Myoendothelial Gap Junction Research Articles

Cl− induces endothelium-dependent mesenteric arteriolar vasorelaxation through the NKCC1/TRPV4/NCX axis

AimsAlthough absorbed NaCl increases intestinal blood flow to facilitate absorption and transportation, it is unclear if it can directly mediate mesenteric arterial relaxation. We aimed to investigate and test our hypothesis that Cl− induces mesenteric arterial vasorelaxation via endothelium-dependent hyperpolarization (EDH). Main methodsWe used wire myograph to study NaCl-induced vasorelaxation of mesenteric arteries isolated from mice. Cl−, Ca2+ and K+ imaging was performed in human vascular endothelial cells pre-treated with pharmacological agents. Key findingsThe Cl− concentration-dependently induced vasorelaxation of mesenteric arteries likely through EDH. The Cl−-induced vasorelaxation was attenuated in TRPV4 KO mice and inhibited by selective blockers of Na+-K+-2Cl− cotransporter 1 (NKCC1) (bumetanide, 10 μM), transient receptor potential vanilloid 4 (TRPV4) (RN-1734, 40 μM), and small conductance Ca2+-activated K+ channels (SKCa) (apamin, 3 μM)/ intermediate conductance Ca2+-activated K+ channels (IKCa) (TRAM-34, 10 μM) and myoendothelial gap junction (18α-glycyrrhetinic acid, 10 μM), but enhanced by a selective activator of IKCa/SKCa (SKA-31, 0.3 μM). Cl− decreased intracellular K+ concentrations in endothelial cells, which was reversed by apamin (200 nM) plus TRAM-34 (500 nM). Extracellular Cl− raised intracellular Cl− concentrations in endothelial cells, which was attenuated by bumetanide (10 μM). Finally, Cl− induced a transient Ca2+ signaling via TRPV4 in endothelial cells, which became sustained when the Ca2+ exit mode of Na+-Ca2+ exchanger (NCX) was blocked. SignificanceCl− induces a pure EDH-mediated vasorelaxation of mesenteric arteries through activation of endothelial NKCC1/TRPV4/NCX axis. We have provided a novel insight into the role of Cl−-induced vasorelaxation via EDH mechanism.

Treatment of preeclamptic vessels with pravastatin enhances vessel function and alters caveolae form and distribution through removal of cellular cholesterol

IntroductionPreeclampsia (PE) causes significant maternal and foetal morbidity and mortality. Maternal vascular endothelial dysfunction contributes to PE resulting in systemic organ dysfunction. Symptoms of PE are most effectively alleviated/managed through pre‐term delivery and/or expectant care, highlighting the need for effective therapies to be identified. The potential clinical applications of pravastatin in the treatment of PE has been reported across multiple studies, however the mechanism(s) behind this is have yet to be elucidated [1, 2].AimsThis study aims to investigate the effect of in vitro pravastatin on uterine microvascular endothelial function in PE, specifically its role in modulating caveolae form and distribution and hence vascular function in PEMethodsMyometrial radial arterioles from caesarean‐section normotensive (NT) and PE patients were incubated with pravastatin (2mM/6h) in vitro. Electron microscopy, immunohistochemistry and pressure myography were used to characterize caveolae and vessel structure and function. Parallel studies conducted with methyl‐β‐cyclodextrin (MβCD) (10mM/1h) examined the effects of removing cholesterol on function.ResultsOverall caveolae density/µm is reduced 24% from 5.5 ± 0.1/µm in NT (n=4, P<0.05) to 4.2 ± 0.01/µm (n=4, P<0.05) in PE vessels, a further 31% to 1.9 ± 0.8/µm (n=4, P<0.05) in PE vessels following treatment with pravastatin. Myoendothelial gap junction density/5mm of vessel length is reduced ~215% from 5.7 ± 1.3/5mm in NT (n=3, P<0.05) to 1.8 ± 0.9/5mm (n=3, P<0.05) in PE vessels. Confocal immunohistochemistry showed decreased caveolin‐1 expression in PE arterioles relative to NT with expression redistributed to endothelial cell borders, following treatment with pravastatin expression was reduced a further ~50% in PE vessels (n=6‐8, P<0.05). Functionally, endothelium‐dependent relaxation was inhibited in arteries from PE compared to NT. Inhibition of NOS/COX reduced bradykinin‐induced dilation in arteries from NT but not PE, suggesting NO/PG‐mediated dilation is lost in PE.. Pravastatin incubation restored endothelium‐dependent relaxation in PE samples to NT levels enhancing the NO and IKCa‐mediated components of relaxation. Incubation with MβCD increased sensitivity (LogEC50 from ‐7.37 to ‐8.24, P<0.05) and maximum dilation to bradykinin in arteries from NT (81.7 ± 4.9.% (n=13) to 94.8 ± 5.2 % (n=8), P<0.05), but had no significant effect in arteries from PE (max 65.6 ± 6.4% (n=17) to 86.5 ± 13.5% (n=3), P>0.05).DiscussionThis data suggests treatment of preeclamptic vessels with pravastatin can be associated with improved vessel function as well as modulation of caveolae form and distribution, specifically though the removal of cellular cholesterol.1. Costantine, M. M. & Cleary, M. K. Obstetrics & Gynecology 121, 349‐353, (2013).2. Esteve‐Valverde, E. et al. Obstetrical & Gynecological Survey 73, 40‐55, (2018).

Roles of Endothelial Motilin Receptor and Its Signal Transduction Pathway in Motilin-Induced Left Gastric Artery Relaxation in Dogs.

Background: Motilin increases left gastric artery (LGA) blood flow in dogs via the endothelial motilin receptor (MLNR). This article investigates the signaling pathways of endothelial MLNR.Methods: Motilin-induced relaxation of LGA rings was assessed using wire myography. Nitric oxide (NO), and cyclic guanosine monophosphate (cGMP) levels were measured using an NO assay kit and cGMP ELISA kit, respectively.Results: Motilin concentration-dependently (EC50=9.1±1.2×10−8M) relaxed LGA rings precontracted with U46619 (thromboxane A2 receptor agonist). GM-109 (MLNR antagonist) significantly inhibited motilin-induced LGA relaxation and the production of NO and cGMP. N-ethylmaleimide (NEM; G-protein antagonist), U73122 [phospholipase C (PLC) inhibitor], and 2-aminoethyl diphenylborinate [2-APB; inositol trisphosphate (IP3) blocker] partially or completely blocked vasorelaxation. In contrast, chelerythrine [protein kinase C (PKC) inhibitor] and H89 [protein kinase A (PKA) inhibitor] had no such effect. Low-calcium or calcium-free Krebs solutions also reduced vasorelaxation. N-nitro-L-arginine methyl ester [L-NAME; nitric oxide synthase (NOS) inhibitor] and ODQ [soluble guanylyl cyclase (sGC) inhibitor] completely abolished vasodilation and synthesis of NO and cGMP. Indomethacin (cyclooxygenase inhibitor), 18α-glycyrrhetinic acid [18α-GA; myoendothelial gap junction (MEGJ) inhibitor], and K+ channel inhibition through high K+ concentrations or tetraethylammonium (TEA-Cl; KCa channel blocker) partially decreased vasorelaxation, whereas glibenclamide (KATP channel blocker) had no such effect.Conclusion: The current study suggests that motilin-induced LGA relaxation is dependent on endothelial MLNR through the G protein-PLC-IP3 pathway and Ca2+ influx. The NOS-NO-sGC-cGMP pathway, prostacyclin, MEGJ, and K+ channels (especially KCa) are involved in endothelial-dependent relaxation of vascular smooth muscle (VSM) cells.

Hypotensive effect of Eugenia dysenterica leaf extract is primarily related to its vascular action: The possible underlying mechanisms.

Eugenia dysenterica (ED) leaves are used in Brazil to treat cardiac diseases; however, there are no scientific data describing the effects of this species on cardiac activity. To investigate the effect of ED aqueous leaf extract (EDLE) on hear rate (HR) and mean arterial pressure (MAP) of anaesthetised rats and its underlying mechanism of action. EDLE was analysed, and its proanthocyanidin composition was determined. After performing dose-effect curves for EDLE on HR and MAP, EDLE-induced hypotension was evaluated before and after atropine (AT), L-N(ω)-nitro-L-arginine methyl ester (L-NAME), hexamethonium (HXT), indomethacin (IND), carbenoxolone (CBX), or nifedipine (NFD) administration. The effect of proanthocyanidin-depleted extract (EDLE/P-) was also determined and compared to that of the EDLE with proanthocyanidins. EDLE decreased the MAP in a dose-dependent manner; HR was decreased only with the highest and most toxic dose. Only CBX and NFD decreased EDLE-induced hypotension. Five polymeric series of proanthocyanidins were identified, which were mainly constituted by procyanidin and prodelphinidin units with B-type linkage and up to 12 flavan-3-ol units. EDLE/P- induced hypotension did not differ from that induced by EDLE. The cardiovascular effects of EDLE were primarily related to its vascular action. EDLE-induced hypotensive effect appeared to involve L-type calcium channel blockage as well as myoendothelial gap junction signalling. The higher molecular weight proanthocyanidins from EDLE are unlikely to contribute to its cardiovascular effect.

Angiopoietin-2 regulates vascular hyporeactivity through myoendothelial gap junction

Objective To observe the mediation role of myoendothelial gap junction (MEGJ) in the regulation of angiopoietin-2 (Ang2) on vascular hyporeactivity after hypoxia, and figure out the responsible Cx. Methods The double-sided cells co-culture model of vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) were set up, the VSMCs contractile was detected via the leakage of fluoresceine isothiocyanate-bovine serum albumin (FITC-BSA), MEGJ formation was observed by immunocytochemistry of phalloidin, and MEGJ communication was reflected by the Sulforhodamine B transfer. Results (1) In the double-sided cells co-culture model, protein expression of iNOS after hypoxia increased more in VSMCs than that in VECs (3.6 times, P=0.003), and the increased iNOS expression in VSMCs could be decreased by Ang2 siRNA (by 60%, P=0.003). (2) The MEGJ non-specific inhibitor 18α-GA, the specific Cx43 siRNA, or angiopoietin receptor 2 (Tie2) siRNA could suppress the iNOS expression in VSMCs (P=0.001, 0.001, and 0.001, respectively), and improved the VSMCs contractile reactivity after hypoxia following Ang2 treatment (P=0.004, 0.009, and 0.001, respectively). (3) Cx43 siRNA or Tie2 siRNA could inhibit the increased MEGJ formation (P=0.005 and 0.005, respectively), and inhibit the increased MEGJ communication after hypoxia following Ang2 treatment (P=0.003 and 0.004, respectively). Conclusion MEGJ mediated VECs-dependent regulation of Ang2 on vascular hyporeactivity after hypoxia, Cx43 was the responsible Cx isoform. Key words: Hypoxia; Vascular hyporeactivity; Angiopoietin-2; Myoendothelial gap junction; Connexin 43

Involvement of connexin 43 phosphorylation and gap junctional communication between smooth muscle cells in vasopressin-induced ROCK-dependent vasoconstriction after hemorrhagic shock.

We examined the roles played by gap junctions (GJs) and the GJ channel protein connexin 43 (Cx43) in arginine vasopressin (AVP)-induced vasoconstriction after hemorrhagic shock and their relationship to Rho kinase (ROCK) and protein kinase C (PKC). The results showed that AVP induced an endothelium-independent contraction in rat superior mesenteric arteries (SMAs). Blocking the GJs significantly decreased the contractile response of SMAs and vascular smooth muscle cells (VSMCs) to AVP after shock and hypoxia. The selective Cx43-mimetic peptide inhibited the vascular contractile effect of AVP after shock and hypoxia. AVP restored hypoxia-induced decrease of Cx43 phosphorylation at Ser262 and gap junctional communication in VSMCs. Activation of RhoA with U-46619 increased the contractile effect of AVP. This effect was antagonized by the ROCK inhibitor Y27632 and the Cx43-mimetic peptide. In contrast, neither an agonist nor an inhibitor of PKC had significant effects on AVP-induced contraction after hemorrhagic shock. In addition, silencing of Cx43 with siRNA blocked the AVP-induced increase of ROCK activity in hypoxic VSMCs. In conclusion, AVP-mediated vascular contractile effects are endothelium and myoendothelial gap junction independent. Gap junctions between VSMCs, gap junctional communication, and Cx43 phosphorylation at Ser262 play important roles in the vascular effects of AVP. RhoA/ROCK, but not PKC, is involved in this process.

P2Y₂ receptor activation decreases blood pressure via intermediate conductance potassium channels and connexin 37.

Nucleotides are important paracrine regulators of vascular tone. We previously demonstrated that activation of P2Y₂ receptors causes an acute, NO-independent decrease in blood pressure, indicating this signalling pathway requires an endothelial-derived hyperpolarization (EDH) response. To define the mechanisms by which activation of P2Y₂ receptors initiates EDH and vasodilation, we studied intermediate-conductance (KCa3.1, expressed in endothelial cells) and big-conductance potassium channels (KCa1.1, expressed in smooth muscle cells) as well as components of the myoendothelial gap junction, connexins 37 and 40 (Cx37, Cx40), all hypothesized to be part of the EDH response. We compared the effects of a P2Y₂/₄ receptor agonist in wild-type (WT) mice and in mice lacking KCa3.1, KCa1.1, Cx37 or Cx40 under anaesthesia, while monitoring intra-arterial blood pressure and heart rate. Acute activation of P2Y₂/₄ receptors (0.01-3 mg kg(-1) body weight i.v.) caused a biphasic blood pressure response characterized by a dose-dependent and rapid decrease in blood pressure in WT (maximal response % of baseline at 3 mg kg(-1) : -38 ± 1%) followed by a consecutive increase in blood pressure (+44 ± 11%). The maximal responses in KCa3.1(-/-) and Cx37(-/-) were impaired (-13 ± 5, +17 ± 7 and -27 ± 1, +13 ± 3% respectively), whereas the maximal blood pressure decrease in response to acetylcholine at 3 μg kg(-1) was not significantly different (WT: -53 ± 3%; KCa3.1(-/-) : -52 ± 3; Cx37(-/-) : -53 ± 3%). KCa1.1(-/-) and Cx40(-/-) showed an identical biphasic response to P2Y2/4 receptor activation compared to WT. The data suggest that the P2Y2/4 receptor activation elicits blood pressure responses via distinct mechanisms involving KCa3.1 and Cx37.

Beneficial effects of platelet-derived growth factor on hemorrhagic shock in rats and the underlying mechanisms.

Studies have shown that local application of platelet-derived growth factor (PDGF) can be used for the treatment of acute and chronic wounds. We investigated if systemic application of PDGF has a protective effect on acute hemorrhagic shock in rats in the present study. Using hemorrhagic shock rats and isolated superior mesenteric arteries, the effects of PDGF-BB on hemodynamics, animal survival, and vascular reactivity as well as the roles of the gap junction proteins connexin (Cx)40 and Cx43, PKC, and Rho kinase were observed. PDGF-BB (1–15 μg/kg iv) significantly improved the hemodynamics and blood perfusion to vital organs (liver and kidney) as well as vascular reactivity and improved the animal survival in hemorrhagic shock rats. PDGF recovering shock-induced vascular hyporeactivity depended on the integrity of the endothelium and myoendothelial gap junction. Cx43 antisense oligodeoxynucleotide abolished these improving effects of PDGF, whereas Cx40 oligodeoxynucleotide did not. Further study indicated that PDGF increased the activity of Rho kinase and PKC as well as vascular Ca2+ sensitivity, whereas it did not interfere with the intracellular Ca2+ concentration in hypoxia-treated vascular smooth muscle cells. In conclusion, systemic application of PDGF-BB may exert beneficial effects on hemorrhagic shock, which are closely related to the improvement of vascular reactivity and hemodynamics. The improvement of PDGF-BB in vascular reactivity is vascular endothelium and myoendothelial gap junction dependent. Cx43, Rho kinase, and PKC play very important role in this process. These findings suggest that PDGF may be a potential measure to treat acute clinical critical diseases such as severe trauma, shock, and sepsis.

Bradykinin induces vascular contraction after hemorrhagic shock in rats

BackgroundBradykinin (BK) has many biological effects in inflammation, allergy, and septic shock. Studies have shown that low doses of BK can induce vascular relaxation and high doses can induce vascular contraction in many pathophysiological conditions, but the role and mechanisms that high doses of BK have on vascular contraction in hemorrhagic shock are not clear. MethodsWith hemorrhagic-shock rats and hypoxia-treated superior mesenteric artery (SMA), we investigated the role and mechanisms of high doses of BK-induced vascular contraction in hemorrhagic shock. ResultsHigh doses of BK (500–50,000 ng/kg in vivo or 10−10 to 10−5 mol/L in vitro) dose dependently induced vascular contraction of SMA and increased the vascular calcium sensitivity in normal and hemorrhagic-shock rats. Less than 10−10 mol/L of BK induced vascular dilation BK-induced increase of vascular contractile response and calcium sensitivity was reduced by denudation of the endothelium, 18α-glycyrrhetic acid (an inhibitor of myoendothelial gap junction) and connexin 43 antisense oligodeoxynucleotide. Further studies found that high concentrations of BK-induced vascular contraction in hemorrhagic shock was closely related to the activation of Rho A–Rho kinase pathway and Protein Kinase C (PKC) α and ε. ConclusionsHigh doses of BK can induce vascular contraction in hemorrhagic shock condition, which is endothelium and myoendothelial gap junction dependent. Cx43-mediated activation of Rho A–Rho kinase and Protein Kinase C (PKC) pathway plays a very important role in this process. This finding provided a new angle of view to the biological role of BK in other pathophysiological conditions such as hemorrhagic shock or hypoxia.

Defective endothelial connexin40 raises blood pressure in mice through impairment to conducted dilation (1079.14)

Global deletion or functional impairment of Cx40 in mice attenuates conducted dilation and causes hypertension. Although impairment of conducted dilation might be expected to increase peripheral resistance, the hypertension is renal in origin. Endothelial‐specific deletion of Cx40 also impairs conducted dilation, without change in blood pressure. However, these latter studies are complicated by coordinate loss of Cx37 from the endothelium. In order to impair Cx40 function selectively in the endothelium, without coordinate loss of Cx37 expression, we have generated a mouse expressing a mutant Cx40 (Cx40T152A) under control of the endothelial specific promoter Tie‐2, alongside native Cx40. We show that this single point mutation impairs both chemical and electrical conduction in vitro, although paradoxically, heterotypic pairings with Cx37 rescue gap junction function. Endothelial Cx37 expression is normal but Cx40T152A mice are hypertensive (105±1mmHg vs wildtype 97±1mmHg; P<0.05) without change in heart rate. While the EDH‐vasodilatory pathway remains intact in Cx40T152A arteries, conducted vasodilation is markedly attenuated. We conclude that myoendothelial gap junction communication remains intact in Cx40T152ATg mice, but impairment to conducted vasodilation arising from defective endothelial Cx40 signalling raises peripheral resistance and blood pressure.

Pregnancy‐induced remodelling and enhanced endothelium‐derived hyperpolarization‐type vasodilator activity in rat uterine radial artery: transient receptor potential vanilloid type 4 channels, caveolae and myoendothelial gap junctions

In pregnancy, the vasculature of the uterus undergoes rapid remodelling to increase blood flow and maintain perfusion to the fetus. The present study determines the distribution and density of caveolae, transient receptor potential vanilloid type 4 channels (TRPV4) and myoendothelial gap junctions, and the relative contribution of related endothelium-dependent vasodilator components in uterine radial arteries of control virgin non-pregnant and 20-day late-pregnant rats. The hypothesis examined is that specific components of endothelium-dependent vasodilator mechanisms are altered in pregnancy-related uterine radial artery remodelling. Conventional and serial section electron microscopy were used to determine the morphological characteristics of uterine radial arteries from control and pregnant rats. TRPV4 distribution and expression was examined using conventional confocal immunohistochemistry, and the contribution of endothelial TRPV4, nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-type activity determined using pressure myography with pharmacological intervention. Data show outward hypertrophic remodelling occurs in uterine radial arteries in pregnancy. Further, caveolae density in radial artery endothelium and smooth muscle from pregnant rats was significantly increased by ~94% and ~31%, respectively, compared with control, whereas caveolae density did not differ in endothelium compared with smooth muscle from control. Caveolae density was significantly higher by ~59% on the abluminal compared with the luminal surface of the endothelium in uterine radial artery of pregnant rats but did not differ at those surfaces in control. TRPV4 was present in endothelium and smooth muscle, but not associated with internal elastic lamina hole sites in radial arteries. TRPV4 fluorescence intensity was significantly increased in the endothelium and smooth muscle of radial artery of pregnant compared with control rats by ~2.6- and 5.5-fold, respectively. The TRPV4 signal was significantly higher in the endothelium compared with the smooth muscle in radial artery of both control and pregnant rats, by ~5.7- and 2.7-fold, respectively. Myoendothelial gap junction density was significantly decreased by ~37% in radial artery from pregnant compared with control rats. Pressure myography with pharmacological intervention showed that NO contributes ~80% and ~30%, and the EDH-type component ~20% and ~70% of the total endothelium-dependent vasodilator response in radial arteries of control and pregnant rats, respectively. TRPV4 plays a functional role in radial arteries, with a greater contribution in those from pregnant rats. The correlative association of increased TRPV4 and caveolae density and role of EDH-type activity in uterine radial artery of pregnant rats is suggestive of their causal relationship. The decreased myoendothelial gap junction density and lack of TRPV4 density at such sites is consistent with their having an integral, albeit complex, interactive role in uterine vascular signalling and remodelling in pregnancy.

Does TRPC3 macrodominate the myoendothelial gap junction microdomain?

This editorial refers to ‘Transient receptor potential canonical type 3 channels facilitate endothelium-derived hyperpolarization-mediated resistance artery vasodilator activity’ by S. Senadheera et al ., pp. 439–447, this issue. In 1998, Griffith and colleagues1 proposed that myoendothelial gap junctions (MEGJs) play a central role in endothelium-dependent hyperpolarization (EDH), the key mechanism coupling the activation of the endothelium by shear stress and blood borne-stimuli to vasodilation in the resistance vasculature. They envisioned that MEGJs, which are found at the ends of endothelial cell (EC) projections that protrude through holes in the internal elastic lamina of arteries and arterioles and link the EC to adjacent vascular smooth muscle cells (VSMCs), enabled EDH by acting as low-resistance pathways allowing the flow of hyperpolarizing current from the endothelium to the adjacent smooth muscle. Subsequently, however, it has become apparent that far from merely providing a locus for MEGJ, myoendothelial projections (MEPs) are unique microdomains that are capable of generating localized Ca2+ signals.2 This may enable them to influence the flow of information between ECs and VSMCs and also probably allows them to play a pivotal active role in triggering EDH ( Figure 1 ). Figure 1 Signalling components and pathways within myoendothelial projections which may contribute to endothelium-dependent hyperpolarization and myoendothelial feedback. The illustrated scheme is speculative, but is consistent with proposals and results described in a number of the references listed. See text for abbreviations and a more detailed explanation. In the current …

Diet-Induced Obesity Impairs Endothelium-Derived Hyperpolarization via Altered Potassium Channel Signaling Mechanisms

BackgroundThe vascular endothelium plays a critical role in the control of blood flow. Altered endothelium-mediated vasodilator and vasoconstrictor mechanisms underlie key aspects of cardiovascular disease, including those in obesity. Whilst the mechanism of nitric oxide (NO)-mediated vasodilation has been extensively studied in obesity, little is known about the impact of obesity on vasodilation to the endothelium-derived hyperpolarization (EDH) mechanism; which predominates in smaller resistance vessels and is characterized in this study.Methodology/Principal FindingsMembrane potential, vessel diameter and luminal pressure were recorded in 4th order mesenteric arteries with pressure-induced myogenic tone, in control and diet-induced obese rats. Obesity, reflecting that of human dietary etiology, was induced with a cafeteria-style diet (∼30 kJ, fat) over 16–20 weeks. Age and sexed matched controls received standard chow (∼12 kJ, fat). Channel protein distribution, expression and vessel morphology were determined using immunohistochemistry, Western blotting and ultrastructural techniques. In control and obese rat vessels, acetylcholine-mediated EDH was abolished by small and intermediate conductance calcium-activated potassium channel (SKCa/IKCa) inhibition; with such activity being impaired in obesity. SKCa-IKCa activation with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) and 1-ethyl-2-benzimidazolinone (1-EBIO), respectively, hyperpolarized and relaxed vessels from control and obese rats. IKCa-mediated EDH contribution was increased in obesity, and associated with altered IKCa distribution and elevated expression. In contrast, the SKCa-dependent-EDH component was reduced in obesity. Inward-rectifying potassium channel (Kir) and Na+/K+-ATPase inhibition by barium/ouabain, respectively, attenuated and abolished EDH in arteries from control and obese rats, respectively; reflecting differential Kir expression and distribution. Although changes in medial properties occurred, obesity had no effect on myoendothelial gap junction density.Conclusion/SignificanceIn obese rats, vasodilation to EDH is impaired due to changes in the underlying potassium channel signaling mechanisms. Whilst myoendothelial gap junction density is unchanged in arteries of obese compared to control, increased IKCa and Na+/K+-ATPase, and decreased Kir underlie changes in the EDH mechanism.

Endothelium-Dependent Vasodilation in Human Mesenteric Artery Is Primarily Mediated by Myoendothelial Gap Junctions Intermediate Conductance Calcium-Activated K+ Channel and Nitric Oxide

Myoendothelial microdomain signaling via localized calcium-activated potassium channel (K(Ca)) and gap junction connexins (Cx) is critical for endothelium-dependent vasodilation in rat mesenteric artery. The present study determines the relative contribution of NO and gap junction-K(Ca) mediated microdomain signaling to endothelium-dependent vasodilation in human mesenteric artery. The hypothesis tested was that such activity is due to NO and localized K(Ca) and Cx activity. In mesenteric arteries from intestinal surgery patients, endothelium-dependent vasodilation was characterized using pressure myography with pharmacological intervention. Vessel morphology was examined using immunohistochemical and ultrastructural techniques. In vessel segments at 80 mm Hg, the intermediate (I)K(Ca) blocker 1-[(2-chlorophenyl)diphenyl-methyl]-1H-pyrazole (TRAM-34; 1 μM) inhibited bradykinin (0.1 nM-3 μM)-induced vasodilation, whereas the small (S) K(Ca) blocker apamin (50 and 100 nM) had no effect. Direct IK(Ca) activation with 1-ethyl-2-benzimidazolinone (1-EBIO; 10-300 μM) induced vasodilation, whereas cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (1-30 μM), the SK(Ca) activator, failed to dilate arteries, whereas dilation induced by 1-EBIO (10-100 μM) was blocked by TRAM-34. Bradykinin-mediated vasodilation was attenuated by putative gap junction block with carbenoxolone (100 μM), with remaining dilation blocked by N-nitro l-arginine methyl ester (100 μM) and [1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (10 μM), NO synthase and soluble guanylate cyclase blockers, respectively. In human mesenteric artery, myoendothelial gap junction and IK(Ca) activity are consistent with Cx37 and IK(Ca) microdomain expression and distribution. Data suggest that endothelium-dependent vasodilation is primarily mediated by NO, IK(Ca), and gap junction Cx37 in this vessel. Myoendothelial microdomain signaling sites are present in human mesenteric artery and are likely to contribute to endothelium-dependent vasodilation via a mechanism that is conserved between species.

Acute shear stress does not increase phosphorylation of eNOS at S1179 in mouse mesenteric arteries

Shear stress provokes vasodilation through activation of eNOS, which produces the vasodilator NO·, and is necessary for healthy endothelial function. In cultured endothelial cells, this activation involves increased phosphorylation of eNOS at S1179, and we hypothesized a similar response in intact arteries during shear stress‐induced dilation. Small mesenteric arteries from male mice (7–13 wks old) were cannulated, pressurized (75 mmHg), and one of four treatments was applied: no treatment, phenylephrine (PE, 10−5 M), PE followed by acetylcholine (ACh, 10−4 M), or PE followed by high luminal flow (60 μl/min, 15 min); then flash frozen for immunoblotting (4 arteries/group/blot, 2 separate blots). Arteries dilated 65% and 32% in response to ACh and flow, respectively (determined by video microscopy). 45% of the dilation to flow at 60 μl/min is due to NO·, as indicated in separate experiments +/− L‐NAME (n=4/group). eNOS phosphorylation at S1179/total eNOS was 3.85‐fold greater in the ACh group (vs. no treatment), but the high flow group was no greater than the PE group (2.4‐fold and 2.47‐fold, respectively, vs. no treatment), despite robust dilation with flow. These data indicate that PE alone induces phosphorylation of eNOS at S1179 (perhaps through myoendothelial gap junction communication), and increased phosphorylation at this site is not required for shear stress‐induced dilation. APS‐UGSRF and NHLBI‐R15

Diabetes-associated Alterations in Volatile Anesthetic Actions on Contractile Response to Norepinephrine in Isolated Mesenteric Resistance Arteries

Clinical concentrations of volatile anesthetics significantly influence contractile response to the sympathetic neurotransmitter norepinephrine although its precise mechanisms remain unclarified. In this study, we investigated its possible alterations in diabetes, as well as its underlying mechanisms. Isometric force was recorded in small mesenteric arteries from streptozotocin-induced diabetic and age-matched control rats. The concentration-response curve for acetylcholine-induced endothelium-dependent relaxation was shifted to the right in diabetic arteries compared with controls. The concentration-response curve for norepinephrine-induced contraction was shifted to the left and upward by both endothelial denudation and diabetic induction. In the presence of endothelium, isoflurane or sevoflurane enhanced norepinephrine-induced contraction in control arteries but not in diabetic arteries; however, in its absence, both anesthetics identically inhibited norepinephrine-induced contraction in both groups. In control arteries, the isoflurane- or sevoflurane-induced enhancement was not affected by adrenomedullin22-52, calcitonin gene-related peptide8-37, 18beta-glycyrrhetinic acid, N-nitro l-arginine, ouabain, Ba, indomethacin, losartan, ketanserin, BQ-123, and BQ-788. In diabetes, vascular responses to acetylcholine, norepinephrine, and volatile anesthetics are altered in mesenteric resistance arteries, presumably reflecting endothelial dysfunction and possibly underlying circulatory instability during administration of either anesthetic. Some endothelial mechanisms that are impaired in diabetes would be involved in the anesthetic-induced enhancement of norepinephrine-induced contraction. However, the vasoregulatory mechanism mediated by adrenomedullin, calcitonin gene-related peptide, myoendothelial gap junction, nitric oxide, endothelium-derived hyperpolarizing factor, cyclooxygenase products, angiotensin II, serotonin, or endothelin-1, all of which have been suggested to be impaired in diabetes, would not be involved in the enhancement.

Arterial internal elastic lamina holes: relationship to function?

Internal elastic lamina (IEL) hole (fenestration) characteristics and myoendothelial gap junction (MEGJ) density were examined in selected resistance and conduit arteries of normal and diseased rat and mouse models, using conventional, ultrastructural and confocal microscopy methods. Selected vessels were those commonly used in functional studies: thoracic aorta, proximal and distal mesenteric, caudal, saphenous, middle-cerebral and caudal cerebellar artery. Rat and mouse strains and treatment groups examined were Dahl, Sprague Dawley, Wistar Kyoto, Wistar, spontaneously hypertensive (SHR), deoxycorticosterone (DOC) treated rat; and apolipoprotein E knockout, C57/BL6 and BALB/c mice. Vessel size (as IEL circumference), IEL hole and MEGJ density were quantified. In mesenteric arteries, the width of IEL holes and the percent of IEL occupied by holes were also determined. IEL hole density varied significantly within and between mesenteric artery beds, even among normotensive rat strains. Among the hypertensive rats (SHR and DOC), hole density in some vessels was higher in the normotensives than in the hypertensives within each strain, whereas in Dahl rats, hole density was similar between hypertensives and normotensives. Hole density was not correlated with the formation of intimal lesions in superior mesenteric artery. There was no positive general correlation between IEL hole and MEGJ density in resistance and conduit vessels. However, there was a positive correlation between the size of some resistance arteries and MEGJ density, although such a relationship did not hold for conduit vessels or during development, and there was no such relationship between vessel size and IEL hole density. Whilst IEL holes are obviously required for MEGJ communication, their presence is not an indication of contact-mediated communication, but rather may be related to the presence of sites for the low resistance passage of diffusion-mediated release of vasoactive endothelial and smooth muscle substances.

REGULATORY EFFECTS OF MYOENDOTHELIAL GAP JUNCTION ON VASCULAR REACTIVITY AFTER HEMORRHAGIC SHOCK IN RATS

Myoendothelial gap junction (MEGJ), one kind of gap junction between vascular endothelial cell and vascular smooth muscle cell, can transmit electrical and chemical signals to keep the electric and machinery activity synchronism of vasculature. After severe trauma or shock, vascular reactivity to vasoconstrictors or vasodilators is greatly reduced. However, whether MEGJ participates in the regulation of vascular reactivity after hemorrhagic shock, what type of MEGJ is involved, and what is the possible mechanism are unknown. With the hemorrhagic shock Sprague-Dawley rats and their superior mesenteric arteries (SMAs), the effects of 18alpha-glycyrrhetic acid, a lipophilic aglycone that disrupts gap junction plaques, on vascular contractile response to norepinephrine (endothelium-independent vascular constrictor), myricetin (endothelium-dependent vasoconstrictor) and relaxation reactivity to sodium nitroprusside (endothelium-independent vasodilator), and acetylcholine (Ach; endothelium-dependent vasodilator) were observed. Meanwhile, the relationship of the mRNA/protein expression of connexins 37, 40, and 43(Cx40 and Cx43) to the changes of vascular reactivity after hemorrhagic shock and the effect of antisense oligodeoxynucleotide of Cx40 or Cx43 on vascular calcium sensitivity and vascular reactivity were investigated. The results indicated that 18alpha-glycyrrhetic acid antagonized myricetin and Ach-induced SMA reactivity, but had no effect on norepinephrine- and sodium nitroprusside-induced vascular response. The mRNA and protein expression of Cx37 and Cx40 of SMA were negatively associated with the vascular reactivity, whereas Cx43 seemed to be a positive relationship to vascular reactivity. Antisense oligodeoxynucleotide of Cx40 significantly increased the calcium sensitivity, myricetin-induced vasoconstriction, and Ach-induced vasodilation, whereas antisense oligodeoxynucleotide of Cx43 depressed them. It was suggested that MEGJ plays an important role in the regulation of endothelium-dependent vascular reactivity after hemorrhagic shock. The involved types were mainly Cx40 and Cx43. The possible mechanism that Cx40/Cx43 regulates the endothelium-dependent vasoconstrictor reactivity may be related to their regulating effects on the calcium sensitivity of vascular smooth muscle cell.

Blockade of gap junction coupling by glycyrrhetinic acids in guinea pig cochlear artery: a whole-cell voltage- and current-clamp study.

Glycyrrhetinic acids (GAs) are widely used as gap junction blockers, but their efficacy and side effects have not been well determined. Whole-cell electrical recordings were made from vascular smooth muscle cells (VSMCs) embedded in or dissociated from, guinea pig cochlear artery segments. 18beta- & 18alpha-GA concentration-dependently increased membrane input resistance (R(in)) of in situ VSMCs, with a maximal input conductance (G(in)=1/R(in)) reduction of 92% & 77% and IC(50) of 2.0 & 4.4 microm, respectively. 18betaGA (30 microM) resulted in a R(in) of 2.2 GOmega and C(in) of 12 pF, comparable to those of freshly dissociated VSMCs (3.1 GOmega & 6.1 pF). The GAs (> or =30 microM) caused a depolarization in VSMCs in situ. In dispersed VSMCs, they both inhibited delayed rectifiers; 18betaGA also activated a non-selective cation conductance while 18alphaGA inactivated a voltage-independent K+-conductance. ACh induced an outward current in VSMCs in situ at -40 mV, with a positive slope I/V relation and a reversal potential near E(K). The ACh-induced current was attenuated by 18beta- & 18alphaGA with an IC(50) of 4.3 & 7.8 microM, respectively. 18betaGA blocked the vascular gap junctions, achieving a complete electrical isolation of the recorded VSMC at > or =30 microM while causing a mild depolarization by a complex conductance alteration. 18betaGA suppressed the ACh-induced current in VSMC by blocking the myoendothelial gap junction and by a non-junctional action. 18alphaGA at 30-100 microM failed to fully block the gap junctions while exerting side actions.

Myoendothelial gap junction frequency does not account for sex differences in EDHF responses in rat MCA

Previous findings from our laboratory have shown that dilations to endothelium-derived hyperpolarizing factor (EDHF) in rat middle cerebral artery (MCA) are less in females compared to males. Myoendothelial gap junctions (MEGJs) appear to mediate the transfer of hyperpolarization between endothelium and smooth muscle in males. In the present study, we hypothesized that MEGJs are the site along the EDHF pathway which is compromised in female rat MCA. Membrane potential in endothelium was measured using the voltage-sensitive dye di-8-ANEPPS and in smooth muscle using intracellular glass microelectrodes in the presence of l-NAME (3 × 10 − 5 M) and indomethacin (10 − 5 M). Electron microscopy was used to assess MEGJ characteristics. In endothelial cells, the di-8-ANEPPS fluorescence ratio change to 10 − 5 M UTP was similar in males (− 2.9 ± 0.5%) and females (− 3.2 ± 0.2%), indicating comparable degrees of endothelial cell hyperpolarization. However, smooth muscle cell hyperpolarization to 10 − 5 M UTP was significantly attenuated in females (0 mV hyperpolarization; − 31 ± 1.5 mV resting) compared to males (8 mV hyperpolarization; − 28 ± 1.7 mV resting). Ultrastructural evidence suggested that MEGJ frequency and area of contact were comparable between males and females. Taken together, our data suggest that in rat MCA, MEGJ frequency does not account for the reduced EDHF responses observed in females compared to males. We conclude that reduced myoendothelial coupling and/or homocellular coupling within the media may account for these differences.