ORCID Profile
0000-0001-8043-3786
Current Organisations
University of Adelaide
,
Philipps-Universität Marburg
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Publisher: Springer Science and Business Media LLC
Date: 19-12-2011
DOI: 10.1007/S00418-010-0772-0
Abstract: Damage to sensory neurons induces neural repair, regrowth and hyperexcitability. The regulation of such responses to injury must be organized in some way by the neurons. Regulation can occur at the post-transcriptional level via microRNAs (miRNAs). miRNAs are small non-coding RNAs that influence the stability or translation of mRNAs and thereby regulate gene expression. Although nociceptive neurons show transcriptional and post-transcriptional regulatory mechanisms at many levels, miRNAs have not yet been systematically investigated in these neurons. Based on our preliminary array data we investigated the presence of miR-1 in dorsal root ganglion (DRG) neurons of mice and humans. We detected miR-1 in total RNA from human and mouse DRG and localised miR-1 in human and murine sensory neurons in situ. In Situ Hybridization detected miR-1 expression by nearly all DRG neurons. In vitro studies of enriched sensory neuron subpopulations from mouse DRG showed higher miR-1 expression levels in I-B4 negative neurons compared with I-B4 positive cells. Culturing of primary sensory neurons reduced the relative miR-1 expression levels independent of the presence or absence of laminin on the culture substrate. Transfection with a miR-1 mimic induced a massive increase in neuronal miR-1 associated with attenuated neurite outgrowth. This first description of miR-1 in sensory neurons including nociceptors suggests that miR-1 has a role in modulating neurite outgrowth.
Publisher: Elsevier BV
Date: 2003
DOI: 10.1016/S1566-0702(02)00258-8
Abstract: The vasculature of the guinea pig tongue is supplied by parasympathetic vasodilator nerve fibres of intrinsic origin. Here, we investigated first to what extent neuropeptides and the synthesizing enzymes of NO, CO and acetylcholine are contained and colocalized within periarterial lingual vasodilator axons of intrinsic origin. Then it was determined whether perivascular innervation by these fibre types changes with vascular diameter, in particular in comparison with the sensory substance P (SP)-positive and sympathetic noradrenergic vascular innervation. To this end, single, double and triple labelling histochemical techniques were performed on control tongues and tongues kept in short-term organotypic culture to induce degeneration of extrinsically originating nerve fibres. Cell bodies of intrinsic microganglia and their periarterial axons contained, simultaneously, NO synthase, vasoactive intestinal peptide and the acetylcholine-synthesizing enzyme choline acetyltransferase. Additionally, neuropeptide Y (NPY) was observed in a small percentage (12%) of neurons that increased to 39% after 36 h of organotypic culture. The CO synthesizing enzyme heme oxygenase-2 was detected only in perikarya but not in periarterial axons. Intrinsic vasodilator fibres were invariably present at arteries down to a luminal diameter of 150 microm, and reached 65% of section profiles of smallest arterioles, while noradrenergic and substance P-positive axons reached 80% of arteriolar profiles. These findings show that the intrinsic lingual vasodilator innervation of the guinea pig is far extending although slightly less developed than that by sensory and sympathetic axons, and differs both in this aspect and in patterns of colocalization from that reported for other organs, e.g. lung and pelvic organs.
Publisher: Informa UK Limited
Date: 04-05-2016
DOI: 10.3109/15412555.2016.1153614
Abstract: Oxidative stress, inflammation, increased bronchial epithelial cell apoptosis, and deficient phagocytic clearance of these cells (efferocytosis) by the alveolar macrophages are present in chronic obstructive pulmonary disease (COPD) and in response to cigarette smoke. We previously showed that the macrophage dysfunction is associated with changes to the sphingosine-1-phosphate (S1P) signalling system. We hypothesized that the antioxidant/anti-inflammatory agent, thymoquinone, would improve macrophage phagocytosis via modulation of the S1P system and protect bronchial epithelial cells from cigarette smoke or lipopolysaccharide (LPS)-induced apoptosis. Phagocytosis was assessed using flow cytometry, S1P mediators by Real-Time PCR, and apoptosis of 16HBE bronchial epithelial cells using flow cytometry and immunohistochemistry. Cigarette smoke and LPS decreased phagocytosis and increased S1P receptor (S1PR)-5 mRNA in THP-1 macrophages. Thymoquinone enhanced efferocytic hagocytic ability, antagonized the effects of cigarette smoke extract and LPS on phagocytosis and S1PR5, and protected bronchial epithelial cells from cigarette smoke-induced apoptosis. Thymoquinone is worth further investigating as a potential therapeutic strategy for smoking-related lung diseases.
Publisher: Bioscientifica
Date: 07-2011
DOI: 10.1530/REP-10-0302
Abstract: The cholinergic system consists of acetylcholine (ACh), its synthesising enzyme, choline acetyltransferase (CHAT), transporters such as the high-affinity choline transporter (SLC5A7 also known as ChT1), vesicular ACh transporter (SLC18A3 also known as VAChT), organic cation transporters (SLC22s also known as OCTs), the nicotinic ACh receptors (CHRN also known as nAChR) and muscarinic ACh receptors. The cholinergic system is not restricted to neurons but plays an important role in the structure and function of non-neuronal tissues such as epithelia and the immune system. Using molecular and immunohistochemical techniques, we show in this study that non-neuronal cells in the parenchyma of rat testis express mRNAs for Chat , Slc18a3 , Slc5a7 and Slc22a2 as well as for the CHRN subunits in locations completely lacking any form of innervation, as demonstrated by the absence of protein gene product 9.5 labelling. We found differentially expressed mRNAs for eight α and three β subunits of CHRN in testis. Expression of the α7-subunit of CHRN was widespread in spermatogonia, spermatocytes within seminiferous tubules as well as within Sertoli cells. Spermatogonia and spermatocytes also expressed the α4-subunit of CHRN. The presence of ACh in testicular parenchyma (TP), capsule and isolated germ cells could be demonstrated by HPLC. Taken together, our results reveal the presence of a non-neuronal cholinergic system in rat TP suggesting a potentially important role for non-neuronal ACh and its receptors in germ cell differentiation.
Publisher: Springer Science and Business Media LLC
Date: 12-2003
DOI: 10.1007/S00429-003-0344-3
Abstract: During ontogenesis the 52 amino acid peptide adrenomedullin is first expressed in the heart and it is essential for normal cardiovascular development. Recent work suggests that most adrenomedullin effects are conveyed via the calcitonin receptor-like receptor (CRLR) in combination with appropriate receptor activity-modifying proteins (RAMPs). Here, we investigated the expression of these components during the development of the rat heart, focusing on the period of coronary vascular development. Using RT-PCR, transcripts for CRLR, RAMP1 and RAMP2 were detected at all stages from E 14 to adulthood. The distribution of CRLR was investigated by immunohistochemistry, and endothelial cells and their precursors identified with monoclonal antibodies against RECA-1 and flk-1. On E 14, intense CRLR immunoreactivity was observed in endothelial cells of the large vessels and the endocardial cushions at the AV-junction. Small CRLR immunoreactive cell clusters were located in the wall of the outflow tract and subepicardially in the ventricular wall. On E 16, tubes of CRLR immunoreactive cells formed a subepicardial plexus, from which they penetrated radially towards the trabecular network and entered at E 18. Smooth muscle cells of coronary arteries gained a moderate CRLR immunoreactivity at E 20 which persisted at this intensity up to P 8 and then decreased. At the same time, CRLR immunoreactivity of endothelial cells in coronary arteries vanished while those of coronary veins still exhibited intense CRLR immunoreactivity. These data suggest multiple functions of the adrenomedullin/CRLR signaling pathway in cardiac development, among which the most prominent appears to be the early outgrowth and proliferation of the immature endothelial cells of the coronary vasculature.
Publisher: Elsevier BV
Date: 11-2009
DOI: 10.1016/J.JACI.2009.06.034
Abstract: Immunologic processes might contribute to the pathogenesis of pulmonary arterial hypertension (PAH), a fatal condition characterized by progressive pulmonary arterial remodeling, increased pulmonary vascular resistance, and right ventricular failure. Experimental allergen-driven lung inflammation evoked morphologic and functional vascular changes that resembled those observed in patients with PAH. Sphingosine kinase 1 (SphK1) is the main pulmonary contributor to sphingosine-1-phosphate (S1P) synthesis, a modulator of immune and vascular functions. We sought to investigate the role of SphK1 in allergen-induced lung inflammation. SphK1-deficient mice and C57Bl/6 littermates (wild-type [WT] animals) were subjected to acute or chronic allergen exposure. After 4 weeks of systemic ovalbumin sensitization and local airway challenge, airway responsiveness increased less in SphK1(-/-) compared with WT mice, whereas pulmonary vascular responsiveness was greatly increased and did not differ between strains. Acute lung inflammation led to an increase in eosinophils and mRNA expression for S1P phosphatase 2 and S1P lyase in lungs of WT but not SphK1(-/-) mice. After repetitive allergen exposure for 8 weeks, airway responsiveness was not augmented in SphK1(-/-) or WT mice, but pulmonary vascular responsiveness was increased in both strains, with significantly higher vascular responsiveness in SphK1(-/-) mice compared with that seen in WT mice. Increased vascular responsiveness was accompanied by remodeling of the small and intra-acinar arteries. : The data support a role for SphK1 and S1P in allergen-induced airway inflammation. However, SphK1 deficiency increased pulmonary vascular hyperresponsiveness, which is a component of PAH pathobiology. Moreover, we show for the first time the dissociation between inflammation-induced remodeling of the airways and pulmonary vasculature.
Publisher: Elsevier BV
Date: 06-2004
Publisher: Oxford University Press (OUP)
Date: 10-2008
DOI: 10.1111/J.1365-2133.2008.08774.X
Abstract: The skin cholinergic signalling system is modulated in atopic dermatitis (AD). To investigate of the role of nicotinic acetylcholine receptors (nAChRs) in the pathogenesis of AD. We investigated the expression and localization of nAChR alpha subunits in AD by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry of biopsies from lesional and nonlesional areas of AD skin and of skin biopsies from healthy control persons. Our data demonstrate the presence of mRNA and protein of the nAChR alpha subunits 3, 5, 7, 9 and 10 in keratinocytes and mast cells in healthy and AD skin. Expression of the alpha subunits 3, 7, 9 and 10 was generally reduced in the skin of patients with AD whereas mast cells in AD but not in healthy skin showed alpha3 and alpha5 subunit immunoreactivity. Differences in the subunit mRNA levels between lesional and nonlesional skin were obtained for the alpha subunits 3, 9 and 10 with higher levels of alpha3 but lower levels of alpha10 subunit mRNA in lesional areas. No differences in the expression of the alpha subunits was found between the groups of extrinsic, intrinsic or mixed AD types, between genders and between smokers and nonsmokers. This supports the idea that the cholinergic system is dysregulated independently from inflammation in AD and that inflammation further modulates in idual nAChR subunits.
Publisher: Elsevier BV
Date: 11-2003
DOI: 10.1016/J.EJPHAR.2003.09.030
Abstract: Calcitonin-gene-related peptide and adrenomedullin have similar and potent vascular effects, which appear to be mediated by the G protein-coupled calcitonin receptor-like (CRL) receptor. Using immunohistochemical and Western blot analyses, we have obtained novel evidence that CRL receptor is expressed in the rat vascular endothelium using an antibody to rat CRL receptor that we have raised and fully characterised. These results are an important basis for further studies aimed at determining the so far ill-defined functional significance of the extensive distribution of CRL receptor in the vascular endothelium.
Publisher: Public Library of Science (PLoS)
Date: 17-02-2011
Publisher: Frontiers Media SA
Date: 19-06-2019
Publisher: Research Square Platform LLC
Date: 02-12-2020
DOI: 10.21203/RS.3.RS-117928/V1
Abstract: Introduction The role inflammasomes play in chronic obstructive pulmonary disease (COPD) is unclear. We hypothesised that the AIM2 inflammasome is activated in the airways of COPD patients, and in response to cigarette smoke. Methods Lung tissue, bronchoscopy-derived alveolar macrophages and bronchial epithelial cells from COPD patients and healthy donors lungs from cigarette smoke-exposed mice and cigarette smoke extract-stimulated alveolar macrophages from healthy controls and HBEC30KT cell line were investigated. AIM2 inflammasome activation was assessed by multi-fluorescence quantitative confocal microscopy of speck foci positive for AIM2, inflammasome component ASC and cleaved IL-1β. Subcellular AIM2 localization was assessed by confocal microscopy, and immunoblot of fractionated cell lysates. Nuclear localization was supported by in-silico analysis of nuclear localization predicted scores of peptide sequences. Nuclear and cytoplasmic AIM2 was demonstrated by immunoblot in both cellular fractions from HBEC30KT cells. Results Increased cytoplasmic AIM2 speck foci, colocalized with cleaved IL-1β, were demonstrated in COPD lungs (n=9) vs. control (n=5), showing significant positive correlations with GOLD stages. AIM2 nuclear-to-cytoplasmic redistribution was demonstrated in bronchiolar epithelium in cigarette-exposed mice and in HBEC30KT cells post 24 hrs stimulation with 5% cigarette smoke extract. Alveolar macrophages from 8 healthy non-smokers responded to cigarette smoke extract with an -fold increase (p .05) of cytoplasmic AIM2 and -fold increase (p .01) of colocalized cleaved IL-1β speck foci, which were also localized with ASC. Conclusion The AIM2 inflammasome is activated in the airway of COPD patients, and in response to cigarette smoke exposure, associated with a nuclear to cytoplasmic shift in the distribution of AIM2.
Publisher: Springer Science and Business Media LLC
Date: 07-2006
Publisher: Elsevier BV
Date: 07-1997
DOI: 10.1016/S0014-2999(97)01050-9
Abstract: Neuropeptide Y and nitric oxide (NO) synthase are colocalized in nervous tissues. We tested the hypothesis whether or not NO might be involved in the release of neuropeptide Y. Neuropeptide Y concentration in the supernatant of PC12 rat pheochromocytoma cells, shown to express NO synthase I by immunohistochemistry, rose threefold in a time- and dose-dependent manner following sodiumnitroprusside and 3-morpholinosydnonimine (SIN-1) incubation. Neuropeptide Y mRNA expression was induced by NO-donors as a function of incubation-time. Neuropeptide Y production rose fivefold with zaprinast, an inhibitor of the phosphodiesterase V and threefold with nerve growth factor (NGF). Combined application of zaprinast and NGF did not further increase neuropeptide Y production while combination of zaprinast and sodiumnitroprusside potentiated the NO effect on neuropeptide Y release. The data suggest that NO regulates neuropeptide Y secretion of PC12 pheochromocytoma cells on the mRNA level.
Publisher: Springer Science and Business Media LLC
Date: 2006
DOI: 10.1385/JMN:30:1:67
Publisher: SAGE Publications
Date: 04-2017
Publisher: Elsevier BV
Date: 02-2001
Publisher: Elsevier BV
Date: 05-2007
DOI: 10.1016/J.LFS.2007.01.026
Abstract: Acetylcholine (ACh), derived both from nerve fibres and from non-neuronal sources such as epithelial cells, is a major regulator of airway function. There is evidence that dysfunction of the neuronal cholinergic system is involved in the pathogenesis of asthma. Here, we asked whether the pulmonary non-neuronal ACh-synthesis and release machinery is altered in a rat and a mouse model of allergic airway disease. Animals were sensitized against ovalbumin, challenged by allergen inhalation, and sacrificed 24 or 48 h later. Targets of investigation were the high-affinity choline transporter-1 (CHT1), that mediates cellular uptake of choline, the ACh-synthesizing enzyme choline acetyltransferase (ChAT), the vesicular ACh transporter (VAChT), and the polyspecific organic cation transporters (OCT1-3), which are able to translocate choline and ACh across the plasma membrane. With cell-type specific distribution patterns, immunohistochemistry identified these proteins in airway epithelial cells and alveolar macrophages. Real-time RT-PCR revealed significant decreases in ChAT-, CHT1-, VAChT-, OCT-mRNA in the lung of sensitized and allergen challenged animals. These data were supported by immunohistochemistry, demonstrating reduced labeling intensity of airway epithelial cells. ChAT-, CHT1-, VAChT-, and OCT1-mRNA were also significantly reduced in cells recovered by bronchoalveolar lavage from sensitized and challenged rats. In conclusion, the pulmonary non-neuronal cholinergic system is down-regulated in acute allergic airway inflammation. In view of the role of ACh in maintenance of cell-cell-contacts, stimulation of fluid-secretion and of ciliary beat frequency, this down-regulation may contribute to epithelial shedding and ciliated cell dysfunction that occur in this pathological condition.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 26-11-2003
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 22-08-2003
DOI: 10.1161/01.RES.0000087643.60150.C2
Abstract: The biological principles that underlie the induction and process of alveolization in the lung as well as the maintenance of the complex lung tissue structure are one of the major obstacles in pulmonary medicine today. Bone marrow–derived cells have been shown to participate in angiogenesis, vascular repair, and remodeling of various organs. We addressed this phenomenon in the lung vasculature of mice in a model of regenerative lung growth. C57BL/6 mice were transplanted with bone marrow from one of three different reporter gene–transgenic strains. flk-1 +/lacZ mice, tie-2/lacZ transgenic mice (both exhibiting endothelial cell–specific reporter gene expression), and ubiquitously enhanced green fluorescent protein (eGFP)-expressing mice served as marrow donors. After hematopoietic recovery, compensatory lung growth was induced by unilateral pneumonectomy and led to complete restoration of initial lung volume and surface area. The lungs were consecutively investigated for bone marrow–derived vascular cells by lacZ staining and immunohistochemistry for phenotype identification of vascular cells. lacZ- or eGFP-expressing bone marrow–derived endothelial cells could not be found in microvascular regions of alveolar septa. Single eGFP-positive endothelial cells were detected in pulmonary arteries at very low frequencies, whereas no eGFP-positive vascular smooth muscle cells were observed. In conclusion, we demonstrate in a model of lung growth and alveolization in adult mice the absence of significant bone marrow–derived progenitor cell contribution to the concomitant vascular growth and remodeling processes.
Publisher: Elsevier BV
Date: 04-2015
DOI: 10.1016/J.NEUROSCIENCE.2015.01.062
Abstract: Chronic pain is a significant burden and much is attributed to back muscles. Back muscles and their associated fasciae make important and distinct contributions to back pain. Peptidergic nociceptors innervating these structures contribute to central transmission and pain modulation by peripheral and central actions. Plastic changes that augment and prolong pain are exhibited by neurons containing calcitonin gene-related peptide (CGRP) following muscle injury. Subpopulations of neurons containing this peptide have been identified in dorsal root ganglia but the distribution of their fibers in skeletal muscles and associated fasciae has not been fully documented. This study used multiple-labeling immunofluorescence and retrograde axonal tracing to identify dorsal root ganglion cells associated with muscle, and to characterize the distribution and density of their nerve fibers in mouse gastrocnemius and back muscles and in the thoracolumbar fascia. Most nerve fibers in these tissues contained CGRP and two major subpopulations of neurons were found: those containing CGRP and substance P (SP) and those containing CGRP but not SP. Innervation density was three times higher in the thoracolumbar fascia than in muscles of the back. These studies show mouse back and leg muscles are predominantly innervated by neurons containing CGRP, an important modulator of pain signal transmission. There are two distinct populations of neurons containing this peptide and their fibers were three times more densely distributed in the thoracolumbar fascia than back muscles.
Publisher: Elsevier BV
Date: 2006
DOI: 10.1016/J.EJPHAR.2005.11.055
Abstract: Cholinergically induced intestinal anion secretion is generally believed to be caused by stimulation of epithelial muscarinic M3 receptors, whereas muscarinic M1 receptors are thought to be localized primarily on enteric neurons. In order to test this assumption, carbachol-stimulated Cl- secretion across distal colon, measured as increase in short-circuit current (I(sc)), was compared between M1-knockout (M1R-KO) and M3-knockout (M3R-KO) mice. Surprisingly, the maximal increase in I(sc) evoked by carbachol was more than twice as large in M3R-KO compared to M1R-KO mice. This difference was not due to a reduced secretory capacity of the epithelium from M3R-KO animals, as forskolin stimulated a similar maximal I(sc) in both types of animals. The neurotoxin tetrodotoxin diminished, but did not abolish the secretory response evoked by carbachol in M3R-KO distal colon, suggesting the existence of epithelial muscarinic receptors other than the type M3. Furthermore, in muscarinic receptor wild-type animals, the muscarinic M1 receptor antagonist pirenzepine inhibited the carbachol-stimulated I(sc) by more than 70% suggesting the presence of epithelial muscarinic M1 receptors a conclusion, which was confirmed by the identification of mRNA for muscarinic M1 receptors in isolated crypts from wild-type colon. Consequently, epithelial muscarinic receptors from the type M1 contribute to cholinergically induced ion secretion in mouse colon.
Publisher: American Thoracic Society
Date: 04-2003
Publisher: Portico
Date: 10-1999
DOI: 10.1076/EJOM.37.4.223.4724
Abstract: Immunohistochemical, biochemical and functional studies have revealed two separate cholinergic systems in the arterial vascular wall. Endothelial cells represent the ubiquitous intrinsic, intimal system they contain the acetylcholine-synthesizing enzyme, choline acetyltransferase, release a choline ester, and contain functional muscarinic receptors. Perivascular autonomic nerve fibres represent the extrinsic, adventitial system. These axons are not ubiquitous but show a highly selective distribution among and even within organs, and utilize co-mediators (NO, neuropeptides) in an organ-specific pattern. We put forward the hypothesis that the intrinsic, intimal system serves as a general regulator of basal vascular tone and wall structure responding to local, luminal stimuli, whereas the perivascular nerve fibres act on top of this basal tone by providing fine tuning in response to reflex activation due to systemic demands.
Publisher: Frontiers Media SA
Date: 19-06-2020
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-2018
DOI: 10.1097/CCM.0000000000002916
Abstract: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia. Controlled, in vitro, ex vivo, and in vivo laboratory study. Female wild-type and SphK1 -deficient mice, 8–10 weeks old. Human postmortem lung tissue, human blood–derived macrophages, and pulmonary microvascular endothelial cells. Wild-type and SphK1 -deficient mice were infected with Streptococcus pneumoniae . Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood–derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae . Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin. Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1 –/– mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate–induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase. Our data suggest that targeting the sphingosine kinase 1–/sphingosine-1-phosphate–/sphingosine-1-phosphate receptor 2–signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.
Publisher: Elsevier BV
Date: 10-2002
DOI: 10.1046/J.1523-1747.2002.00182.X
Abstract: Choline is an essential component in acetylcholine biosynthesis, and is involved in cell signaling. It is unable to permeate the cell membrane and requires a transporter to enter the cell. Neurons that synthesize acetylcholine take up choline by a recently cloned high-affinity choline transporter (choline transporter 1) that is Na+-dependent and can be blocked by hemicholinium-3. The aim of this study was to determine the expression and to analyze the distribution of choline transporter 1 in human and rat skin. The mRNA for choline transporter 1 was detected in rat and human skin and in the human keratinocyte cell line HaCaT. A polyclonal anti-serum was developed against the N-terminal region of the human and rat protein. In rat and human skin, choline transporter 1 immunoreactivity was present in nerve fibers. In addition, keratinocytes, HaCaT cells and cells of the internal root sheath of the hair follicle contained choline transporter 1 immunoreactivity. The labeling patterns of nonconfluent vs confluent cultured cells and the distribution of choline transporter 1 along the epidermal layer suggest an association of choline transporter 1 with keratinocyte differentiation. In conclusion, this study shows the presence of the high-affinity choline transporter choline transporter 1 in nerve fibers and epithelial cells in the human and rat skin supporting the pivotal role of this transporter in both the neuronal and non-neuronal cholinergic system of the skin.
Publisher: Elsevier BV
Date: 03-2003
DOI: 10.1016/S0024-3205(03)00067-5
Abstract: We investigated the occurrence and distribution of the ligand-binding alpha-subunits of nicotinic acetylcholine receptors in the rat arterial system in situ by means of RT-PCR and immunohistochemistry. Except the alpha9-subunit, all other mammalian non-muscular alpha-subunits were expressed in the arterial wall--either in endothelial or in smooth muscle cells--suggesting it as a direct target of nicotine and endogenous acetylcholine. The distribution pattern of alpha-subunits found in smooth muscle cells varied considerably among the in idual elastic, muscular and intraparenchymal arteries investigated, suggesting that non-neuronal cholinergic signalling via nicotinic receptors in the vascular wall includes components that are highly specific for in idual arteries.
Publisher: Elsevier BV
Date: 2004
Publisher: Georg Thieme Verlag KG
Date: 12-2000
DOI: 10.1055/S-2000-9189
Abstract: It is not clear whether surgical intervention during lung transplantation which includes cutting vegetative nerves, lymphatic vessels and bronchial arteries, leads to alterations in immune responses. Thus, it was studied in an animal model whether an induced pulmonary immune reaction after syngenic lung transplantation was impaired without the influence of immunosuppression and rejection. The recruitment of leukocytes and the status of reinnervation was examined. Syngenic transplantation of the left lung was performed in Lewis rats without rejection and therefore without immunosuppressive therapy. In a subgroup of animals host and donor leukocytes were distinguished. An ovalbumin (OVA)-specific pulmonary immune response was induced four months after transplantation. Bronchoalveolar lavage (BAL) and interstitial leukocytes were examined using flow cytometry and immunocytology, comparing the right lung and the grafted left lung. Immunohistology was performed to detect nerve fibers on cryostat sections. An induced cellular inflammation was observed in the right host lung as well as in the grafted left lung. However, the CD4 T cell numbers in the BAL were increased in the left lung. Single donor-type leukocytes could still be observed four months after transplantation. A partial reinnervation was found. The recruitment of immune cells into the lung interstitium and bronchoalveolar space of grafted lungs is not impaired. The incomplete reinnervation has no influence on leukocyte recruitment.
Publisher: Elsevier BV
Date: 05-1999
DOI: 10.1016/S0304-3940(99)00300-6
Abstract: The occurrence and distribution of the muscarinic M2-receptor subtype (M2R) was investigated in rat thoracic dorsal root ganglia (DRG). Messenger RNA for M2R was demonstrated by RT-PCR in total RNA from DRG. Immunoreactivity to M2R-protein was localized to 26% of sensory neurons, the majority of them (85%) belonging to the size class of 25-40 microm in diameter. Double-labeling (immuno)histochemistry revealed that all M2R-immunoreactive neurons bind the lectin, I-B4, whereas they are generally devoid of substance P-immunoreactivity. These data show the presence of M2R on a subpopulation of presumably nociceptive primary afferent neurons, thereby extending previous pharmacological and electrophysiological studies that indicated a role of M2R and/or M4R in inhibition of calcium channel currents in rat sensory neurons (Wanke, E., Bianchi, L., Mantegazza, M., Guatteo, E., Macinelli, E. and Ferroni, A., Muscarinic regulation of Ca2+ currents in rat sensory neurons: channel and receptor types, dose-response relationships and cross-talk pathways. Eur. J. Neurosci., 6 (1994) 381-391).
Publisher: Springer Science and Business Media LLC
Date: 23-02-2010
DOI: 10.1007/S00441-010-0927-2
Abstract: Although the water channel protein aquaporin-1 (AQP1) is widely observed outside the rat brain in continuous, but not fenestrated, vascular endothelia, it has not previously been observed in any endothelia within the normal rat brain and only to a limited extent in the human brain. In this immunohistochemical study of rat brain, AQP1 has also been found in microvessel endothelia, probably of the fenestrated type, in all circumventricular organs (except the subcommissural organ and the vascular organ of the lamina terminalis): in the median eminence, pineal, subfornical organ, area postrema and choroid plexus. The majority of microvessels in the median eminence, pineal and choroid plexus, known to be exclusively fenestrated, are shown to be AQP1-immunoreactive. In the subfornical organ and area postrema in which many, but not all, microvessels are fenestrated, not all microvessels are AQP1-immunoreactive. In the AQP1-immunoreactive microvessels, the AQP1 probably facilitates water movement between blood and interstitium as one component of the normal fluxes that occur in these specialised sensory and secretory areas. AQP1-immunoreactive endothelia have also been seen in a small population of blood vessels in the cerebral parenchyma outside the circumventricular organs, similar to other observations in human brain. The proposed development of AQP1 modulators to treat various brain pathologies in which AQP1 plays a deleterious role will necessitate further work to determine the effect of such modulators on the normal function of the circumventricular organs.
Publisher: Frontiers Media SA
Date: 07-11-2016
Publisher: SAGE Publications
Date: 30-07-2020
Abstract: This study examined the stratified anatomy of the traditional acupuncture point Jingbi and the neuroanatomical relationship between Jingbi and the brachial plexus, and investigated neural pathways that could be affected by acupuncture stimulation at Jingbi. Twelve dissected specimens were used to study the pathway of an acupuncture needle inserted at Jingbi. The stratified anatomy and the neuroanatomical relationship between Jingbi and the brachial plexus were studied. Our s les were grouped by gender and cause of death for comparative analysis. All needles ( n = 24, on both sides of a total of 12 cadavers) punctured the anterior scalene muscle medial to the brachial plexus and external jugular vein, lateral to the phrenic nerve and internal jugular vein, and superior to the clavicle and subclavian artery/vein. The depth of needle insertion at Jingbi on the right side of male s les was 28.0 (interquartile range (IQR), 22.5–30.8) mm, which was approximately 8 mm deeper than for female subjects ( p 0.05). The needle was 3.0 (IQR, 2.0–5.0) mm and 7.0 (IQR, 5.5–8.0) mm medial to the brachial plexus on the left and right sides, respectively. Deep needle insertion at Jingbi can puncture the anterior scalene muscle. The mechanism of action of acupuncture stimulation at Jingbi might be related to its close relationship with the brachial plexus. Significant differences in needling depth were observed when our s les were grouped by gender. More studies are needed.
Publisher: Public Library of Science (PLoS)
Date: 20-10-2015
Publisher: Springer Science and Business Media LLC
Date: 09-2003
DOI: 10.1007/S00418-003-0550-3
Abstract: In dorsal root ganglia (DRG) intraganglionic communication takes place both among neurons and between neurons and satellite cells. One diffusible substance involved in this signalling is nitric oxide (NO), and acetylcholine (ACh) is a candidate for the stimulation of intraganglionic NO synthesis. DRG neurons react to ACh-receptor stimulation with NO-dependent cGMP production. Here, we investigated the role of the alpha 7-subunit containing Ca(2+)-permeable nicotinic ACh receptors (nAChR) in this process. The alpha 7-nAChR mRNA and the protein were expressed in virtually all lumbar DRG neurons as evidenced by laser-assisted cell picking and oligo cell RT-PCR, in situ hybridisation and immunohistochemistry. Strong alpha 7-nAChR immunoreactivity was present in vanilloid receptor 1-immunoreactive, i.e. nociceptive, neurons. A neuronal production of NO in response to nicotine could be demonstrated in DRG slice preparations utilising the NO-sensitive fluorescent indicator diaminofluorescein diacetate (DAF-2DA). This stimulation of NO production was sensitive to inhibition of alpha 7-nAChR by mecamylamine and alpha-bungarotoxin, to inhibition of nitric oxide synthase (NOS) with L-NAME and L-NMMA, and to the blockade of voltage-operated Ca(2+) channels by verapamil. The results show the presence of the alpha 7-nAChR subunit in nociceptive rat DRG neurons and provide evidence for its coupling to NOS activation, indicating a role of this pathway in the intraganglionic communication in sensory ganglia.
Publisher: European Respiratory Society (ERS)
Date: 12-02-2009
Publisher: Frontiers Media SA
Date: 03-08-2015
Publisher: Springer Science and Business Media LLC
Date: 03-2002
DOI: 10.1007/S00441-002-0520-4
Abstract: The rate-limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline via a high-affinity transporter. We have generated antisera against the recently identified transporter CHT1 to investigate its distribution in rat motor neurons and skeletal muscle and have used these antisera in combination with (1) antisera against the vesicular acetylcholine transporter (VAChT) to identify cholinergic synapses and (2) Alexa-488-labelled alpha-bungarotoxin to identify motor endplates. In the motor unit, immunohistochemistry and RT-PCR have demonstrated that CHT1 is restricted to motoneurons and absent from the non-neuronal ACh-synthesizing elements, e.g. skeletal muscle fibres. In addition, CHT1 is also present in parasympathetic neurons of the tongue, as evidenced by immunohistochemistry and RT-PCR. CHT1 immunoreativity is principally found at all segments (perikaryon, dendrites, axon) of the motoneuron but is enriched at neuro-neuronal and neuro-muscular synapses. This preferential localisation matches well with its anticipated pivotal role in synaptic transmitter recycling and synthesis.
Publisher: Wiley
Date: 20-11-2016
DOI: 10.1111/RESP.12949
Abstract: We previously showed that alveolar macrophages from COPD patients are defective in their ability to phagocytose apoptotic cells ('efferocytosis') and that this defect is potentially linked to the sphingosine-1 phosphate (S1P) system, in particular the sphingosine-1 phosphate receptor 5 (S1PR5). In alveolar macrophages from COPD patients, S1PR5 mRNA expression levels increased and were correlated with both lung function and efferocytosis. However, it us unknown whether these changes are under epigenetic control via DNA methylation or whether DNA methylation directly modulates macrophage function. Bisulfite sequencing was used to assess DNA methylation levels at CpG islands associated with genes encoding selected S1P system components, including sphingosine kinase 1 (SPHK1), S1PR1 and S1PR5, in alveolar macrophages from 20 COPD patients, 7 healthy smokers and 10 healthy non/ex-smokers) by methyl quantitative real-time PCR (methyl qPCR). The effect of the DNA methyltransferase inhibitor, 5-azacytidine on the efferocytosis capacity of THP-1 macrophages was assessed using flow cytometry. Among the S1P system genes examined, S1PR5 was the single target that showed significant changes in DNA methylation between patient groups. Alveolar macrophages isolated from COPD patients showed lower methylation levels in the same region compared to macrophages from non/ex-smokers. in vitro studies using THP-1 macrophages showed that DNA demethylation with 5-azacytidine increased the efferocytosis capacity and dose-dependently rescued the cells from the cigarette smoke-induced defect in efferocytosis. Macrophage function can be modulated epigenetically. Reduced methylation may underlie the increased expression of the S1PR5 gene in alveolar macrophages and associated defective efferocytosis in COPD.
Publisher: Elsevier BV
Date: 02-2018
DOI: 10.1016/J.NEUROSCIENCE.2017.12.026
Abstract: Vulvodynia is a prevalent chronic pain disorder associated with high medical costs and often ineffective treatments. The major pathological feature is proliferation of vaginal nerve fibers. This study aimed to develop a highly reproducible animal model to study neuroproliferation in the vagina and aid the identification of appropriately targeted treatments for conditions such as vulvodynia. Mild chronic inflammation was induced using microinjection of complete Freund's adjuvant in the distal vagina of C57Bl/6 mice. Control mice received saline. Inflammation and innervation density were assessed at 7 and 28 days after a single administration or 14 days following repeated administration of complete Freund's adjuvant or saline. Histochemistry and blinded-analysis of images were used to assess vaginal morphology (H & E) and abundance of macrophages (CD68-labeling), mast cells (toluidine blue staining, mast cell tryptase-immunoreactivity), blood vessels (αSMA-immunoreactivity) and nerve fibers immunoreactive for the pan-neuronal marker PGP9.5. Subpopulations of nerve fibers were identified using immunoreactivity for calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY). Single administration of complete Freund's adjuvant resulted in vaginal swelling, macrophage infiltration, vascular proliferation and increased abundance of nerve fibers immunoreactive for CGRP, SP, VIP and/or PGP9.5 but not NPY, evident at seven days. Inflammation further increased following repeated administration of complete Freund's adjuvant but nerve fiber proliferation did not. Nerve fiber proliferation continued to be evident at 28 days. The inter-in idual differences within each treatment group were small, indicating that this model may be useful to study mechanisms underlying vaginal nerve fiber proliferation associated with inflammation.
Publisher: Elsevier BV
Date: 03-2006
DOI: 10.1016/J.NEULET.2005.10.078
Abstract: Afferent information from the lung is conveyed both to the brainstem and to the spinal cord by primary afferent fibres originating from vagal sensory (jugular-nodose ganglion complex=JNC) and dorsal root ganglion (DRG) neurons, respectively. Most interest, so far, has been paid to the vagal pathway while much less is known about spinal afferents. Here we provide the first direct comparison of rat pulmonary spinal and vagal pulmonary afferent neurons with respect to structural (soma size) and two neurochemical characteristics (binding of lectin IB4, immunoreactivity to calcitonin gene-related peptide=CGRP). After retrograde labelling from the lung, all possible combinations of CGRP-immunoreactivity and IB4-binding were observed, and the neurochemically defined subpopulations occurred in the same order of frequency in DRG and JNC: (1) IB4(-)/CGRP(+) (DRG: 48%, JNC: 47%) (2) IB4(-)/CGRP(-) (DRG: 35%, JNC: 29%) (3) IB4(+)/CGRP(+) (DRG: 12%, JNC: 21%) and (4) IB4(+)/CGRP(-) (DRG: 5%, JNC: 3%). In the IB4(-)/CGRP(-) population, pulmonary DRG neurons were slightly, but significantly larger than those in JNC (mean diameter: 33 microm versus 30 microm). This group is likely to contain slowly and rapidly adapting mechanoreceptors, which may be differently distributed among rat vagal and spinal afferent pathways. In rat DRG, labelling patterns IB4(-)/CGRP(+), IB4(+)/CGRP(+) and IB4(+)/CGRP(-) are generally characteristic for different nociceptor subtypes. With respect to these features and soma size, no further distinction between spinal and vagal afferents became obvious, although this does not exclude elicitation of entirely different responses when these pathways are stimulated.
Publisher: Frontiers Media SA
Date: 10-10-2017
Publisher: Elsevier BV
Date: 03-2007
Abstract: Intermedin (IMD), also called adrenomedullin-2, is a peptide that belongs to the calcitonin/calcitonin gene-related peptide/amylin peptide family. IMD exerts many effects on the cardiovascular system, gastrointestinal tract, and central nervous system. Here, we analyzed the expression of the IMD peptide in human skin of healthy controls, in biopsies from lesional and non-lesional areas of atopic dermatitis (AD) skin, in cultured human keratinocytes, and in the HaCaT keratinocyte cell line at the transcriptional (quantitative reverse transcription-PCR) and translational (immunohistochemistry) level. IMD messenger RNA (mRNA) and protein could be detected in keratinocytes and human skin. Keratinocytes, nerve fibers, periglandular cells, arterial/arteriolar smooth muscle cells, and pericytes of dermal microvessels were intensely IMD-immunoreactive. The IMD mRNA was, compared to healthy skin, significantly reduced in lesional and non-lesional areas of AD skin. This was accompanied by a reduction of IMD immunoreactivity in pericytes of the upper dermis indicating that skin from AD patients is generally affected, and downregulation of IMD in AD skin is not a secondary phenomenon caused by acute inflammation but is a general characteristic of AD skin. These data further point to a role of IMD expressed by pericytes in conferring higher susceptibility of the skin of AD patients to inflammatory stimuli.
Publisher: Elsevier BV
Date: 03-2003
DOI: 10.1016/S0024-3205(03)00088-2
Abstract: Uptake of choline by the high-affinity choline transporter CHT1 is the rate-limiting step in neuronal acetylcholine (ACh) synthesis. Here, we investigated by RT-PCR, in-situ hybridisation, immunohistochemistry, and Western blotting whether CHT1 is also expressed in cholinergic epithelia. CHT1-mRNA and -protein were detected in keratinocytes of human skin, rat skin and tongue, the human keratinocyte cell line HaCaT, and the ciliated cells of the rat tracheal epithelium. Immunohistochemically, CHT1 was predominantly localized to the epithelial cell membranes, in case of ciliated tracheal cells it was restricted to the apical membrane. This is the first study to demonstrate the expression of CHT1 in non-neuronal cells. The close apposition of CHT1 to reported sites of localization of choline acetyltransferase in these cells is strongly in favour of ACh synthesis being fuelled by choline uptake via CHT1 in these epithelia.
Publisher: Frontiers Media SA
Date: 06-08-2019
Publisher: The Endocrine Society
Date: 11-2005
DOI: 10.1210/ME.2004-0191
Abstract: Calcitonin, α- and β-calcitonin gene-related peptides, amylin, and adrenomedullin belong to a unique group of peptide hormones important for homeostasis maintenance. We recently identified intermedin (IMD) as a novel member of the calcitonin/calcitonin gene-related peptide family expressed in the pituitary, digestive tract, and other organs of vertebrates. Real-time PCR and immunohistochemical analysis of pituitaries from rats at different stages of development showed that IMD is expressed in the intermediate lobe and select adrenocorticotrophs in the anterior lobe, suggesting that IMD could function as a paracrine factor regulating anterior pituitary hormone secretion. In support of a paracrine role for IMD in the pituitary, quantitative and in situ hybridization analyses showed the expression of IMD receptor transcripts including the calcitonin receptor-like receptor and receptor activity-modifying proteins in the pituitary. Treatment with IMD leads to a dose-dependent increase of prolactin release in cultured rat pituitary cells. In contrast, IMD treatment has negligible effects on the release of GH, FSH, or ACTH. Likewise, in vivo treatment with IMD leads to an elevation of plasma prolactin levels in conscious rats. Based on these functional characteristics, we hypothesized that IMD could represent one of the intermediate lobe-derived prolactin-releasing factors important for prolactin regulation during reproduction. In support of this hypothesis, studies of IMD expression in lactating and ovariectomized rats showed that pituitary IMD transcripts in lactating animals increased to more than 2-fold over nonlactating controls whereas ovariectomy leads to a 90% reduction of IMD expression in the pituitary. Of importance, subsequent treatment with 17β-estradiol or diethylstilbestrol increased pituitary IMD expression in ovariectomized rats. In addition, analysis of the proximate region of the IMD gene promoter showed that the IMD gene promoter contains consensus estrogen response element sequences, and estrogen treatments up-regulate the promoter reporter activity in transfected pituitary cells. Collectively, the present study indicates that IMD represents a novel estrogen-dependent intermediate lobe-derived prolactin-releasing factor and could play important roles in the regulation of prolactin release during reproduction in females.
Publisher: Springer Science and Business Media LLC
Date: 22-05-2021
DOI: 10.1186/S12950-021-00286-4
Abstract: The role inflammasomes play in chronic obstructive pulmonary disease (COPD) is unclear. We hypothesised that the AIM2 inflammasome is activated in the airways of COPD patients, and in response to cigarette smoke. Lung tissue, bronchoscopy-derived alveolar macrophages and bronchial epithelial cells from COPD patients and healthy donors lungs from cigarette smoke-exposed mice and cigarette smoke extract-stimulated alveolar macrophages from healthy controls and HBEC30KT cell line were investigated. AIM2 inflammasome activation was assessed by multi-fluorescence quantitative confocal microscopy of speck foci positive for AIM2, inflammasome component ASC and cleaved IL-1β. Subcellular AIM2 localization was assessed by confocal microscopy, and immunoblot of fractionated cell lysates. Nuclear localization was supported by in-silico analysis of nuclear localization predicted scores of peptide sequences. Nuclear and cytoplasmic AIM2 was demonstrated by immunoblot in both cellular fractions from HBEC30KT cells. Increased cytoplasmic AIM2 speck foci, colocalized with cleaved IL-1β, were demonstrated in COPD lungs ( n = 9) vs. control ( n = 5), showing significant positive correlations with GOLD stages. AIM2 nuclear-to-cytoplasmic redistribution was demonstrated in bronchiolar epithelium in cigarette-exposed mice and in HBEC30KT cells post 24 h stimulation with 5% cigarette smoke extract. Alveolar macrophages from 8 healthy non-smokers responded to cigarette smoke extract with an 8-fold increase ( p 0.05) of cytoplasmic AIM2 and 6-fold increase ( p 0.01) of colocalized cleaved IL-1β speck foci, which were also localized with ASC. The AIM2 inflammasome is activated in the airway of COPD patients, and in response to cigarette smoke exposure, associated with a nuclear to cytoplasmic shift in the distribution of AIM2.
Publisher: American Thoracic Society
Date: 08-2008
Publisher: Wiley
Date: 20-10-2017
DOI: 10.1002/NAU.23434
Abstract: Peptidergic nerve fibers provide important contributions to urethral function. Urethral innervation of female mice is not well documented. To determine the distribution and projection sites of nerve fibers immunoreactive for vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY) in the urethra of wild-type control mice and compare innervation characteristics between the proximal and distal urethra of young nullipara and older multipara mice. Furthermore, to identify the location and neurochemical coding of the spinal afferent nerve endings in the urethra, whose sensory neurons reside in lumbosacral dorsal root ganglia (DRG). Multiple labeling immunohistochemistry of urethral sections of nulliparous (6-8 weeks old), and multiparous (9-12 months old) mice, and anterograde axonal tracing from L5-S2 (DRG) in vivo. Abundant VIP-, CGRP-, SP-, and NPY-immunoreactive nerve fibers were identified in the adventitia, muscularis, and lamina propria of proximal and distal segments of the urethra. A proportion of fibers were closely associated with blood vessels, glands, and cells immunoreactive for PGP9.5. The epithelium contained abundant nerve fibers immunoreactive for CGRP and/or SP. Epithelial innervation was increased in the distal urethra of multipara mice. Abundant fibers were traced from L5-S2 DRG to all urethral regions. We present the first identification of spinal afferent endings in the urethra. Peptidergic nerve fibers, including multiple populations of spinal afferents, provide rich innervation of the female mouse urethra. The morphology of fibers in the epithelium and other regions suggests multiple nerve-cell interactions impacting on urethral function.
Publisher: SAGE Publications
Date: 2011
Abstract: Unmyelinated primary afferent nociceptors are commonly classified into two main functional types: those expressing neuropeptides, and non-peptidergic fibers that bind the lectin IB4. However, many small diameter primary afferent neurons neither contain any known neuropeptides nor bind IB4. Most express high levels of vesicular glutamate transporter 2 (VGluT2) and are assumed to be glutamatergic nociceptors but their terminations within the spinal cord are unknown. We used in vitro anterograde axonal tracing with Neurobiotin to identify the central projections of these putative glutamatergic nociceptors. We also quantitatively characterised the spatial arrangement of these terminals with respect to those that expressed the neuropeptide, calcitonin gene-related peptide (CGRP). Neurobiotin-labeled VGluT2-immunoreactive (IR) terminals were restricted to lamina I, with a medial-tolateral distribution similar to CGRP-IR terminals. Most VGluT2-IR terminals in lateral lamina I were not labeled by Neurobiotin implying that they arose mainly from central neurons. 38 ± 4% of Neurobiotin-labeled VGluT2-IR terminals contained CGRP-IR. Conversely, only 17 ±4% of Neurobiotin-labeled CGRP-IR terminals expressed detectable VGluT2-IR. Neurobiotin-labeled VGluT2-IR or CGRP-IR terminals often aggregated into small clusters or microdomains partially surrounding intrinsic lamina I neurons. The central terminals of primary afferents which express high levels of VGluT2-IR but not CGRP-IR terminate mainly in lamina I. The spatial arrangement of VGluT2-IR and CGRP-IR terminals suggest that lamina I neurons receive convergent inputs from presumptive nociceptors that are primarily glutamatergic or peptidergic. This reveals a previously unrecognized level of organization in lamina I consistent with the presence of multiple nociceptive processing pathways.
Publisher: S. Karger AG
Date: 2002
DOI: 10.1159/000068365
Abstract: i Background/Aims: /i Pharmacological and morphological studies suggest that the gut mucosal immune system and local neuropeptide-containing neurones interact. We aimed to determine whether gut immune cells are targets for calcitonin gene-related peptide (CGRP), which has potent immune regulatory properties. i Methods: /i Using density gradient centrifugation, rat lamina propria mononuclear cells (LP-MNCs) and intra-epithelial lymphocytes (IELs) were isolated. RT-PCR was employed for the detection of mRNA of rat calcitonin receptor-like receptor (CRLR), which is considered to represent the pharmacologically defined CGRP receptor-1 subtype, as well as mRNA of the receptor activity-modifying proteins, which are essential for CRLR function and determine ligand specificity. A radioreceptor assay was employed for the detection of specific CGRP binding sites. i Results: /i RT-PCR and DNA sequencing showed that LP-MNCs and IELs express CRLR. Incubation of isolated LP-MNCs with radiolabelled αCGRP revealed the existence of specific binding sites for CGRP. i Conclusion: /i These novel data indicate that mucosal immune cells of the rat gut are a target for CGRP and provide significant evidence that CGRP functions as an immune regulator in the gut mucosa.
Publisher: Oxford University Press (OUP)
Date: 07-2011
DOI: 10.1111/J.1743-6109.2011.02258.X
Abstract: The structural and neurochemical characterization of the sensory innervation of the external genitalia of females is poorly known. To immunohistochemically map the sensory innervation of external genitalia and surrounding structures of female guinea pigs and mice. Large-diameter sensory fibers, presumably mechanoreceptors, were identified by their immunoreactivity to neuron-specific enolase (NSE) or vesicular glutamate transporter 1 (VGluT1). Peptidergic sensory fibers, presumably unmyelinated nociceptors, were identified by their immunoreactivity to calcitonin gene-related peptide (CGRP), substance P, or both. Multiple-labelled tissues were examined with high-resolution confocal microscopy. Microscopic identification of sensory endings, including potential nociceptors, characteristic of the external genitalia. Large complex nerve endings immunoreactive for NSE and VGluT1 were abundant in dermal papillae of the clitoris. Each large ending was accompanied by one or two fine fibers immunoreactive for CGRP but neither substance P nor VGluT1. More simple NSE-immunoreactive endings occurred within dermal papillae in non-hairy skin of the labia and anal canal but were rare in pudendal or perineal hairy skin. Fine intra-epithelial fibers immunoreactive for NSE but not CGRP were abundant in hairy skin but rare in non-hairy genital skin and the clitoris. Only fine varicose fibers immunoreactive for both CGRP and substance P occurred in connective tissue underlying the mucosal epithelium of cervix and endometrium. Compared with surrounding tissues, the sensory innervation of the clitoris is highly specialized. The coactivation of nociceptors containing CGRP but not substance P within each mechanoreceptor complex could be the explanation of pain disorders of the external genitalia.
Publisher: American Diabetes Association
Date: 07-02-2017
DOI: 10.2337/DB16-0837
Abstract: Pancreatic islet transplantation is a promising clinical treatment for type 1 diabetes, but success is limited by extensive β-cell death in the immediate posttransplant period and impaired islet function in the longer term. Following transplantation, appropriate vascular remodeling is crucial to ensure the survival and function of engrafted islets. The sphingosine kinase (SK) pathway is an important regulator of vascular beds, but its role in the survival and function of transplanted islets is unknown. We observed that donor islets from mice deficient in SK1 (Sphk1 knockout) contain a reduced number of resident intraislet vascular endothelial cells. Furthermore, we demonstrate that the main product of SK1, sphingosine-1-phosphate, controls the migration of intraislet endothelial cells in vitro. We reveal in vivo that Sphk1 knockout islets have an impaired ability to cure diabetes compared with wild-type controls. Thus, SK1-deficient islets not only contain fewer resident vascular cells that participate in revascularization, but likely also a reduced ability to recruit new vessels into the transplanted islet. Together, our data suggest that SK1 is important for islet revascularization following transplantation and represents a novel clinical target for improving transplant outcomes.
Publisher: Elsevier BV
Date: 1999
DOI: 10.1016/S0304-3940(98)00926-4
Abstract: The occurrence and distribution of the preferred receptor for the neuropeptide, substance P (SP), the neurokinin-1 receptor (NK1R) was investigated in the vascular supply of the rat sciatic nerve. Messenger RNA for NK1R was demonstrated by RT-PCR in the epineurial layer where the majority of small arteries and arterioles feeding the endoneurial vasculature are located. Immunoreactivity to NK1 R-protein was localized on the smooth muscle cells of these arterial vessels by means of immunofluorescence using a polyclonal NK1R antiserum. This muscular localization of NK1R explains the previously reported [Zochodne, D.W. and Ho, L.T., J. Physiol., 444 (1991) 615-630] moderate vasoconstrictor rather than vasodilator effects of SP in this vascular bed.
Publisher: Elsevier BV
Date: 05-2004
Publisher: Springer Science and Business Media LLC
Date: 31-10-2008
Publisher: Springer Science and Business Media LLC
Date: 20-04-2023
DOI: 10.1007/S00441-023-03770-W
Abstract: Dorsal root ganglia (DRG) contains thousands of sensory neurons that transmit information about our external and internal environment to the central nervous system. This includes signals related to proprioception, temperature, and nociception. Our understanding of DRG has increased tremendously over the last 50 years and has established the DRG as an active participant in peripheral processes. This includes interactions between neurons and non-neuronal cells such as satellite glia cells and macrophages that contribute to an increasingly complex cellular environment that modulates neuronal function. Early ultrastructural investigations of the DRG have described subtypes of sensory neurons based on differences in the arrangement of organelles such as the Golgi apparatus and the endoplasmic reticulum. The neuron-satellite cell complex and the composition of the axon hillock in DRG have also been investigated, but, apart from basic descriptions of Schwann cells, ultrastructural investigations of other cell types in DRG are limited. Furthermore, detailed descriptions of key components of DRG, such as blood vessels and the capsule that sits at the intersection of the meninges and the connective tissue covering the peripheral nervous system, are lacking to date. With rising interest in DRG as potential therapeutic targets for aberrant signalling associated with chronic pain conditions, gaining further insights into DRG ultrastructure will be fundamental to understanding cell–cell interactions that modulate DRG function. In this review, we aim to provide a synopsis of the current state of knowledge on the ultrastructure of the DRG and its components, as well as to identify areas of interest for future studies.
Publisher: Springer Science and Business Media LLC
Date: 31-05-2005
Publisher: Elsevier BV
Date: 11-2011
DOI: 10.1016/J.LFS.2011.08.018
Abstract: Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. Despite its importance, treatment methods are limited and restricted to symptomatic care, highlighting the urgent need for new treatment options. Tissue damage in COPD is thought to result from an inability of the normal repair processes with accumulation of apoptotic material and impaired clearance of this material by macrophages in the airways. Lung inflammation involves the bioactive sphingolipid sphingosine 1-phosphate (S1P). We investigated lung tissue s les from 55 patients (25 with COPD) undergoing lobectomies for management of cancer. We analysed the sphingosine-kinase (SphK) mRNA expression profile, SphK enzyme activity as well as the localisation and expression of in idual proteins related to the SphK-signalling system. We show in this study for the first time a comprehensive expression profile of all synthesising enzymes, receptors and degrading enzymes of the SphK-signalling system in the human lung. Multivariate ANOVA showed that the relative mRNA expression of S1P receptor (S1PR) subtype 5 was reduced in COPD. There were strong positive correlations between the mRNA expression of S1PR5 and S1PR1 and S1PR3, and between S1PR3 and S1PR2. A significant negative correlation was found between S1PR1 and SphK protein activity. The correlations between expression levels of receptors and enzymes involved in the sphingosine kinase signalling system in the lung suggest common regulatory mechanisms. Our findings of reduced S1PR5 in COPD and the correlation with other S1P receptors in COPD identify S1PR5 as a possible novel target for pharmacotherapy.
Publisher: American Institute of Mathematical Sciences (AIMS)
Date: 2015
Publisher: Wiley
Date: 27-04-2011
DOI: 10.1002/ART.30282
Abstract: In primary Sjögren's syndrome (SS), impairment of the gastrointestinal (GI) tract is common, and includes reduced esophageal motor function, delayed gastric emptying, and abnormalities in colonic motility the pathogenesis is as yet unknown. We undertook this study to investigate the role of functional antibodies to the type 3 muscarinic receptor (M3R) in GI dysfunction associated with primary SS. Muscle strip and whole-organ functional assays were used to determine whether IgG with anti-M3R activity from patients with primary SS disrupted neurotransmission in tissue from throughout the mouse GI tract. Specificity of the autoantibody for the M3R was determined using knockout mice that were deficient in the expression of muscarinic receptor subtypes. Functional antibodies to the M3R inhibited neuronally mediated contraction of smooth muscle from throughout the GI tract and disrupted complex contractile motility patterns in the colon. The autoantibodies were not active on tissue from mice that lacked the M3R, providing compelling evidence of the direct interaction of patient autoantibodies with the M3R. Our results indicate that anti-M3R autoantibodies have the potential to mediate multiple dysfunctions of the GI tract in primary SS, ranging from reduced esophageal motor activity to altered colonic motility. We hypothesize that altered GI motility forms part of a broader autonomic dysfunction mediated by pathogenic anti-M3R autoantibodies in primary SS.
Publisher: Medknow
Date: 2018
Publisher: Frontiers Media SA
Date: 18-01-2022
DOI: 10.3389/FCIMB.2021.784972
Abstract: Improved understanding of vestibulodynia pathophysiology is required to develop appropriately targeted treatments. Established features include vulvovaginal hyperinnervation, increased nociceptive signalling and hypersensitivity. Emerging evidence indicates macrophage-neuron signalling contributes to chronic pain pathophysiology. Macrophages are broadly classified as M1 or M2, demonstrating pro-nociceptive or anti-nociceptive effects respectively. This study investigates the impact of clodronate liposomes, a macrophage depleting agent, on nociceptive signalling in a mouse model of vestibulodynia. Microinjection of complete Freund’s adjuvant (CFA) at the vaginal introitus induced mild chronic inflammation in C57Bl/6J mice. A subgroup was treated with the macrophage depleting agent clodronate. Control mice received saline. After 7 days, immunolabelling for PGP9.5, F4/80+CD11c+ and F4/80+CD206+ was used to compare innervation density and presence of M1 and M2 macrophages respectively in experimental groups. Nociceptive signalling evoked by vaginal distension was assessed using immunolabelling for phosphorylated MAP extracellular signal-related kinase (pERK) in spinal cord sections. Hyperalgesia was assessed by visceromotor response to graded vaginal distension. CFA led to increased vaginal innervation (p & 0.05), increased pERK-immunoreactive spinal cord dorsal horn neurons evoked by vaginal-distension (p & 0.01) and enhanced visceromotor responses compared control mice (p & 0.01). Clodronate did not reduce vaginal hyperinnervation but significantly reduced the abundance of M1 and M2 vaginal macrophages and restored vaginal nociceptive signalling and vaginal sensitivity to that of healthy control animals. We have developed a robust mouse model of vestibulodynia that demonstrates vaginal hyperinnervation, enhanced nociceptive signalling, hyperalgesia and allodynia. Macrophages contribute to hypersensitivity in this model. Macrophage-sensory neuron signalling pathways may present useful pathophysiological targets.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 04-2005
DOI: 10.1097/00001756-200504040-00012
Abstract: The novel alpha-conotoxin Vc1.1 is a potential analgesic for the treatment of painful neuropathic conditions. In the present study, the effects of Vc1.1 were tested on the nicotine-induced increase in excitability of unmyelinated C-fiber axons in isolated segments of peripheral human nerves. Vc1.1 in concentrations above 0.1 microM antagonized the increase in axonal excitability produced by nicotine the maximal inhibition was observed with 10 microM. We also demonstrate immunoreactivity for alpha 3 and alpha 5 subunits of neuronal nicotinic receptors on unmyelinated peripheral human axons. Blockade of nicotinic receptors on unmyelinated peripheral nerve fibers may be helpful in painful neuropathies affecting unmyelinated sympathetic and/or sensory axons.
Publisher: Elsevier BV
Date: 03-2016
Publisher: Public Library of Science (PLoS)
Date: 27-04-2012
Publisher: Springer Science and Business Media LLC
Date: 12-04-2006
Abstract: It has been proposed that serotonin (5-HT)-mediated constriction of the murine trachea is largely dependent on acetylcholine (ACh) released from the epithelium. We recently demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells. Hence, the hypothesis emerged that 5-HT evokes bronchoconstriction by inducing release of ACh from epithelial cells via OCTs. We tested this hypothesis by analysing bronchoconstriction in precision-cut murine lung slices using OCT and muscarinic ACh receptor knockout mouse strains. Epithelial ACh content was measured by HPLC, and the tissue distribution of OCT isoforms was determined by immunohistochemistry. Epithelial ACh content was significantly higher in OCT1/2 double-knockout mice (42 ± 10 % of the content of the epithelium-denuded trachea, n = 9) than in wild-type mice (16.8 ± 3.6 %, n = 11). In wild-type mice, 5-HT (1 μM) caused a bronchoconstriction that slightly exceeded that evoked by muscarine (1 μM) in intact bronchi but amounted to only 66% of the response to muscarine after epithelium removal. 5-HT-induced bronchoconstriction was undiminished in M 2 /M 3 muscarinic ACh receptor double-knockout mice which were entirely unresponsive to muscarine. Corticosterone (1 μM) significantly reduced 5-HT-induced bronchoconstriction in wild-type and OCT1/2 double-knockout mice, but not in OCT3 knockout mice. This effect persisted after removal of the bronchial epithelium. Immunohistochemistry localized OCT3 to the bronchial smooth muscle. The doubling of airway epithelial ACh content in OCT1/2 -/- mice is consistent with the concept that OCT1 and/or 2 mediate ACh release from the respiratory epithelium. This effect, however, does not contribute to 5-HT-induced constriction of murine intrapulmonary bronchi. Instead, this activity involves 1) a non-cholinergic epithelium-dependent component, and 2) direct stimulation of bronchial smooth muscle cells, a response which is partly sensitive to acutely administered corticosterone acting on OCT3. These data provide new insights into the mechanisms involved in 5-HT-induced bronchoconstriction, including novel information about non-genomic, acute effects of corticosteroids on bronchoconstriction.
Publisher: Elsevier BV
Date: 09-2005
DOI: 10.1016/J.AANAT.2005.05.003
Abstract: The present immunohistochemical study set out to determine the extent of perivascular innervation in the rat heart, using markers for noradrenergic sympathetic fibres (tyrosine hydroxylase = TH), cholinergic parasympathetic fibres (vesicular acetylcholine transporter = VAChT), nitrergic fibres (neuronal NO synthase = nNOS), and peptidergic sensory fibres (calcitonin gene-related peptide = CGRP). For each of these antigens, the vascular innervation density was assessed separately in the atria, the basal and the apical parts of the ventricles, and was correlated to the inner vascular diameter. The four major findings are: (1) Each of these neurochemically defined populations shows an in idual distribution pattern significantly different from the others with respect to correlation with vascular diameter and occurrence along atrial versus ventricular vessels. (2) Among autonomic efferent axons, nNOS-containing fibres are far less numerous than cholinergic and noradrenergic fibres. (3) Autonomic efferent axons (noradrenergic, cholinergic, nitrergic) are much more abundant around atrial than ventricular vessels, whereas perivascular CGRP-immunoreactive sensory nerve fibres are equally distributed in the various parts of the heart. (4) Noradrenergic and cholinergic axons preferentially innervate small-diameter vessels (negative linear correlation between index of innervation and vascular diameter), whereas the supply with CGRP-immunoreactive sensory nerve fibres does not change with vascular diameter. Collectively, the present study shows in idual distribution patterns for each of the neurochemically defined populations of perivascular axons along the atrial and ventricular coronary arteries, indicating a highly differentiated nervous regulation of atrial versus ventricular, and large-diameter versus resistance vessels.
Publisher: Springer Science and Business Media LLC
Date: 2006
DOI: 10.1385/JMN:30:1:55
Publisher: Springer Science and Business Media LLC
Date: 11-2011
DOI: 10.1007/S00441-011-1263-X
Abstract: The unpleasant sensory and emotional experience of pain is initiated by excitation of primary afferent nociceptive neurons. Nerve damage or inflammation induces changes in nociceptive DRG neurons which contribute to both peripheral and central sensitization of pain-sensitive pathways. Recently, blockade of microRNA synthesis has been found to modulate the response of nociceptive neurons to inflammatory stimuli. However, little is known about the contributions of in idual miRNAs to painful conditions. We compared miRNA expression in mouse sensory neurons and focussed on the localisation and control of miR-143. Using miRNA-arrays we compared the microRNA expression profile of intact lumbar DRG with one-day-old DRG cultures and found that nine miRNAs including miR-143 showed lower expression levels in cultures. Subsequent RT-qPCR confirmed array data and in-situ hybridisation localised miR-143 in the cytosol of sensory DRG neurons in situ and in vitro. Analysis of microbead-enriched neuron cultures showed significantly higher expression levels of miR-143 in isolectin B4 (I-B4) binding sensory neurons compared with neurons in the I-B4 negative flow-through fraction. In animal models of peripheral inflammation (injection of Complete Freund's Adjuvant, CFA) and nerve damage (transection of the sciatic nerve), we found that expression levels of miR-143 were significantly lower in DRGs ipsilateral to CFA injection or after nerve damage. Taken together, our data demonstrate for the first time miR-143 expression in nociceptive neurons. Since expression levels of miR-143 were higher in I-B4 positive neurons and declined in response to inflammation but not axotomy, miR-143 could selectively contribute to mRNA regulation in specific populations of nociceptors.
Publisher: Society for Neuroscience
Date: 06-02-2013
DOI: 10.1523/JNEUROSCI.4479-12.2013
Abstract: The biolipid sphingosine-1-phosphate (S1P) is an essential modulator of innate immunity, cell migration, and wound healing. It is released locally upon acute tissue injury from endothelial cells and activated thrombocytes and, therefore, may give rise to acute post-traumatic pain sensation via a yet elusive molecular mechanism. We have used an interdisciplinary approach to address this question, and we find that intradermal injection of S1P induced significant licking and flinching behavior in wild-type mice and a dose-dependent flare reaction in human skin as a sign of acute activation of nociceptive nerve terminals. Notably, S1P evoked a small excitatory ionic current that resulted in nociceptor depolarization and action potential firing. This ionic current was preserved in “cation-free” solution and blocked by the nonspecific Cl − channel inhibitor niflumic acid and by preincubation with the G-protein inhibitor GDP-β-S. Notably, S1P 3 receptor was detected in virtually all neurons in human and mouse DRG. In line with this finding, S1P-induced neuronal responses and spontaneous pain behavior in vivo were substantially reduced in S1P 3 −/− mice, whereas in control S1P 1 floxed (S1P 1 fl/fl ) mice and mice with a nociceptor-specific deletion of S1P 1 −/− receptor (SNS-S1P 1 −/− ), neither the S1P-induced responses in vitro nor the S1P-evoked pain-like behavior was altered. Therefore, these findings indicate that S1P evokes significant nociception via G-protein-dependent activation of an excitatory Cl − conductance that is largely mediated by S1P 3 receptors present in nociceptors, and point to these receptors as valuable therapeutic targets for post-traumatic pain.
Publisher: Springer Science and Business Media LLC
Date: 29-07-2008
DOI: 10.1007/S00418-008-0477-9
Abstract: Transient receptor potential (TRP) channels of the TRPV, TRPA, and TRPM subfamilies play important roles in somatosensation including nociception. While particularly the Thermo TRPs have been extensively investigated in sensory neurons, the relevance of the subclass of "canonical" TRPC channels in primary afferents is yet elusive. In the present study, we investigated the presence and contribution to Ca(2+) transients of TRPC channels in dorsal root ganglion neurons. We found that six of the seven known TRPC subtypes were expressed in lumbar DRG, with TRPC1, C3, and C6 being the most abundant. Microfluorimetric calcium measurements showed Ca(2+) influx induced by oleylacylglycerol (OAG), an activator of the TRPC3/C6/C7 subgroup. Furthermore, OAG induced rises in [Ca(2+)](i) were inhibited by SKF96365, an inhibitor of receptor and store operated calcium channel. OAG induced calcium transients were also inhibited by blockers of diacylglycerol (DAG) lipase, lipoxygenase or cyclooxygenase and, intriguingly, by inhibitors of the capsaicin receptor TRPV1. Notably, SKF96365 did not affect capsaicin-induced calcium transients. Taken together, our findings suggest that TRPC are functionally expressed in subpopulations of DRG neurons. These channels, along with TRPV1, contribute to calcium homeostasis in rat sensory neurons.
Publisher: Springer Science and Business Media LLC
Date: 2006
DOI: 10.1385/JMN:30:1:15
Publisher: Elsevier BV
Date: 2002
DOI: 10.1016/S0196-9781(01)00586-1
Abstract: Calcitonin gene-related peptide and adrenomedullin exert potent effects in skin but their cellular targets are unknown. This study aimed to identify the cellular location of calcitonin receptor-like receptor (CRLR) which is pharmacologically identical to CGRP receptor-1, a putative molecular target of CGRP and adrenomedullin. RT-PCR analysis of human hairy skin revealed the presence of CRLR mRNA and immunohistochemical analysis, employing a previously characterized polyclonal antibody raised to CRLR, provided novel evidence of the cellular distribution of CRLR. Extensive and specific CRLR-immunostaining was detected in arteriolar smooth muscle and venular endothelium and is consistent with CGRP's putative role in neurogenic inflammation. Novel targets for CGRP and/or adrenomedullin were identified, including capillary endothelium, hair follicles and sweat glands.
Start Date: 2013
End Date: End date not available
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2017
End Date: 2019
Funder: Australian and New Zealand College of Anaesthetists
View Funded ActivityStart Date: 2022
End Date: 2026
Funder: National Health and Medical Research Council
View Funded Activity