotensin II (AngII) induces heart growth via cardiomyocyte hypertrophy, and central to this is the capacity of the type 1 AngII receptor (AT1R) to "transactivate" epidermal growth factor receptors (EGFRs)--a family with four main subtypes (HER1-4)--although the exact molecular mechanism remains unresolved. In this study, the pharmacological inhibition of AngII-stimulated ERK1/2 activation and cardiomyocyte hypertrophy by increasing concentrations of an EGFR inhibitor, AG1478, indicated that other EGFR subtypes, in addition to HER1, may be involved. We constructed expression vectors and adenoviruses expressing truncated mutant versions of HER1, HER2, and HER4 and determined their capacity to act as dominant-negative inhibitors when co-transfected with full-length EGFRs. It is surprising that adenoviral-mediated expression of these truncated EGFRs in cardiomyocytes led to paradoxical, ligand-independent increases in cardiomyocyte hypertrophy and unusual morphological changes. These results challenge our perception of AT1R-mediated EGFR transactivation and imply that truncated EGFRs may affect cell function through unconventional mechanisms.
G protein coupling and second messenger generation are indispensable for metalloprotease-dependent, heparin-binding epidermal growth factor shedding through angiotensin II type-1 receptor
Publisher: Elsevier BV
Date: 07-2005
Modular transient nanoclustering of activated β2-adrenergic receptors revealed by single-molecule tracking of conformation-specific nanobodies
Publisher: Proceedings of the National Academy of Sciences
Date: 19-11-2020
Abstract: Proteins moving freely on the plasma membrane can become transiently trapped in functionally essential clusters. This capability is likely to be influenced by subtle conformational states of the protein promoting or preventing such confinement. The downside of conventional imaging of overexpressed tagged proteins is that it precludes selective tracking of inherently minor albeit functionally essential conformer populations. Intracellular expression of single-chain nanobodies allowed us to track endogenous proteins in highly specific conformational states in live cells and small organisms. We unveiled the full scope of nanoclustering behavior of β 2 -adrenergic receptors in various conformations, along with their transient nature. This technique is broadly applicable to other proteins and will help unravel essential dynamics and organization of nanoclusters.
Angiotensin receptors: Interpreters of pathophysiological angiotensinergic stimulis
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 27-08-2015
Research use and publishing diversity: The role of organisation research publishing for policy and practice
Publisher: Wiley
Date: 11-2023
Abstract: Digital technologies have enhanced the capacity for organisations across many sectors to produce, publish, and disseminate research in a variety of formats, and a great deal of it is sought and used in public policy and practice‐related research, yet this ersity is often overlooked in studies of research use. While the need for erse research sources and formats for public policy and practice is increasingly acknowledged, there have been few studies which articulate and categorise what this ersity looks like in practice, and how research is filtered and selected based on genre, source, and other facets. This article reports on a large‐scale online survey and semi‐structured interviews with research users across multiple sectors in Australia on the materials they access and use for policy and practice work. The results indicate that research users are active information seekers who require online access to erse genres and formats produced by a range of sources and sectors. However, respondents also faced many barriers to research use, including the cost of subscriptions for academic journals, discoverability of reports and data, poor management of publications by organisations including government, and the time required for filtering and evaluation. Based on these findings I argue that policy research requires a far greater variety of genres and sources than is generally recognised with implications for the way research use and the research publishing system is understood and managed in Australia. Policy research and implementation requires erse online sources and resources from multiple sectors, including reports, discussion papers, evaluations, and data, produced by organisations (grey literature), as well as journals and books. However, this paper finds there are major barriers to discovery, filtering, and access to erse research publications for practitioners, resulting in poor productivity and policy outcomes. To improve the use of evidence for policy and practice, we must invest in efficient discovery, access, and management systems for erse research publications.
Double trouble for type 1 angiotensin receptors in atherosclerosis
Publisher: Massachusetts Medical Society
Date: 03-02-2005
Angiotensin receptors: Form and function and distribution
Publisher: Elsevier BV
Date: 06-2003
DOI: 10.1016/S1357-2725(02)00263-7
Abstract: The peptide hormone, angiotensin II, acts primarily via type I (AT(1)) and type II (AT(2)) angiotensin receptors. Proteolytic fragments of angiotensin II also have biological activity via these and other receptors, with actions that may mimic or antagonise angiotensin II. Most notably, a high affinity-binding site for angiotensin IV (the Val(3)-Phe(8) fragment of angiotensin II) has recently been identified as the insulin-regulated aminopeptidase (IRAP). While AT(1) and AT(2) receptors are seven transmembrane-spanning, G protein-coupled receptors with some well-established features of relevance to health and disease, the existence of separate receptor systems for angiotensin fragments offers exciting possibilities for new therapeutics to target the erse actions of the angiotensin peptides.
Therapeutic Inhibition of Acid-Sensing Ion Channel 1a Recovers Heart Function After Ischemia-Reperfusion Injury.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 21-09-2021
DOI: 10.1161/CIRCULATIONAHA.121.054360
Abstract: Ischemia–reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell–derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell–derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.
Integrating the GPCR transactivation-dependent and biased signalling paradigms in the context of PAR1 signalling
Publisher: Wiley
Date: 16-02-2016
DOI: 10.1111/BPH.13398
Oxytocin receptors in the ovine corpus luteum
Publisher: Bioscientifica
Date: 04-1989
Abstract: There is inconclusive evidence that oxytocin acts directly on the corpus luteum and affects steroidogenesis. Since any such action would probably be mediated by oxytocin receptors, these should be present in luteal tissue. In this study, homogenates of corpora lutea from both pregnant and non-pregnant ewes were examined for oxytocin receptors by radio-receptor assay. Specific oxytocin binding was not observed in luteal tissue during the oestrous cycle. However specific binding was found in the corpora lutea of pregnant ewes appearing at a fetal head length of approximately 0·65 cm (about 30 days of pregnancy) and persisting to a head size of 11 cm, the largest size examined in this study. The affinity ( K d ) of the receptor was calculated as 2·9 ± 0·3 nmol/l ( s.e.m. n = 9), a value similar to that obtained for the uterus. The receptor number ranged from a low of 8·7± 3·2 fmol/mg protein ( n = 6) at a head size of ·65 cm, to a maximum of 40·1 ± 6·5 fmol/mg protein ( n = 25) at a head size of 2·5–3·75 cm. These values were lower than our estimate of 588 ± 39 fmol/mg protein ( n = 5) for the uterus. It is concluded that a direct action of oxytocin on the corpus luteum is possible but only after the first month of pregnancy and not in the corpus luteum of the oestrous cycle. Journal of Endocrinology (1989) 121, 117–123
Heritable pathologic cardiac hypertrophy in adulthood is preceded by neonatal cardiac growth restriction
Publisher: American Physiological Society
Date: 03-2009
DOI: 10.1152/AJPREGU.90919.2008
Abstract: The identification of genetic factors influencing cardiac growth independently of increased load is crucial to an understanding of the molecular and cellular basis of pathological cardiac hypertrophy. The central aim of this investigation was to determine how pathological hypertrophy in the adult can be linked with disturbances in cardiomyocyte growth and viability in early neonatal development. The hypertrophic heart rat (HHR) model is derived from the spontaneously hypertensive rat and exhibits marked cardiac hypertrophy, in the absence of a pressure load at maturity. Hearts were harvested from male HHR, and control strain normal heart rats (NHR), at different stages of postnatal development [neonatal (P2), 4 wk, 6 wk, 8 wk, 12 wk, 20 wk]. Isolated neonatal cardiomyocytes were prepared to evaluate cell size, number, and binucleation. At postnatal day 2, HHR hearts were considerably smaller than control NHR (4.3 ± 0.2 vs. 5.0 ± 0.1 mg/g, P 0.05). Cardiac growth restriction in the neonatal HHR was associated with reduced myocyte size (length and width) and an increased proportion of binucleated cardiomyocytes. Furthermore, the number of cardiomyocytes isolated from HHR neonatal hearts was significantly less (∼29%) than NHR. We also observe that growth stress in the neonate is associated with accentuated PI3K and suppressed MAPK activation, although these signaling pathways are normalized in the adult heart exhibiting established hypertrophy. Thus, using the HHR model, we identified novel molecular and cellular mechanisms involving premature exit from the cell cycle, reduced cardiomyocyte endowment, and dysregulated trophic signaling during early development, which are implicated in the etiology of heritable cardiac hypertrophy in the adult.
Urotensin II: The old kid in town
Publisher: Elsevier BV
Date: 05-2004
Taste and Hypertension in Humans: Targeting Cardiovascular Disease
Publisher: Bentham Science Publishers Ltd.
Date: 27-04-2016
DOI: 10.2174/1381612822666160216151545
Abstract: The association between salty taste and NaCl intake with hypertension is well-established, although it is far from completely understood. Other taste types such as sweet, umami or bitter have also been related to alterations in blood pressure. Here, we review the mutual relationship between taste and hypertension to identify potential avenues to better control blood pressure. This review focuses on published data involving humans, with the exception of a section on molecular mechanisms. There is compelling evidence to suggest that changes in salty taste sensitivity can be used to predict the onset of hypertension. This goes hand in hand with the medical concept of sodium sensitivity, which also increases with age, particularly in hypertensive patients. The association of hypertension with the loss of taste acuity less definitive with some data/conclusions masked by the use of anti-hypertensive drugs. In fact, this group of therapeutic agents can reduce food taste perception resulting in mild to severe hypogeusia and dysgeusia. In the elderly, antihypertensive drugs may lead to a loss of appetite, thus, selecting treatments with low or no impact on taste perception should be advised. Pharmacological approaches to mitigate cardiovascular disease (CVD) could well take a different spin in the future following the discovery of taste receptors (TAS1R and TAS2R) in the cardiovascular system. Finally, long-term dietary strategies to minimize the risk of development of hypertension and CVD are discussed identifying several nutrients and public health policies with relevant potential.
Urotensin II promotes hypertrophy of cardiac myocytes via mitogen-activated protein kinases
Publisher: The Endocrine Society
Date: 09-2004
DOI: 10.1210/ME.2003-0309
When 6 is 9: 'Uncoupled' AT1 receptors turn signalling on its head
Publisher: Springer Science and Business Media LLC
Date: 11-2004
DOI: 10.1007/S00018-004-4245-2
Abstract: The type 1 angiotensin receptor (AT(1)) activates an array of intracellular signalling pathways that control cell and tissue responses to the peptide hormone angiotensin II (AngII). The capacity of AT(1) receptors to initiate and maintain such signals has typically been explained on the basis of conventional heterotrimeric guanine nucleotide binding protein (G protein) activation, specifically G(q/11). Accumulating evidence from studies utilising a variety of AT(1) receptor mutants and AngII analogues indicates that some important downstream effects of AT(1) receptors are independent of classical G protein coupling. Importantly, AT(1) receptor-mediated endocytosis, tyrosine phosphorylation signalling and mitogen-activated protein kinase activation as well as transactivation of the epidermal growth factor receptor can occur in G(q/11)-uncoupled receptor mutants. These observations point to a functional partitioning of AT(1) receptor signals that permits separation of short-term AngII actions (e.g., vasoconstriction) from more extended events, such as pathological cell growth in heart and blood vessels, and may open up new avenues for selective antagonism.
A peptide toxin in ant venom mimics vertebrate EGF-like hormones to cause long-lasting hypersensitivity in mammals
Publisher: Proceedings of the National Academy of Sciences
Date: 07-02-2022
Abstract: The targeting of mammalian ErbB receptor signaling by a venom toxin to cause hypersensitivity is a mode of action that has not previously been described. Natural selection of a defensive toxin to target ErbB signaling provides compelling independent evidence for a fundamental role of this receptor and its ligands in mammalian pain. The evolution of a toxin in ant venom to mimic a vertebrate nociceptive hormone serves as an ex le of both convergent evolution and molecular mimicry, illustrating how natural selection can shape the gene product of one organism to resemble that of another.
Akt acts as a switch for GPCR transactivation of the TGF‐β receptor type 1
Publisher: Wiley
Date: 18-12-2022
DOI: 10.1111/FEBS.16297
Abstract: Transforming growth factor (TGF)‐β signalling commences with the engagement of TGF‐β ligand to cell surface TGF‐β receptors (TGFBR) stimulating Smad2 carboxyl‐terminal phosphorylation (phospho‐Smad2C) and downstream biological responses. In several cell models, G protein‐coupled receptors (GPCRs) transactivate the TGF‐β receptors type‐1 (TGFBR1) leading to phospho‐Smad2C, however, we have recently published that in keratinocytes thrombin did not transactivate the TGFBR1. The bulk of TGFBRs reside in the cytosol and in response to protein kinase B (Akt phosphorylation) can translocate to the cell surface increasing the cell's responsiveness to TGF‐β. In this study, we investigate the role of Akt in GPCR transactivation of the TGFBR1. We demonstrate that angiotensin II and thrombin do not phosphorylate Smad2C in human vascular smooth muscle cells and in keratinocytes respectively. We used Akt agonist, SC79 to sensitise the cells to Akt and observed that Ang II and thrombin phosphorylate Smad2C via Akt/AS160‐dependent pathways. We show that SC79 rapidly translocates TGFBRs to the cell surface thus increasing the cell's response to the GPCR agonist. These findings highlight novel mechanistic insight for the role of Akt in GPCR transactivation of the TGFBR1.
Epidermal Growth Factor Receptor Transactivation: Mechanisms, Pathophysiology, and Potential Therapies in the Cardiovascular System
Publisher: Annual Reviews
Date: 06-01-2016
DOI: 10.1146/ANNUREV-PHARMTOX-070115-095427
Abstract: Epidermal growth factor receptor (EGFR) activation impacts the physiology and pathophysiology of the cardiovascular system, and inhibition of EGFR activity is emerging as a potential therapeutic strategy to treat diseases including hypertension, cardiac hypertrophy, renal fibrosis, and abdominal aortic aneurysm. The capacity of G protein–coupled receptor (GPCR) agonists, such as angiotensin II (AngII), to promote EGFR signaling is called transactivation and is well described, yet delineating the molecular processes and functional relevance of this crosstalk has been challenging. Moreover, these critical findings are dispersed among many different fields. The aim of our review is to highlight recent advancements in defining the signaling cascades and downstream consequences of EGFR transactivation in the cardiovascular renal system. We also focus on studies that link EGFR transactivation to animal models of the disease, and we discuss potential therapeutic applications.
Caveolin interacts with the angiotensin II type I receptor during exocytic transport but not at the plasma membrane
Publisher: Elsevier BV
Date: 06-2003
Heteromerization of angiotensin receptors changes trafficking and arrestin recruitment profiles
Publisher: Elsevier BV
Date: 11-2011
DOI: 10.1016/J.CELLSIG.2011.06.011
Abstract: The cardiovascular hormone angiotensin II (AngII) exerts its actions via two G protein-coupled receptor (GPCR) subtypes, AT(1) and AT(2), which often display antagonistic functions. Methodological constraints have so far precluded detailed analyses of the ligand-dependency, cellular localization, and functional relevance of AngII receptor interactions in live cells. In this study, we utilize a protein-fragment complementation assay (PCA) and GPCR-Heteromer Identification Technology (GPCR-HIT) to provide the first detailed investigation of the ligand-dependency and cellular localization of AngII receptor interactions in human embryonic kidney 293 cells. Fluorescent-tagged receptor constructs for PCA and GPCR-HIT displayed normal affinity and selectivity for AngII (AT(1): IC(50)=1.0-1.6nM AT(2): IC(50)=2.0-3.0nM). Well-characterized angiotensin receptor interactions were used as positive and negative controls to demonstrate the sensitivity and specificity of these fluorescence-based assays. We report that AT(1)-AT(2) receptor heteromers form constitutively, are localized to the plasma membrane and perinuclear compartments, and do not internalize following AngII stimulation despite arrestin being recruited specifically to the heteromer. Our findings using novel fluorescence-based technologies reveal a previously unrecognized mechanism of angiotensin receptor cross-talk involving cross-inhibition of AT(1) receptor internalization through heteromerization with the AT(2) receptor subtype.
Purification of rat angiotensinogen
Publisher: Informa UK Limited
Date: 03-1986
DOI: 10.1080/00327488608062458
Abstract: A method of purifying rat angiotensinogen in three chromatography steps with a yield 3-4 times better than previous methods is described. Using chromatofocusing media for two steps and DEAE-affigel blue for the third step it was possible to separate angiotensinogen into three major peaks with pI of 5.25 (peak B), 4.80 (peak C) and 4.50 (peak D). Peaks B and C were completely purified with recoveries of 12% and 17% and specific activities of 21.8 and 20.0 micrograms AI/mg protein respectively. Analytical SDS-PAGE showed a 53,000 dalton band in both peaks with additional 51,000 and 57,000 dalton bands in peak C.
Role of Angiotensin II Type 1A Receptor Phosphorylation, Phospholipase D, and Extracellular Calcium in Isoform-specific Protein Kinase C Membrane Translocation Responses
Publisher: Elsevier BV
Date: 09-2006
A High-Fat Diet Increases Influenza A Virus-Associated Cardiovascular Damage
Publisher: Oxford University Press (OUP)
Date: 04-04-2020
Abstract: Influenza A virus (IAV) causes a wide range of extrarespiratory complications. However, the role of host factors in these complications of influenza virus infection remains to be defined. Here, we sought to use transcriptional profiling, virology, histology, and echocardiograms to investigate the role of a high-fat diet in IAV-associated cardiac damage. Transcriptional profiling showed that, compared to their low-fat counterparts (LF mice), mice fed a high-fat diet (HF mice) had impairments in inflammatory signaling in the lung and heart after IAV infection. This was associated with increased viral titers in the heart, increased left ventricular mass, and thickening of the left ventricular wall in IAV-infected HF mice compared to both IAV-infected LF mice and uninfected HF mice. Retrospective analysis of clinical data revealed that cardiac complications were more common in patients with excess weight, an association which was significant in 2 out of 4 studies. Together, these data provide the first evidence that a high-fat diet may be a risk factor for the development of IAV-associated cardiovascular damage and emphasizes the need for further clinical research in this area.
Surface plasmon resonance spectroscopy in the study of membrane-mediated cell signalling
Publisher: Wiley
Date: 2003
DOI: 10.1002/PSC.439
Abstract: Peptide-membrane interactions contribute to many important biological processes such as cellular signaling, protein trafficking and ion-channel formation. During receptor-mediated signalling, activated intracellular signalling molecules are often recruited into receptor-induced signaling complexes at the cytoplasmic surface of the cell membrane. Such recruitment can depend upon protein-protein and protein-lipid interactions as well as protein acylation. A wide variety of biophysical techniques have been combined with the use of model membrane systems to study these interactions and have provided important information on the relationship between the structure of these proteins involved in cell signalling and their biological function. More recently, surface plasmon resonance (SPR) spectroscopy has also been applied to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. This article provides an overview of these recent applications, which demonstrate the potential of SPR to enhance our molecular understanding of membrane-mediated cellular signalling.
Beta-arrestin 2 is required for complement C1q expression in macrophages and constrains factor-independent survival
Publisher: Elsevier BV
Date: 12-2009
DOI: 10.1016/J.MOLIMM.2009.09.012
Abstract: The beta-arrestins (ARRB1 and ARRB2) regulate G-protein coupled receptor (GPCR) dependent- and independent-signaling pathways and are ubiquitously expressed. Here we show that ARRB2 mRNA and protein expression is enriched in macrophages, and that it regulates complement C1q expression and cell survival. Basal and Toll-like receptor (TLR) inducible expression of mRNAs encoding the complement subcomponents C1qa, C1qb and C1qc was greatly reduced in bone marrow-derived macrophages (BMM) from ARRB2-deficient, but not ARRB1-deficient mice, while factor-independent survival of ARRB2(-/-) BMM was enhanced compared to wildtype BMM. TatARRB2(23), a cell-permeable peptide that contains the MAPK JNK-binding motif from within the ARRB2 C-domain, impaired ARRB2 interaction with JNK3, down-regulated C1q expression and permitted factor-independent survival in BMM, thus suggesting that this peptide antagonises ARRB2 function in macrophages. In addition, TatARRB2(23) transiently activated the phosphorylation of JNK and ERK, but not p38 in BMM. These data imply that ARRB2 acts to limit JNK/ERK activation and survival in macrophages, but is required for basal and TLR-inducible complement C1q expression. Given that loss of C1q function is strongly associated with the development of systemic lupus erythematosus, ARRB2 may act to limit the development of autoimmune disease.
Immunocytochemical Localization of Angiotensinogen and Angiotensin II in the Rat Pituitary
Publisher: Wiley
Date: 06-1990
DOI: 10.1111/J.1365-2826.1990.TB00408.X
Abstract: Abstract The presence and distribution of angiotensinogen and angiotensin II (All) were demonstrated in rat pituitary by immunocytochemical staining with the avidin-biotin-peroxidase method, using primary polyclonal antibodies specific for angiotensinogen and All. Silver enhancement of the reaction product was used to intensify lightly stained areas. Attempts were made to identify immunopositive cells by colocalization studies with antisera against luteinizing hormone, prolactin and S-100, a glial cell protein. In the anterior pituitary, angiotensinogen-immunoreactivity was observed in cells lining follicle-like structures. These cells, which were irregularly shaped and had processes extending between the glandular cells, did not colocalize with any of the reference antisera and are therefore of unknown cell type. The follicular endothelium was also immunopositive for angiotensinogen. After silver intensification, dispersed immunoreactive glandular cells were consistently observed in the anterior lobe. A proportion of these costained for luteinizing hormone, but not prolactin or S-100, indicating their identity as gonadotrophs. In the posterior pituitary, angiotensinogen immunostaining was associated only with the vasculature, while groups of immunopositive cells were observed in the medial region of the intermediate lobe after silver enhancement. All-immunoreactivity was observed in large cells preferentially located at the poles of the anterior pituitary which also costained for luteinizing hormone. No staining was observed in either the posterior or intermediate lobes. The presence of immunoreactive angiotensinogen in all three lobes of the pituitary suggests that there are sites, in addition to gonadotrophs, at which the intracellular production of All could occur.
Regulation of angiotensin II type 1 (AT1 ) receptor function
Publisher: Elsevier BV
Date: 1999
DOI: 10.1016/S0167-0115(98)00140-2
Abstract: The type 1 angiotensin receptor (AT1) mediates the important biological actions of the peptide hormone, angiotensin II (AngII), by activating an array of intracellular signaling pathways. The unique temporal arrangement and duration of AngII-stimulated signals suggests a hierarchy of post-AT1 receptor binding events that permits activation of selective effector pathways. Moreover, it predicts that the coupling of AT1 receptors is tightly regulated, allowing cells to differentiate acute responses from those requiring longer periods of stimulation. Recent studies have concentrated on delineating the molecular processes involved in modulating AT1 receptor activity. In addition to AT1 receptor modification (phosphorylation), trafficking (internalization and degradation) and interaction with regulatory intracellular proteins, other processes may include receptor dimerization, cross-regulation by other receptor systems, and receptor isomerization between activated and non-activated forms. This review focuses on recent advances in this area of research, highlighting directions for future investigation.
Stable expression of a functional rat angiotensin II (AT1A) receptor in CHO-K1 cells: Rapid desensitization by angiotensin II
Publisher: Springer Science and Business Media LLC
Date: 1995
DOI: 10.1007/BF00926885
Mutations in the NPxxY motif stabilize pharmacologically distinct conformational states of the α
Publisher: American Association for the Advancement of Science (AAAS)
Date: 12-03-2019
DOI: 10.1126/SCISIGNAL.AAS9485
Abstract: G protein-coupled receptors (GPCRs) convert extracellular stimuli to intracellular responses that regulate numerous physiological processes. Crystallographic and biophysical advances in GPCR structural analysis have aided investigations of structure-function relationships that clarify our understanding of these dynamic receptors, but the molecular mechanisms associated with activation and signaling for in idual GPCRs may be more complex than was previously appreciated. Here, we investigated the proposed water-mediated, hydrogen-bonded activation switch between the conserved NPxxY motif on transmembrane helix 7 (TMH7) and a conserved tyrosine in TMH5, which contributes to α
Immunccytochemical Localization of Angiotensinogen in Rat Brain: Dependence of Neuronal Immunoreactivity on Method of Tissue Processing
Publisher: Wiley
Date: 12-1991
DOI: 10.1111/J.1365-2826.1991.TB00330.X
Abstract: Abstract There is disagreement between laboratories on the presence and location of angiotensinogen immunostaining in neuronal cells. We examined this issue by using different antisera and histological procedures to stain for angiotensinogen in brains from normal, colchicine-treated and nephrectomized rats. Five different antisera from three laboratories were used to stain sections of paraffin-embedded tissue, frozen sections and Vibratome sections of cerebral cortex, thalamus, hypothalamus, brainstem and cerebellum. All five antisera and all three tissue treatments were effective in showing angiotensinogen staining in glial cells, with the most intense staining being achieved in Vibratome sections. All five antisera gave identical results. Neuronal staining was also found with all antisera but mostly in paraffin-embedded sections, with occasional light staining in frozen sections. No neuronal staining was observed in Vibratome sections. Neuronal staining was frequently perivascular, tended to have a more variable anatomical localization, and to occasionally lack bilateral symmetry in coronal sections. These results provide an explanation for the disagreement between laboratories on the presence and location of angiotensinogen immunostaining in neuronal cells. Taken together with the limited concordance between published sites of angiotensinogen and angiotensin II staining, and the recent demonstration by hybridization in situ of a specifically glial cell localization of angiotensinogen mRNA, our own results suggest a need for caution in the interpretation of neuronal staining with anti-angiotensinogen antisera.
Effect of intrauterine growth restriction on the number of cardiomyocytes in rat hearts
Publisher: Springer Science and Business Media LLC
Date: 06-2005
Structural determinants for binding to angiotensin converting enzyme 2 (ACE2) and angiotensin receptors 1 and 2
Publisher: Frontiers Media SA
Date: 30-01-2015
AngiotensinII mediates cardiomyocyte hypertrophic growth pathways via MMP-dependent HB-EGF liberation
Publisher: Springer Science and Business Media LLC
Date: 09-2003
DOI: 10.1007/BF02442574
Ligand-supported purification of the urotensin-II receptor.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 20-07-2010
Abstract: A crucial limitation for structural and biophysical analysis of G protein-coupled receptors (GPCRs) is the inherent challenge of purifying and stabilizing these receptors in an active (agonist-bound) conformation. Peptide ligands, such as the vasoactive, cyclic hormone urotensin-II (U-II), may provide new purification tools, via high affinity, pseudo-irreversible binding suitable for ligand-based affinity purification. We show that the U-II receptor (UT) is resistant to desensitization as a result of low phosphorylation and diminished endocytosis. UT also displays an unusual proclivity to remain active with vasoconstriction sustained despite extensive washout of the ligand. To exploit these properties for ligand-supported purification, we modified the U-II ligand by attaching a biotin moiety and spacer arm to the N terminus, creating a novel affinity ligand (Bio-U-II) to interface with streptavidin media. Bio-U-II bound to UT with pharmacological properties analogous to those of the unmodified U-II ligand (high-affinity, pseudo-irreversible binding). The prebinding of Bio-U-II to UT (before exposure to detergent) facilitated specific capture of UT by stabilizing the receptor structure during solubilization with detergent. Solubilization of UT with the most compatible detergent, n-dodecyl β-d-maltoside, was dependent on the critical micelle concentration, and Gα(q/11) protein was copurified with captured Bio-U-II-UT complexes. Furthermore, captured Bio-U-II-UT complexes were resistant to dissociation at elevated temperatures, suggesting that UT is relatively thermostable, making it an ideal candidate for future structural and biophysical studies. This work demonstrates the utility of pseudo-irreversible ligands to support the purification of a GPCR during detergent extraction, resulting in the first successful purification of the UT.
Protein kinase C regulates the cell surface activity of endothelin-converting enzyme-1
Publisher: Springer Science and Business Media LLC
Date: 19-05-2006
Electronic documents in a print world: grey literature and the internet
Publisher: Media International Australia
Date: 2012
Differential Participation of Angiotensin II Type 1 and 2 Receptors in the Regulation of Cardiac Cell Death Triggered by Angiotensin II
Publisher: Oxford University Press (OUP)
Date: 19-03-2009
DOI: 10.1038/AJH.2009.32
Abstract: The Angiotensin II (Ang II) type 1 (AT(1)R) and type 2 (AT(2)R) receptors are increased in the heart following myocardial infarction and dilated cardiomyopathy, yet their contribution at a cellular level to compensation and/or failure remains controversial. We ectopically expressed AT(1)R and AT(2)R in cultured adult rat cardiomyocytes and cardiac fibroblasts to investigate Ang II-mediated cardiomyocyte hypertrophy and cardiac cell viability. In adult rat cardiomyocytes, Ang II did not induce hypertrophy via the AT(1)R, and no effect of Ang II on cell viability was observed following AT(1)R or AT(2)R expression. In adult rat cardiac fibroblasts, Ang II stimulated cell death by apoptosis via the AT(1)R (but not the AT(2)R), which required the presence of extracellular calcium, and induced a rapid dissipation of mitochondrial membrane potential, which was significant from 8 h. We conclude that Ang II/AT(1)R triggers apoptosis in adult rat cardiac fibroblasts, which is dependent on Ca2+ influx.
Angiotensin II enhances noradrenaline release from sympathetic nerves of the rat prostate via a novel angiotensin receptor: Implications for the pathophysiology of benign prostatic hyperplasia
Publisher: Bioscientifica
Date: 10-2001
Abstract: The renin-angiotensin system (RAS) is present in the human prostate and may be activated in benign prostatic hyperplasia (BPH), possibly contributing to the pathophysiology of this disorder by enhancing local sympathetic tone and cell growth. The functional role of the RAS in the prostate, however, is unknown. The present study was undertaken to determine whether angiotensin (Ang) II enhances sympathetic transmission in the prostate. The neuronal stores of the rat prostate were labelled with [(3)H]noradrenaline (NA). Ang II and Ang I enhanced [(3)H]NA release in a concentration-dependent manner. The Ang II receptor subtype 1 (AT(1) receptor) antagonist losartan and the AT(2) receptor antagonist PD123319 inhibited this facilitatory effect of Ang II and Ang I, whereas the other AT(2) receptor antagonist, CGP42112, was without effect. Bradykinin also increased [(3)H]NA release, which was inhibited by the B(2) receptor antagonist Hoe140. The angiotensin-converting enzyme inhibitor captopril inhibited the effect of Ang I, but potentiated that of bradykinin. Interestingly, captopril alone produced an increase in [(3)H]NA release which was inhibited by Hoe140. Losartan, but not PD123319 or CGP42112, inhibited [(125)I]-Ang II binding in Chinese hamster ovary cells transfected with the AT(1a) or AT(1b) receptor. In contrast, in cells expressing the AT(2) receptor, PD123319 and CGP42112, but not losartan, inhibited [(125)I]-Ang II binding. In conclusion, Ang II enhances the release of NA from sympathetic nerves of the rat prostate via a novel functional receptor distinct from the cloned AT(1a), AT(1b) or AT(2). These data provide direct evidence in support of a functional role for the local RAS in modulating sympathetic transmission in the prostate, which may have important implications for the pathophysiology of BPH.
Regulation of rat brain angiotensin II (AII) receptors by intravenous AII and low dietary Na+
Publisher: Elsevier BV
Date: 10-0009
DOI: 10.1016/0006-8993(85)90835-2
Abstract: Previous studies have shown the presence of specific AII receptors at several areas of the brain. The purpose of this study was to examine by radioreceptor assay the effect of intravenous AII infusion (5 or 25 ng/kg/min) and low dietary Na+ (less than 8 mmol/100 g) on AII receptors in five brain regions: the olfactory lobes (OLF), hypothalamus/thalamus/septum (HTS), midbrain (MID), cerebellum (CER) and medulla (MED). Scatchard analysis of binding data from control rats showed significant (P less than 0.01 ANOVA) differences between brain areas in both Ka (1.54 OLF, 1.87 HTS, 1.25 MID, 1.33 MED, 0.77 CER x 10(9) M-1) and Ro (321 OLF, 224 HTS, 203 MID, 145 MED, 41 CER fmol/g tissue). Following the i.v. infusion of AII for 4-7 days, marked changes were observed in the areas with a porous BBB, the HTS and MED. Both the Ka [3.20 (HTS) and 0.67 (MED) x 10(9) M-1] and Ro [116 (HTS) and 249 (MED) fmol/g tissue] changed. In addition, decreases in Ro were also observed in the OLF (241 fmol/g tissue) and CER (21 fmol/g tissue), areas which have not been considered as being accessible to blood-borne AII. A low Na+ diet for 21-30 days changed the Ka and Ro in all five regions but not in similar directions. Furthermore, with the exception of the OLF the direction of change was not similar to that caused by i.v. infusion of AII. It was concluded that AII receptor sites in the rat brain differ from each other in both receptor properties in their response to such regulatory factors as AII Na+ depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
Identification of a Ca2+/calmodulin-binding domain within the carboxyl-terminus of the angiotensin II (AT(1A)) receptor
Publisher: Wiley
Date: 23-07-1999
DOI: 10.1016/S0014-5793(99)00904-7
Abstract: To identify regulators of the type 1A angiotensin II receptor (AT1A), we investigated the interaction of cellular proteins with a fusion protein containing the rat AT1A receptor carboxyl-terminus. An approximately 20 kDa cytoplasmic protein interacted with the fusion protein in a Ca2+-dependent manner and was identified as calmodulin. A control peptide with high affinity for Ca2+/calmodulin and a peptide corresponding to a membrane proximal portion of the AT1A receptor carboxyl-terminus with analogy to known calmodulin-binding sequences were synthesised and tested for calmodulin-binding. Using in vitro binding assays combined with gel shift analysis, we demonstrated the formation of complexes between calmodulin and both peptides, which were Ca2+-dependent and of 1:1 stoichiometry. Affinity gels produced from these peptides also purified calmodulin from cell extracts. These results suggest a novel feedback regulation of the AT1A receptor by Ca2+/calmodulin and identify the membrane proximal region of the carboxyl-terminus as a focal point for interactions important for AT1A receptor function.
Angiotensinogen Secretion by Single Rat Pituitary Cells: Detection by a Reverse Haemolytic Plaque Assay and Cell Identification by Immunocytochemistry
Publisher: S. Karger AG
Date: 1992
DOI: 10.1159/000126130
Abstract: A reverse haemolytic plaque assay (RHPA) for angiotensinogen was developed in rat hepatoma H4 cells and applied to investigate the possible secretion of angiotensinogen from rat pituitary cells in primary culture. Over a 24-hour incubation period in Cunningham chambers plaques with a mean area of 2,800 +/- 430 and 590 +/- 220 microns2 laque (SD, n = 6) formed around all viable H4 cells and 2.8 +/- 0.59% of viable pituitary cells respectively. As a positive control PRL secretion from lactotrophs was routinely checked by the RHPA and shown to form plaques with a mean area of 4,050 +/- 1,850 microns2 laque after a 4-hour incubation. By comparing plaque size in H4 cells with angiotensinogen release in cell culture, as quantified by radioimmunoassay, the secretion rate of angiotensinogen from pituitary cells was calculated as 22 +/- 8 ng/10(6) cells/24 h. Plaque-forming cells consisted of two morphologically distinct populations 78% being small cells (less than 6 microns diameter) containing little cytoplasm and 22% were large (greater than 9 microns diameter) cells with an abundant cytoplasm. Immunocytochemical staining of pituitary cells after formation of plaques with anti-angiotensinogen, anti-LH and anti-PRL antiserum showed that the large plaque-forming cells were gonadotrophs and none were lactotrophs. All plaque-forming cells stained for angiotensinogen but only 44% of the viable cells which stained for angiotensinogen actually formed plaques. The possibility that cellular angiotensinogen was imported from extracellular sources was investigated by incubation of pituitary cells with pure 125I-angiotensinogen for periods up to 24 h. No uptake of the radiolabelled protein was found.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of angiotensinogen by angiotensin II in mouse primary astrocyte cultures
Publisher: Wiley
Date: 22-08-2011
DOI: 10.1111/J.1471-4159.2011.07406.X
Abstract: Astrocytes are the major source of angiotensinogen in the brain and play an important role in the brain renin-angiotensin system. Regulating brain angiotensinogen production alters blood pressure and fluid and electrolyte homeostasis. In turn, several physiological and pathological manipulations alter expression of angiotensinogen in brain. Surprisingly, little is known about the factors that regulate astrocytic expression of angiotensinogen. There is evidence that angiotensinogen production in both hepatocytes and cardiac myocytes can be positively regulated via the angiotensin type 1 receptor, but this effect has not yet been studied in astrocytes. Therefore, the aim of this project was to establish whether angiotensin II modulates angiotensinogen production in brain astrocytes. Primary astrocyte cultures, prepared from neonatal C57Bl6 mice, expressed angiotensinogen measured by immunocytochemistry and real-time PCR. Using a variety of approaches we were unable to identify angiotensin receptors on cultured astrocytes. Exposure of cultured astrocytes to angiotensin II also did not affect angiotensinogen expression. When astrocyte cultures were transduced with the angiotensin type 1A receptor, using adenoviral vectors, angiotensin II induced a robust down-regulation (91.4% ± 1.8%, p < 0.01, n = 4) of angiotensinogen gene expression. We conclude that receptors for angiotensin II are present in extremely low levels in astrocytes, and that this concurs with available data in vivo. The signaling pathways activated by the angiotensin type 1A receptor are negatively coupled to angiotensinogen expression and represent a powerful pathway for decreasing expression of this protein, potentially via signaling pathways coupled to Gα(q/11) .
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