ORCID Profile
0000-0003-0848-6044
Current Organisation
University of Oxford
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
Publisher: S. Karger AG
Date: 2003
DOI: 10.1159/000074549
Abstract: Endothelium-dependent hyperpolarization of vascular smooth muscle provides a major pathway for relaxation in resistance arteries. This can occur due to direct electrical coupling via myoendothelial gap junctions (MEGJs) and/or the release of factors (EDHF). Here we provide evidence for the existence of functional MEGJs in the same, defined branches of BALB/C mouse mesenteric arteries which show robust EDHF-mediated smooth muscle relaxation. Cyclopiazonic acid (CPA, 10 µ i M /i ) was used to stimulate EDHF in arteries mounted under isometric conditions and constricted with phenylephrine. Simultaneous measurement of smooth muscle membrane potential and tension demonstrated that CPA caused a hyperpolarization of around 10 mV, reversing the depolarization to phenylephrine by 94% and the associated constriction by 66%. The relaxation to CPA was endothelium dependent, associated with the opening of Ca sup + /sup -activated K channels, and only in part due to the release of nitric oxide (NO). In the presence of the NO synthase inhibitor, i L /i -NAME (100 µ i M /i ), the relaxation to CPA could be almost completely inhibited with the putative gap junction uncoupler, carbenoxolone (100 µ i M /i ). Inhibition of the synthesis of prostaglandins or metabolites of arachidonic acid had no effect under the same conditions, and small rises in exogenous K sup + /sup failed to evoke consistent or marked smooth muscle relaxation, arguing against a role for these molecules and ions as EDHF. Serial section electron microscopy revealed a high incidence of MEGJs, which was correlated with heterocellular dye coupling. Taken together, these functional and morphological data from a defined mouse resistance artery suggest that the EDHF response in this vessel may be explained by extensive heterocellular coupling through MEGJs, enabling spread of hyperpolarizing current.
Publisher: Elsevier BV
Date: 05-1994
DOI: 10.1016/0306-3623(94)90218-6
Abstract: 1. This study examined the ability of various nitro-vasodilators, 8-bromo cyclic guanosine 3':5' monophosphate (8-BrcGMP) and forskolin to relax rings of rat thoracic aorta pre-contracted with either noradrenaline (0.1 microM) or the protein kinase C activators, phorbol 12,13-dibutyrate (PDB, 0.1 microM) or phorbol 12-myristate 13-acetate (PMA, 0.5 microM). 2. In noradrenaline pre-contracted rings, acetylcholine (10 nM-10 microM), sodium nitroprusside (1 nM-0.5 microM), the calcium ionophore A23187 (10 nM-10 microM) and 8-BrcGMP (10 mM) totally reversed the smooth muscle contraction. In PDB-contracted aortic rings acetylcholine, sodium nitroprusside and 8-BrcGMP-induced relaxation was reduced compared to that in noradrenaline-contracted aortic rings, but A23187 and forskolin-induced relaxations were unaffected. Both acetylcholine and A23187-induced relaxations in PDB-contracted rings were abolished in the presence of the nitric oxide synthesis inhibitor N omega-nitro-L-arginine (NOLA, 100 microM). 3. Acetylcholine and sodium nitroprusside were even less potent in their ability to relax PMA-contracted aortic rings compared with noradrenaline and PDB-contracted rings. A23187-induced relaxation was also inhibited in PMA-contracted rings. 4. These results show that protein kinase C activation reduces the ability of agents which liberate nitric oxide to induce smooth muscle relaxation, and also inhibits the biochemical pathways which are subsequently activated by nitric oxide and lead to vascular smooth muscle relaxation.
Publisher: Wiley
Date: 02-1994
DOI: 10.1111/J.1476-5381.1994.TB14769.X
Abstract: 1. Small strips from third-order branches of rabbit mesenteric artery (approximately 150-200 microM wide) contracted in response to noradrenaline (10 microM) or 5-hydroxytryptamine (5-HT 10 microM) in oxygenated Krebs solution containing 2.5 mM Ca2+. In a Ca(2+)-free mock intracellular solution (0 Ca2+ plus 0.2 mM EGTA), noradrenaline (10 microM) and caffeine (10 mM) induced only a single, transient contraction in artery strips, while 5-HT (10 microM) failed to induce any response. 2. In strips of mesenteric artery which had been permeabilized with Staphylococcus alpha-toxin and bathed in Ca(2+)-free mock intracellular solution, noradrenaline (10 microM), caffeine (10 mM) and D-myo-inositol (1,4,5)-trisphosphate (IP3, 100 microM), but not 5-HT (10 or 100 microM) induced a transient contraction. In contrast to the non-permeabilized strips, contractions to noradrenaline, caffeine and IP3 were restored by prior incubation (10 min) in solution containing 0.08 microM Ca2+. The contractions to noradrenaline and IP3 in permeabilized muscle strips required the presence of 100 microM guanosine 5'-triphosphate (GTP), although in the absence of Ca2+. GTP alone did not induce contraction. 3. Exposure of permeabilized mesenteric artery strips to IP3 significantly reduced the subsequent contractile responses to caffeine. Contractile responses to caffeine and IP3 were abolished by the Ca(2+)-ATPase inhibitor, thapsigargin (1 microM). 4. Ca2+ (0.1-10 microM) induced concentration-dependent contraction in permeabilized artery strips. In strips which were submaximally contracted with 0.5 microM Ca2+/100 microM GTP, the subsequent addition of 5-HT (10 microM) stimulated further contraction. The protein kinase C inhibitor, H-7 (1 microM) abolished the 5-HT/GTP-induced contraction, but did not alter the contraction to Ca2+. 5. In non-permeabilized, endothelium-denuded segments of rabbit mesenteric artery bathed in Ca2+-replete Krebs solution, noradrenaline (10 microM) stimulated a rapid, transient accumulation of IP3. 5-HT(100 microM) failed to stimulate IP3 accumulation during exposure periods of up to 5 min. 5-HT (100 microM)did stimulate IP3 accumulation if the external K+ concentration was raised (to around 25 mM). This concentration of K+ alone did not stimulate IP3 production and the 5-HT-stimulated IP3 accumulation in the presence of elevated extracellular [K+] was abolished by the alpha l-adrenoceptor antagonist, prazosin(O.1 microM).6. These results suggest that intracellular Ca2+ release does not play an important role in 5-HT-induced smooth muscle contraction in the rabbit mesenteric artery. This is despite the fact that a significant intracellular Ca2+ pool is present in these cells, which can be discharged by either noradrenaline or IP3.However, 5-HT did stimulate smooth muscle contraction in the presence of raised intracellular calcium,suggesting that a component of the contraction to 5-HT will reflect an increase in myofilament Ca2+sensitivity, possibly due to the activation of protein kinase C.
Publisher: Elsevier BV
Date: 07-1995
DOI: 10.1016/0922-4106(95)90027-6
Abstract: The present study investigated the characteristics of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding sites in crude membrane preparations of rabbit aortic smooth muscle. A particular aim was to demonstrate if increases in cytoplasmic cyclic guanosine 3':5' monophosphate (cGMP), which mediates the effect of nitrovasodilators, may cause smooth muscle relaxation in part by the displacement of Ins(1,4,5)P3 binding. Negligible Ins(1,4,5)P3 binding was observed at pH < 7, while maximum binding occurred over the pH range 8-9. Saturation analysis of isotopic dilution binding data revealed an apparently homogenous population of Ins(1,4,5)P3 binding sites with a KD of 4.02 +/- 0.53 nM and a Bmax of 27.7 +/- 4.6 fmol/mg protein. Heparin, an Ins(1,4,5)P3 receptor antagonist, inhibited binding with an IC50 of 11.43 +/- 2.81 micrograms/ml. The ability of other polyphosphate compounds to inhibit Ins(1,4,5)P3 binding in this preparation was also examined. D-myo-Inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), adenosine 5'-triphosphate (ATP) and guanosine 5'-triphosphate (GTP) inhibited Ins(1,4,5)P3 binding, although each was significantly less potent that Ins(1,4,5)P3. In contrast, cyclic guanosine 3':5' monophosphate (cGMP) did not significantly alter Ins(1,4,5)P3 binding in rabbit aortic smooth muscle. This observation suggests that competitive inhibition of Ins(1,4,5)P3 receptor binding is not an important consideration in cGMP-mediated vascular smooth muscle cell relaxation.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 19-08-2005
DOI: 10.1161/01.RES.0000178008.46759.D0
Abstract: In resistance arteries, spread of hyperpolarization from the endothelium to the adjacent smooth muscle is suggested to be a crucial component of dilation resulting from endothelium-derived hyperpolarizing factor (EDHF). To probe the role of endothelial gap junctions in EDHF-mediated dilation, we developed a method, which was originally used to load membrane impermeant molecules into cells in culture, to load connexin (Cx)-specific inhibitory molecules rapidly (≈15 minutes) into endothelial cells within isolated, pressurized mesenteric arteries of the rat. Validation was achieved by luminally loading cell-impermeant fluorescent dyes selectively into virtually all the arterial endothelial cells, without affecting either tissue morphology or function. The endothelial monolayer served as an effective barrier, preventing macromolecules from entering the underlying smooth muscle cells. Using this technique, endothelial cell loading either with antibodies to the intracellular carboxyl-terminal region of Cx40 (residues 340 to 358) or mimetic peptide for the cytoplasmic loop (Cx40 residues 130 to 140) each markedly depressed EDHF-mediated dilation. In contrast, multiple antibodies directed against different intracellular regions of Cx37 and Cx43, and mimetic peptide for the intracellular loop region of Cx37, were each without effect. Furthermore, simultaneous intra- and extraluminal incubation of pressurized arteries with inhibitory peptides targeted against extracellular regions of endothelial cell Cxs ( 43 Gap 26, 40 Gap 27, and 37,43 Gap 27 300 μmol/L each) for 2 hours also failed to modify the EDHF response. High-resolution immunohistochemistry localized Cx40 to the end of endothelial cell projections at myoendothelial gap junctions. These data directly demonstrate a critical role for Cx40 in EDHF-mediated dilation of rat mesenteric arteries.
Publisher: Wiley
Date: 24-10-2006
Publisher: Elsevier BV
Date: 07-1995
DOI: 10.1016/0922-4106(95)90026-8
Abstract: This study examined the ability of 5-hydroxytryptamine and noradrenaline to stimulate inositol 1,4,5-trisphosphate (IP3) mass accumulation in segments of the rabbit basilar artery. 5-Hydroxytryptamine (5-HT, 100 microM) failed to stimulate any significant accumulation of IP3 during the 5 min period following its application. In the presence of prazosin, 5-HT (300 microM) caused a rapid, transient decrease in IP3 accumulation which was significant after 5 s but had increased to pre-stimulation levels within 15 s. In contrast, noradrenaline (10 microM) stimulated a rapid, transient accumulation of IP3 which was significant after 5 s but had declined to basal levels after 60 s. In basilar artery segments bathed in Krebs solution containing 25.7 mM K+ (normal concentration 5.7 mM), the basal IP3 concentration was significantly elevated. The IP3 accumulation stimulated by either 5-HT or raised K+ was not reduced by the presence of the alpha 1-adrenoceptor antagonist, prazosin (0.1 microM). In the presence of raised K+, 5-hydroxytryptamine caused a rapid, transient inhibition of the K(+)-induced IP3 accumulation, which was maximal after 5 s but had increased to pre-stimulation levels within 30 s in the continued presence of 5-hydroxytryptamine. Noradrenaline did not affect the IP3 accumulation induced by raised extracellular [K+]. These results provide further evidence that IP3 is not involved in 5-hydroxytryptamine-induced smooth muscle contraction in the rabbit basilar artery, but support a role for this second messenger in the contraction induced in response to noradrenaline.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 05-2006
DOI: 10.1161/01.STR.0000217307.71231.43
Abstract: Background and Purpose— Endothelium-derived hyperpolarizing factor responses in the rat middle cerebral artery are blocked by inhibiting IK Ca channels alone, contrasting with peripheral vessels where block of both IK Ca and SK Ca is required. As the contribution of IK Ca and SK Ca to endothelium-dependent hyperpolarization differs in peripheral arteries, depending on the level of arterial constriction, we investigated the possibility that SK Ca might contribute to equivalent hyperpolarization in cerebral arteries under certain conditions. Methods— Rat middle cerebral arteries (≈175 μm) were mounted in a wire myograph. The effect of K Ca channel blockers on endothelium-dependent responses to the protease-activated receptor 2 agonist, SLIGRL (20 μmol/L), were then assessed as simultaneous changes in tension and membrane potential. These data were correlated with the distribution of arterial K Ca channels revealed with immunohistochemistry. Results— SLIGRL hyperpolarized and relaxed cerebral arteries undergoing variable levels of stretch-induced tone. The relaxation was unaffected by specific inhibitors of IK Ca (TRAM-34, 1 μmol/L) or SK Ca (apamin, 50 nmol/L) alone or in combination. In contrast, the associated smooth-muscle hyperpolarization was inhibited, but only with these blockers in combination. Blocking nitric oxide synthase (NOS) or guanylyl cyclase evoked smooth-muscle depolarization and constriction, with both hyperpolarization and relaxation to SLIGRL being abolished by TRAM-34 alone, whereas apamin had no effect. Immunolabeling showed SK Ca and IK Ca within the endothelium. Conclusions— In the absence of NO, IK Ca underpins endothelium-dependent hyperpolarization and relaxation in cerebral arteries. However, when NOS is active SK Ca contributes to hyperpolarization, whatever the extent of background contraction. These changes may have relevance in vascular disease states where NO release is compromised and when the levels of SK Ca expression may be altered.
Publisher: Elsevier BV
Date: 04-2018
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Christopher Garland.