Vascular gap junctions and implications for hypertension

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. Endothelial cells are well coupled by large gap junctions expressing Cx37, Cx40 and, to a lesser extent, Cx43, whose expression may be more subject to regulation by physical factors. Smooth muscle cells are more heterogeneously coupled by gap junctions that are small and rare. The identity of the Cx expressed in the media may vary among different arteries. Myoendothelial gap junctions are small and more common in resistance arteries with fewer layers of smooth muscle cells. Given the small size of these gap junctions and the rapid turnover rate of Cxs, homocellular coupling in the media and heterocellular coupling between the cell layers may be subject to more dynamic control than coupling in the endothelium. Vascular gap junctions have been implicated in a number of vasomotor responses that may regulate vascular tone and blood pressure. These include the mechanism of action of the vasodilator, endothelium-derived hyperpolarizing factor (EDHF), the myogenic constriction to intramural pressure increase, the spontaneous or agonist-induced vasomotion of arteries and arterioles and the spreading vasodilation and constriction observed in microcirculatory networks. Few data are available on Cx expression in the media of resistance arteries during hypertension. Changes in the expression of Cx43 described in the media of the aorta of hypertensive rats vary with the hypertensive model studied and are likely to represent adaptations to structural changes in the vascular wall. In contrast, in the endothelium of the caudal and mesenteric arteries of spontaneously hypertensive rats, expression of Cxs is significantly decreased compared with arteries from normotensive rats and this decrease is reversed by inhibitors of the renin-angiotensin system. During hypertension, the activity of EDHF is decreased in the mesenteric artery, but this occurs much later than the initial increase in blood pressure and the decrease in endothelial Cxs, suggesting that changes in EDHF may not be causally related to hypertension or to the changes in endothelial Cxs. Upregulation of the myogenic response and the incidence of vasomotion has been reported in hypertension. Little is currently known of the effects of hypertension on spreading vasomotor responses. Deletion of specific Cxs in genetically modified mice is complicated by neonatal lethality or coordinate regulation and compensatory changes in the remaining Cxs. Nevertheless, mice in which Cx40 has been deleted are hypertensive and spreading vasodilatory responses are significantly impaired. Determination of a role for specific Cxs in the control of blood pressure must await the development of animals in which Cx expression can be modulated in a more complex temporal and tissue-specific manner.     

Connexin-mimetic peptides dissociate electrotonic EDHF-type signalling via myoendothelial and smooth muscle gap junctions in the rabbit iliac artery

Synthetic peptides corresponding to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40 and Cx43), designated as (43)Gap 26, (40)Gap 27, (37,40)Gap 26 and (37,43)Gap 27 according to Cx homology, were used to investigate the role of gap junctions in the spread of endothelial hyperpolarizations evoked by cyclopiazonic acid (CPA) through the wall of the rabbit iliac artery. Immunostaining and confocal microscopy demonstrated that gap junction plaques constructed from Cx37 and Cx40 were abundant in the endothelium, whereas Cx43 was the dominant Cx visualized in the media. None of the Cx-mimetic peptides affected endothelial hyperpolarizations evoked by CPA directly. When administered individually, (40)Gap 27, (37,40)Gap 26 and (37,43)Gap 27, but not (43)Gap 26, attenuated endothelium-dependent subintimal smooth muscle hyperpolarization. By contrast, only (43)Gap 26 and (37,43)Gap 27 reduced the spread of subintimal hyperpolarization through the media of the rabbit iliac artery. The site of action of the peptides therefore correlated closely with the expression of their target Cxs in detectable gap junction plaques. The findings provide further evidence that the EDHF phenomenon is electrotonic in nature, and highlight the contribution of myoendothelial and homocellular smooth muscle communication via gap junctions to arterial function.     

缝隙连接及其连接蛋白对血管加压素诱导的失血性休克大鼠血管收缩的作用研究

摘要：目的 观察缝隙连接（GJ）及其组成亚单位连接蛋白（Cx）在血管加压素（AVP）诱导失血性休克大鼠血管收缩中的作用。方法 采用失血性休克大鼠模型和缺氧培养血管平滑肌细胞（VSMC）,观察GJ阻断剂CBX和octanol、以及各Cx亚型反义寡核苷酸（AODN）对AVP诱导的血管收缩反应的影响,随后进一步观察参与AVP作用的Cx37和Cx43对AVP调节休克血管钙敏感性和缺氧VSMC内钙离子浓度的影响。结果 GJ阻断剂CBX和octanol明显抑制了AVP诱导的休克血管的收缩反应。在所有血管中表达的连接蛋白中,Cx37AODN和Cx43AODN明显抑制了AVP的血管收缩作用。进一步结果显示,Cx43AODN、而不是Cx37AODN,可拮抗AVP升高休克血管钙敏感性的作用。此外, AVP处理和干扰Cx37及Cx43对缺氧VSMC内钙离子浓度无明显影响。结论 缝隙连接在休克后AVP介导的血管收缩调节中有重要作用,Cx37和Cx43参与了这一过程,其中Cx43可能通过影响AVP介导的血管钙敏感性调节途径来发挥作用,而Cx37可能通过其它机制来参与AVP的血管调节作用。     

Activation of endothelial IKCa channels underlies NO-dependent myoendothelial feedback

Agonist-induced vasoconstriction triggers a negative feedback response whereby movement of charged ions through gap junctions and/or release of endothelium-derived (NO) limit further reductions in diameter, a mechanism termed myoendothelial feedback. Recent studies indicate that electrical myoendothelial feedback can be accounted for by flux of inositol trisphosphate (IP₃) through myoendothelial gap junctions resulting in localized increases in endothelial Ca^2 + to activate intermediate conductance calcium-activated potassium (IK_Ca) channels, the resultant hyperpolarization then conducting back to the smooth muscle to attenuate agonist-induced depolarization and tone. In the present study we tested the hypothesis that activation of IK_Ca channels underlies NO-mediated myoendothelial feedback. Functional experiments showed that block of IP₃ receptors, IK_Ca channels, gap junctions and transient receptor potential canonical type-3 (TRPC3) channels caused endothelium-dependent potentiation of agonist-induced increase in tone which was not additive with that caused by inhibition of NO synthase supporting a role for these proteins in NO-mediated myoendothelial feedback. Localized densities of IK_Ca and TRPC3 channels occurred at the internal elastic lamina/endothelial-smooth muscle interface in rat basilar arteries, potential communication sites between the two cell layers. Smooth muscle depolarization to contractile agonists was accompanied by IK_Ca channel-mediated endothelial hyperpolarization providing the first demonstration of IK_Ca channel-mediated hyperpolarization of the endothelium in response to contractile agonists. Inhibition of IK_Ca channels, gap junctions, TRPC3 channels or NO synthase potentiated smooth muscle depolarization to agonists in a non-additive manner. Together these data indicate that rather being distinct pathways for the modulation of smooth muscle tone, NO and endothelial IK_Ca channels are involved in an integrated mechanism for the regulation of agonist-induced vasoconstriction.     

粉防己碱和依那普利逆转高血压大鼠血管重构粉防己碱和依那普利逆转高血压大鼠血管重构

目的观察粉防己碱(Tet)和依那普利(Ena)对高血压大鼠血管重构的逆转作用。方法用两肾一夹(2K1C)法制备肾血管性高血压(RH)大鼠病理模型,组织切片图像分析法评价大动脉、小动脉和阻力血管结构改变及药物的影响。结果RH大鼠主动脉、尾动脉和股动脉湿重增加;腹主动脉、尾动脉、心脏冠脉小血管、肾小动脉和肠系膜3级小动脉横截面积、中膜厚度和壁/腔比值均明显增大,肾小动脉管壁平滑肌细胞(VSMC)数明显增加。2K1C术后9周给予Tet或Ena连续9周,上述血管变化明显减轻。结论RH大鼠各级动脉均呈肥厚性重构,肾小动脉管壁伴有VSMC增生;Tet和Ena可有效逆转RH大鼠血管重构。     

Role of membrane potential in endothelium-dependent relaxation of isolated mouse main pulmonary artery

The physiology of smooth muscle and endothelial cells of a particular vascular bed and from different species differs from each other. Acetylcholine causes an endothelium-dependent relaxation of preconstricted pulmonary arteries from the rat. This relaxation is mediated by nitric oxide (NO) plus a yet-unidentified endothelium-derived hyperpolarizing factor, which relaxes the smooth muscles by hyperpolarizing them. Our aim is to test whether these observations could be generalized to the smooth muscle cells from the mouse pulmonary artery. Smooth muscle or endothelial cell membrane potential of strips of murine pulmonary artery were measured simultaneously with the force developed by the strip. Acetylcholine hyperpolarized the endothelial cells. However, acetylcholine did not induce an endothelium-dependent hyperpolarization of the smooth muscle, while it relaxed the strip in an endothelium-dependent manner. This relaxation was abolished by an inhibitor of NO synthesis, nitro-L-arginine. Moreover, nitroglycerin relaxed the strips without changing the membrane potential of the smooth muscle cells. Injection of Lucifer yellow into the endothelial cells and the smooth muscle cells did not show heterocellular dye coupling. Furthermore, electron microscopy did not show gap junction plate at the myoendothelial junctions. We conclude that in the mouse main pulmonary artery, NO alone is responsible for the acetylcholine-induced endothelium-dependent vasodilatation, whereas the phenomenon called endothelium-derived hyperpolizing factor is not present. Therefore, caution should be taken when comparing different animal models to study pulmonary circulation and its reactivity.     

Effects of connexin-mimetic peptides on gap junction functionality and connexin expression in cultured vascular cells

1. We have investigated the effects of connexin-mimetic peptides homologous to the Gap 26 and Gap 27 domains of Cxs 37, 40 and 43 against gap junctional communication and connexin expression in rat aortic endothelial cells (RAECs) and A7r5 myocytes. 2. Immunostaining and Western blot analysis confirmed the presence of gap junction plaques containing Cx43, but not Cx40, in RAECs, whereas plaques containing Cxs 40 and 43 were evident in A7r5 cells. Expression of Cx37 was limited in RAECs and absent from A7r5 cells. 3. Under control conditions calcein-loaded RAECs transferred dye to approximately 70% of subjacent A7r5 cells after coculture for 4-5 h. Dye transfer was inhibited by a peptide targeted to Cxs 37 and 43 ((37,43)Gap 27), but minimally affected by peptides targeted to Cxs 37 and 40 ((37,40)Gap 26 and (40)Gap 27). These findings suggest that the myoendothelial gap junctions that couple RAECs and A7r5 cells are constructed principally from Cx43. 4. Inhibition of dye transfer from RAECs to A7r5 cells cocultured in the presence of (37,43)Gap 27 plus (37,40)Gap 26 for 5 h was fully reversible. 5. In A7r5 cells, endogenous expression of Cx40 and Cx43 was unaffected by incubation with (37,43)Gap 27, (37,40)Gap 26, either individually or in combination, and the peptide combination did not impair connexin trafficking or the de novo formation of gap plaques in A7r5 cells transfected to express Cx43-GFP. 6. Treatment of A7r5 cells with (37,43)Gap 27 plus (37,40)Gap 26 abolished synchronized oscillations in intracellular [Ca2+] induced by the alpha1-adrenoceptor agonist phenylephrine. 7. The reversibility and lack of effect of the peptides on plaque formation suggests that they may be considered ideal probes for functional studies of connexin-mediated communication in the vascular wall.     

Networks of pacemaker cells for gastrointestinal motility

In the wall of the digestive tract, there are pacemaker and conduction systems which can be compared with those in the heart. The introduction of c-Kit as a specific marker of the cells, ICCs, have dramatically clarified morphological and functional understanding of the cells. Mutant animals that lack c-Kit lose or decrease intestinal motility. Four classes of ICCs have been identified and these are distributed along the digestive tract in an organ- and tissue-specific manner: 1) IC-MY locate along the myenteric plexus; 2) IC-DMP, along the deep muscular plexus of small intestine; 3) IC-SMP, along the interface between the submucosa and circular muscle layer of large intestine; and 4) IC-IM, within the muscular layer of the stomach and large intestine. Basically, IC-MY and IC-SMP have pacemaker functions, whereas IC-DMP and IC-IM link signals between the enteric nervous system and smooth muscle cells (SMC). All classes of the cells are connected by gap junctions. Immunocytochemical observations using specific antibodies against various gap junction proteins, connexins (Cx), revealed that Cx43 was localized in the gap junctions between SMC and ICCs, whereas Cx45 was specifically expressed in IC-DMP as it is in the cardiac conduction systems. Mutant animals that we produced enabled us to show cells expressing Cx45 mRNA by replacing the Cx45 locus with a LacZ reporter gene and revealed that most of SMC express Cx45, where so far gap junctions were not demonstrated by electron microscopy or immunocytochemistry, probably due to their small size.     

Junctional and nonjunctional effects of heptanol and glycyrrhetinic acid derivates in rat mesenteric small arteries

1 Heptanol, 18alpha-glycyrrhetinic acid (18alphaGA) and 18beta-glycyrrhetinic acid (18betaGA) are known blockers of gap junctions, and are often used in vascular studies. However, actions unrelated to gap junction block have been repeatedly suggested in the literature for these compounds. We report here the findings from a comprehensive study of these compounds in the arterial wall. 2 Rat isolated mesenteric small arteries were studied with respect to isometric tension (myography), [Ca2+]i (Ca(2+)-sensitive dyes), membrane potential and--as a measure of intercellular coupling--input resistance (sharp intracellular glass electrodes). Also, membrane currents (patch-clamp) were measured in isolated smooth muscle cells (SMCs). Confocal imaging was used for visualisation of [Ca2+]i events in single SMCs in the arterial wall. 3 Heptanol (150 microm) activated potassium currents, hyperpolarised the membrane, inhibited the Ca2+ current, and reduced [Ca2+]i and tension, but had little effect on input resistance. Only at concentrations above 200 microm did heptanol elevate input resistance, desynchronise SMCs and abolish vasomotion. 4 18betaGA (30 microm) not only increased input resistance and desynchronised SMCs but also had nonjunctional effects on membrane currents. 18alphaGA (100 microm) had no significant effects on tension, [Ca2+]i, total membrane current and synchronisation in vascular smooth muscle. 5 We conclude that in mesenteric small arteries, heptanol and 18betaGA have important nonjunctional effects at concentrations where they have little or no effect on intercellular communication. Thus, the effects of heptanol and 18betaGA on vascular function cannot be interpreted as being caused only by effects on gap junctions. 18alphaGA apparently does not block communication between SMCs in these arteries, although an effect on myoendothelial gap junctions cannot be excluded.     

PKCɛ mediates serine phosphorylation of connexin43 induced by lysophosphatidylcholine in neonatal rat cardiomyocytes

Lysophosphatidylcholine (LPC) is a potent pro-arrhythmic derivative of the membrane phosphotidylcholine, which is accumulated in heart tissues during cardiac ischemia. However, the cellular mechanism underlying LPC-induced cardiomyocyte damage remains to be elucidated. This study focuses on the effects of LPC on cardiomyocyte gap junction. At 30 μM, LPC decreased the spontaneous contraction rates of cardiomyocytes, and caused arrhythmic contraction without affecting cell viability. Connexin43 (Cx43) was seen as large plaques at cell junctions in control cells, whereas upon LPC treatment, the intensity of Cx43 staining was decreased in a concentration-sensitive manner and Cx43 staining appeared as tiny dots at cell junctions with a corresponding increase in cytoplasmic punctate staining. This distributional change of Cx43 was accompanied by an impairment of the gap junction intercellular communication (GJIC). Further, LPC treatment induced protein kinase C (PKC) activation, and PKC-dependent Cx43 phosphorylation at serine (Ser) 368. Pre-treatment with a specific PKC? inhibitor, eV1-2, prevented the LPC-induced Cx43 phosphorylation at Ser368 and the loss of Cx43 from gap junctions, both of which may disturb GJIC functions. Furthermore, siRNA knockdown of PKC? in H9c2 cells prevented LPC-induced serine phosphorylation of Cx43, confirming the role of PKC? in Cx43 serine phosphorylation. Double labeling immunofluorescence showed that LPC increased the colocalization of Cx43 with ubiquitin, and pretreatment with MG132 effectively prevented LPC-induced gap junction disassembly. LPC increased the ubiquitination of Cx43, which was blocked by eV1-2 pretreatment, suggesting that LPC accelerated the intracellular degradation of Cx43 via the ubiquitin-proteasomal pathway. It can be concluded that LPC destroyed the structure and function of gap junctions via PKC?-mediated serine phosphorylation of Cx43. PKC? inhibitors might therefore be effective in prevention of LPC-related diseases.     

Gap junctions synchronize vascular tone within the microcirculation

Gap junctions are formed in the cardiovascular system by connexin40 (Cx40), Cx37, Cx43, and Cx45. These low resistance channels allow the transfer of ions and small molecules between cells. The longitudinal coupling of endothelial and smooth muscle cells via gap junctions allows the spread of changes in membrane potential along the vascular wall and hence provides conduction pathways within the vessel itself. Functionally, this tight coupling is reflected by the spread of locally initiated vasomotor responses along the arteriole which are termed conducted responses. Conducted dilations are initiated by the application of endothelium-dependent stimuli which result in local hyperpolarization. This signal spreads along the wall, most likely along the endothelial cell layer, to elicit a coordinated dilation of the arteriole over a considerable distance. Likewise, the opposite signal (depolarization) spreads along the vessel giving rise to a conducted constriction. The latter response is however most likely transmitted along the smooth muscle cell layer. Thus, conducted responses reflect the synchronized behavior of the cells of the vascular wall. It is assumed that conducted responses are critical for the matching of oxygen delivery and tissue needs because they contribute to an ascending dilation which lowers resistance along the length of the arterioles and upstream vessels in a well-tuned fashion. Herein, Cx40 is of special importance because it is critically required for intact signal transduction along the endothelial cell layer. In addition, Cx40 mediates pressure feedback inhibition on renin synthesis in the kidney. Both, vascular and renal function of Cx40, may be involved in the hypertension that is observed in Cx40-deficient animals. In this review, we will summarize physiologic function of connexins in arterioles and briefly address their role in the kidney with respect to renin secretion.     

C-type natriuretic peptide: a new endothelium-derived hyperpolarizing factor?

Vascular relaxation mediated by endothelium-derived hyperpolarizing factor (EDHF) is important for resistance artery function and is underpinned by hyperpolarization of the smooth muscle cells of the blood vessel wall. Debate surrounds the identity of EDHF and its mechanism of action, with the consensus being that there is no universal EDHF. Regional differences in vascular function reflect the complex mechanisms of EDHF. Two primary mechanistic pathways are implicated: (i) myoendothelial gap junctions mediating the spread of endothelial cell hyperpolarization or small signaling molecules (or both) to the smooth muscle; and (ii) diffusible mediators released from the endothelium, including K+ and epoxyeicosatrienoic acids. Here, we discuss the evidence for and against C-type natriuretic peptide (CNP), the latest candidate for a diffusible mediator.     

Vasorelaxant properties of norbormide, a selective vasoconstrictor agent for the rat microvasculature.

1. The effects of norbormide on the contractility of endothelium-deprived rat, guinea-pig, mouse, and human artery rings, and of freshly isolated smooth muscle cells of rat caudal artery were investigated. In addition, the effect of norbormide on intracellular calcium levels of A7r5 cells was evaluated. 2. In resting rat mesenteric, renal, and caudal arteries, norbormide (0.5-50 microM) induced a concentration-dependent contractile effect. In rat caudal artery, the contraction was very slowly reversible on washing, completely abolished in the absence of extracellular calcium, and antagonized by high concentrations (10-800 microM) of verapamil. The norbormide effect persisted upon removal of either extracellular Na+ or K+. The contractile effect of norbormide was observed also in single, freshly isolated smooth muscle cells from rat caudal artery. 3. In resting rat and guinea-pig aortae, guinea-pig mesenteric artery, mouse caudal artery, and human subcutaneous resistance arteries, norbormide did not induce contraction. When these vessels were contracted by 80 mM KCl, norbormide (10-100 microM) caused relaxation. Norbormide inhibited the response to Ca2+ of rat aorta incubated in 80 mM KCl/Ca2(+)-free medium. Norbormide (up to 100 microM) was ineffective in phenylephrine-contracted guinea-pig and rat aorta. 4. In A7r5 cells, a cell line from rat aorta, norbormide prevented high K(+)- but not 5-hydroxytryptamine-induced intracellular calcium transients. 5. These findings indicate that in vitro, norbormide induces a myogenic contraction, selective for the rat small vessels, by promoting calcium entry in smooth muscle cells, presumably through calcium channels. In rat aorta and arteries from other mammals, norbormide behaves like a calcium channel entry blocker.     

Role of gap junctions and EETs in endothelium-dependent hyperpolarization of porcine coronary artery

1. The effects of endothelium-derived hyperpolarizing factor (EDHF: elicited using substance P or bradykinin) were compared with those of 11,12-EET in pig coronary artery. Smooth muscle cells were usually impaled with microelectrodes through the adventitial surface. 2. Substance P (100 nM) and 11,12-EET (11,12-epoxyeicosatrienoic acid; 3 microM) hyperpolarized endothelial cells in intact arteries. These actions were unaffected by 100 nM iberiotoxin but were abolished by charybdotoxin plus apamin (each 100 nM). 3. Substance P (100 nM) and bradykinin (30 nM) hyperpolarized intact artery smooth muscle; Substance P had no effect after endothelium removal. 11,12-EET hyperpolarized de-endothelialized vessels by 12.6+/-0.3 mV, an effect abolished by 100 nM iberiotoxin. 4. 11,12-EET hyperpolarized intact arteries by 18.6+/-0.8 mV, an action reduced by iberiotoxin, which was ineffective against substance P. Hyperpolarizations to 11, 12-EET and substance P were partially inhibited by 100 nM charybdotoxin and abolished by further addition of 100 nM apamin. 5. 30 microM barium plus 500 nM ouabain depolarized intact artery smooth muscle but responses to substance P and bradykinin were unchanged. 500 microM gap 27 markedly reduced hyperpolarizations to substance P and bradykinin which were abolished in the additional presence of barium plus ouabain. 6. Substance P-induced hyperpolarizations of smooth muscle cells immediately below the internal elastic lamina were unaffected by gap 27, even in the presence of barium plus ouabain. 7. In pig coronary artery, 11,12-EET is not EDHF. Smooth muscle hyperpolarizations attributed to 'EDHF' are initiated by endothelial cell hyperpolarization involving charybdotoxin- (but not iberiotoxin) and apamin-sensitive K(+) channels. This may spread electrotonically via myoendothelial gap junctions but the involvement of an unknown endothelial factor cannot be excluded.     

Resveratrol Enhances Chemosensitivity in Mouse Melanoma Model Through Connexin 43 Upregulation

Although current studies indicate that resveratrol exhibits potential antitumor activities, the precise mechanisms of its beneficial effects combined with chemotherapy are not fully understood. This work is warranted to elucidate the underlying mechanism of antitumor effects by the combination therapy of resveratrol and cisplatin. The presence of functional gap junctions is highly relevant for the success of chemotherapy. Gap junctions mediate cell communication by allowing the passage of molecules from one cell to another. Connexin (Cx) 43 is ubiquitous and reduced in a variety of tumor cells. Cx43 may influence the response of tumor cells to treatments by facilitating the passage of antitumor drugs or death signals between neighboring tumor cells. Following resveratrol treatment, dose‐dependent upregulation of Cx43 expressions was observed. In addition, gap junction intercellular communication was increased. To study the mechanism underlying these resveratrol‐induced Cx43 expressions, we found that resveratrol induced a significant increase in mitogen‐activated protein kinases (MAPK) signaling pathways. The MAPK inhibitors significantly reduced the expression of Cx43 protein after resveratrol treatment. Specific knockdown of Cx43 resulted in a reduction of cell death after resveratrol and cisplatin treatment. Our results suggest that treatment of resveratrol in tumor leads to increase Cx43 gap junction communication and enhances the combination of resveratrol and cisplatin therapeutic effects. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 877–886, 2015.     

Endothelium‐Derived Hyperpolarizing Factor: Is There A Novel Chemical Mediator?

1. Endothelium-derived hyperpolarization (EDH) has been reported in many vessels and an extensive literature suggests that a novel, non-nitric oxide and non-prostanoid, endothelium-derived factor(s) may be synthesized in endothelial cells. 2. The endothelium-dependent hyperpolarizing factor, or EDHF, is synthesized by the putative EDHF synthase and mediates its cellular effects by either, directly or indirectly, opening K channels on vascular smooth muscle cells or, via hyperpolarization of the endothelial cell, by facilitating electrical coupling between the endothelial and the vascular smooth muscle cell. 3. The question of the chemical identity of EDHF has received considerable attention; however, no consensus has been reached. Tissue and species heterogeneity exists that may imply there are multiple EDHF. Leading candidate molecules for EDHF include an arachidonic acid product, possibly an epoxygenase product, or an endogenous cannabinoid, or simply an increase in extracellular K+. 4. An increasing body of evidence suggests that EDH, notably in the resistance vasculature, may be mediated via electrical coupling through myoendothelial gap junctions and the existence of electrical coupling may negate the need to hypothesize the existence of a true endothelium-derived chemical mediator. 5. In this paper we review the evidence that supports and refutes the existence of a novel EDHF versus a hyperpolarization event mediated solely by myoendothelial gap junctions.     

Inhibitory effects of cadralazine and its metabolite, ISF-2405, on contractions and the level of cytosolic Ca2+ in vascular smooth muscle

The inhibitory effects of a hypotensive agent, cadralazine and its metabolite, ISF-2405, on the level of cytosolic Ca2+ ([Ca2+]cyt) and on contractions were examined in isolated vascular smooth muscle. Cadralazine slightly inhibited the transient norepinephrine-induced contraction in rabbit aorta and canine femoral, renal and mesenteric arteries and saphenous vein, and prostaglandin F2 alpha-induced contractions in canine basilar and coronary arteries. In contrast, ISF-2405 inhibited the contractions induced by prostaglandin F2 alpha in canine basilar and coronary arteries and those induced by norepinephrine in canine renal and femoral arteries and rabbit aorta. In aorta, ISF-2405 inhibited the increase in [Ca2+]cyt and muscle tension caused by norepinephrine. A Ca2+ channel blocker, verapamil, inhibited the norepinephrine-stimulated increase in [Ca2+]cyt more potently than it inhibited the increase in muscle tension, and ISF-2405 inhibited the verapamil-resistant part of the contraction. In Ca2(+)-free solution, norepinephrine induced transient increases in [Ca2+]cyt and muscle tension. ISF-2405 inhibited these changes. However, ISF-2405 did not inhibit the transient contraction induced by caffeine in the aorta. These results suggest that cadralazine is metabolized to ISF-2405 and inhibits vascular smooth muscle contraction by inhibiting receptor-mediated Ca2+ influx, Ca2+ release and Ca2+ sensitization of contractile elements.     

Mechanisms underlying diabetes enhancement of endothelin-1-induced contraction in rabbit basilar artery

The influence of alloxan-induced diabetes on the reactivity of rabbit basilar artery to endothelin-1 was examined. Endothelin-1 induced concentration-dependent contraction of basilar arteries that was higher in diabetic than in control rabbits. Endothelium removal produced a higher enhancement of the endothelin-1-induced contraction in control than in diabetic rabbits. N(G)-nitro-L-arginine (L-NOArg) enhanced the maximal contraction induced by endothelin-1 in control rabbits and potentiated this response in diabetic rabbits. Endothelin ETA receptor antagonist, cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), inhibited endothelin-1-induced contraction in both rabbit groups. Endothelin ETB receptor antagonist, 2,6-Dimethylpiperidinecarbonyl-gamma-Methyl-Leu-Nin-(Methoxycarbonyl)-D-Trp-D-Nle (BQ-788), enhanced endothelin-1-induced contraction in control rabbits and decreased the potency of endothelin-1 in diabetic rabbits. Sodium nitroprusside-induced relaxation of basilar arteries was lower in diabetic than in control rabbits. These results suggest that mechanisms underlying rabbit basilar artery hyperreactivity to endothelin-1 include decreased endothelial modulation of endothelin-1-induced contraction, with impaired endothelial endothelin ETB receptor activity; decreased sensitivity to nitric oxide (NO) in vascular smooth muscle; and enhanced participation of muscular endothelin ETA and ETB receptors.     

In vitro studies of release of adenine nucleotides and adenosine from rat vascular endothelium in response to alpha 1-adrenoceptor stimulation.

1. Noradrenaline-induced release of endogenous adenine nucleotides (ATP, ADP, AMP) and adenosine from both rat caudal artery and thoracic aorta was characterized, using high-performance liquid chromatography with fluorescence detection. 2. Noradrenaline, in a concentration-dependent manner, increased the overflow of ATP and its metabolites from the caudal artery. The noradrenaline-induced release of adenine nucleotides and adenosine from the caudal artery was abolished by bunazosin, an alpha 1-adrenoceptor antagonist, but not by idazoxan, an alpha 2-adrenoceptor antagonist. Clonidine, an alpha 2-adrenoceptor agonist, contracted caudal artery smooth muscle but did not induce release of adenine nucleotides or adenosine. 3. Noradrenaline also significantly increased the overflow of ATP and its metabolites from the thoracic aorta in the rat; however, the amount of adenine nucleotides and adenosine released from the aorta was considerably less than that released from the caudal artery. 4. Noradrenaline significantly increased the overflow of ATP and its metabolites from cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the aorta was also much less than that from cultured endothelial cells from the caudal artery. In cultured smooth muscle cells from the caudal artery, a significant release of ATP or its metabolites was not observed. 5. These results suggest that there are vascular endothelial cells that are able to release ATP by an alpha 1-adrenoceptor-mediated mechanism, but that these cells are not homogeneously distributed in the vasculature.