Nicole M. Rummery

Myoendothelial gap junctions may provide the pathway for EDHF in mouse mesenteric artery

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 µM) 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 Ca2+-activated K channels, and only in part due to the release of nitric oxide (NO). In the presence of the NO synthase inhibitor, L-NAME (100 µM), the relaxation to CPA could be almost completely inhibited with the putative gap junction uncoupler, carbenoxolone (100 µM). Inhibition of the synthesis of prostaglandins or metabolites of arachidonic acid had no effect under the same conditions, and small rises in exogenous K+ 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.

Heterogeneity in the distribution of vascular gap junctions and connexins:implications for function

1. Gap junctions, which are comprised of members of a family of membrane proteins called connexins (Cx), permit the transfer of electrical and chemical information between adjacent cells in a wide variety of tissues. The aim of the present study was to compare the expression of Cx37, 40 and 43 in the smooth muscle and endothelium of a large elastic artery and two smaller muscular arteries of the rat. Serial section electron microscopy was also used to determine the presence of pentalaminar gap junctions in the smooth muscle and the incidence of myoendothelial gap junctions between the smooth muscle and endothelial cells in muscular arteries of different size.

Vascular gap junctions and implications for hypertension

1. Four connexin (Cx) molecules, namely Cx37, Cx40, Cx43 and Cx45, are expressed in the gap junctions that exist within and between the cellular layers of arteries.

Attenuation of conducted vasodilatation in rat mesenteric arteries during hypertension: role of inwardly rectifying potassium channels

The present study was designed to elucidate whether the conduction of vasomotor responses mediated by endothelium‐derived hyperpolarizing factor (EDHF) in rat mesenteric arteries is altered during hypertension. Iontophoresed acetylcholine (ACh; 500 ms) caused EDHF‐mediated hyperpolarization and vasodilatation at the local site and these responses spread through the endothelium to remote sites in 12‐week‐old Wistar‐Kyoto rats (WKY). Conducted responses were significantly attenuated in age‐matched spontaneously hypertensive rats (SHR) although the rate of decay with distance did not change. Inhibition of inwardly rectifying potassium (Kir) channels (30 μm barium) eliminated the difference between WKY and SHR by attenuating conducted responses in WKY but not SHR. At the local site, barium (30 μm) significantly reduced the duration but not the amplitude of ACh‐induced hyperpolarization in WKY only. Barium had no effect when the iontophoretic stimulus was reduced to 350 ms. After blockade of EDHF in SHR, ACh elicited a depolarization which our indirect data suggest spreads along the vessel in the endothelium. Messenger RNA expression of Kir2.0 genes did not differ between the strains nor did the amplitude of K+‐induced hyperpolarization, which was abolished by disruption of the endothelium. Immunohistochemistry revealed a decrease in connexin (Cx)37 but not Cx40 or Cx43 protein in endothelial cells of SHR compared to WKY. Results suggest that conduction of EDHF‐mediated responses in WKY, but not in SHR, is facilitated by activation of Kir channels at the site of ACh application and not by differences in endothelial connexin expression. Lack of Kir channel involvement in hypertension may result from reduction in the duration of the hyperpolarization due to the development of ACh‐mediated depolarization, rather than to any difference in Kir subunit expression or function.

Developmental changes in myoendothelial gap junction mediated vasodilator activity in the rat saphenous artery

A role for myoendothelial gap junctions (MEGJs) has been proposed in the action of the vasodilator endothelium‐derived hyperpolarizing factor (EDHF). EDHF activity varies in disease and during ageing, but little is known of the role of EDHF during development when, in many organ systems, gap junctions are up‐regulated. The aims of the present study were therefore to determine whether an up‐regulation of heterocellular gap junctional coupling occurs during arterial development and whether this change is reflected functionally through an increased action of EDHF. Results demonstrated that in the saphenous artery of juvenile WKY rats, MEGJs were abundant and application of acetylcholine (ACh) evoked EDHF‐mediated hyperpolarization and relaxation in the presence of Nω‐nitro‐l‐arginine methyl ester (L‐NAME) and indomethacin to inhibit nitric oxide and prostaglandins, respectively. Responses were blocked by a combination of charybdotoxin plus apamin, or 1‐[(2‐chlorophenyl)diphenylmethyl]‐1H‐pyrazole (TRAM‐34) plus apamin, or by blockade of gap junctions with the connexin (Cx)‐mimetic peptides, 43Gap26, 40Gap27 and 37,43Gap27. On the other hand, we found no evidence for the involvement of the putative chemical mediators of EDHF, eicosanoids, L‐NAME‐insensitive nitric oxide, hydrogen peroxide or potassium ions, since 14,15‐epoxyeicosa‐5(Z)‐enoic acid (14,15‐EEZE), hydroxocobalamin, catalase or barium and ouabain were without effect. In contrast, in the adult saphenous artery, MEGJs were rare, EDHF‐mediated relaxation was absent and hyperpolarizations were small and unstable. The present study demonstrates that MEGJs and EDHF are up‐regulated during arterial development. Furthermore, the data show for the first time that this developmentally regulated EDHF is dependent on direct electrotonic coupling via MEGJs.

Connexin37 Is the Major Connexin Expressed in the Media of Caudal Artery

Objective—To determine the connexins (Cxs) involved in intercellular coupling within vascular muscle, the present study has quantified mRNA and protein expression for Cx37, Cx40, Cx43, and Cx45 in the caudal artery (CA) and thoracic aorta (ThA) of the rat. Methods and Results—Real-time polymerase chain reaction and immunohistochemistry identified Cx37 as the most abundantly expressed Cx in the CA, with fine punctate staining observed in the media. Conversely, mRNA for Cx43 was 40-fold greater in the ThA than in the CA, with punctate staining in the endothelium and media of the ThA but confined to the endothelium in the CA. Western blotting confirmed the differences in the relative amounts of Cx43 between the 2 vessels. For both arteries, Cx45 was expressed to a lesser degree in the media but not in the endothelium, whereas Cx40 was found only in the endothelium. Cx37, Cx40, and Cx43 were expressed in the endothelium of both vessels, although the density of Cx40 plaques was significantly greater in the CA. Conclusions—The demonstration of Cx37 as the dominant Cx in the media of the CA highlights the potential heterogeneity in Cx involvement in vascular smooth muscle.

Decreased endothelial size and connexin expression in rat caudal arteries during hypertension

Objectives Hypertension is accompanied by endothelial dysfunction. The present study has investigated endothelial cell morphology and connexin expression in the caudal artery of the rat during the development of hypertension. Methods A significant increase in systolic blood pressure was detected from 9 weeks of age in spontaneously hypertensive male rats (SHR) compared to normotensive Wistar–Kyoto (WKY) rats, reaching a maximum by 11–12 weeks of age. Immunohistochemistry was used to quantify cell size and expression of connexins (Cxs) 37, 40 and 43 in the endothelium of prehypertensive (3-week-old) and hypertensive (12-week-old) rats. Results At 12 weeks, the size of endothelial cells and the expression of all three Cxs per endothelial cell were significantly less in SHR than WKY rats. At 3 weeks, there was no significant difference in cell size nor in the expression of Cxs 37 or 43; however, expression of Cx40 was significantly lower in SHR than in WKY rats. Between 3 and 12 weeks in WKY rats, there was no change in endothelial cell size, nor in the expression of Cxs 37, 40 and 43. In SHR, both cell size and Cx expression per endothelial cell were significantly decreased during the same developmental period, with a significant decrease in the density of Cx40 plaques. Conclusion The development of hypertension in the SHR is accompanied by significant decreases in endothelial cell size and expression of Cx40, which may contribute to the endothelial dysfunction present in hypertension.

Structure, Function, and Endothelium-Derived Hyperpolarizing Factor in the Caudal Artery of the SHR and WKY Rat

Objective—To quantify structural and functional characteristics of the caudal artery from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats with particular reference to endothelium-derived hyperpolarizing factor (EDHF). Methods and Results—Ultrastructural studies showed that the number of myoendothelial gap junctions, smooth muscle cell (SMC) layers, and medial cross-sectional area were significantly greater in SHR than WKY. Intracellular dye labeling demonstrated hyperplasia of SMCs in SHR. Analysis of nerve-mediated excitatory junction potentials recorded in SMCs at the adventitial and luminal borders demonstrated decreased radial coupling of SMCs in SHR. In both SHR and WKY, in the presence of NG-nitro-l-arginine methyl ester and indomethacin, acetylcholine-elicited EDHF was abolished by charybdotoxin and apamin, while iberiotoxin had no effect, implicating the involvement of small and intermediate, but not large, calcium-activated potassium channels. EDHF was abolished by Gap-mimetic peptides, 18&bgr;-glycyrrhetinic acid, and endothelial removal but not affected by the NO scavengers hydroxocobalamin and carboxy-PTIO. Conclusions—Significant differences in SMC morphology and homocellular and heterocellular coupling exist between the caudal artery of SHR and WKY rats. In the caudal artery of SHR, significantly greater heterocellular coupling compensates for other structural changes in the media to maintain a functional role for EDHF.

Angiotensin-converting enzyme inhibition restores endothelial but not medial connexin expression in hypertensive rats.

Objective and design Remodelling in the media and decreases in connexin (Cx) expression and size of endothelial cells occur in the caudal artery of spontaneously hypertensive rats (SHR). The objective of this study was to determine whether similar changes are found in the aorta and whether effects in both aorta and caudal artery are present in the pre-hypertensive period or can be reversed by antihypertensive treatment. Methods and results In the aorta of SHR, there was no difference in endothelial cell size although Cxs 37 and 40 were decreased, compared with normotensive Wistar-Kyoto rats. Cxs 37 and 43 were also reduced in the media. These differences were not apparent in pre-hypertensive SHR. Inhibition of angiotensin-converting enzyme (ACE) in SHR decreased blood pressure and restored Cx expression in the endothelium of both aorta and caudal artery. The decreased endothelial cell size in the caudal artery or the reduced Cxs in the media of the aorta of SHR were unaffected by ACE inhibition. Conclusion We conclude that cellular coupling is reduced in the endothelium of arteries of SHR, but this can be restored by inhibition of the renin-angiotensin system. Decreased cellular coupling in the media or decreased endothelial size in SHR were not reversed by this antihypertensive treatment.