Although the chemical nature of endothelium-derived hyperpolarizing factor (EDHF) remains elusive, electrophysiological evidence exists for electrical communication between smooth muscle cells and endothelial cells suggesting that electrotonic propagation of hyperpolarization may explain the failure to identify a single chemical factor as EDHF. Anatomical evidence for myoendothelial gap junctions, or the sites of electrical coupling, is, however, rare. In the present study, serial-section electron microscopy and reconstruction techniques have been used to examine the incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat where EDHF responses have been reported to vary. Myoendothelial gap junctions were found to be very small in the mesenteric arteries, the majority being <100 nm in diameter. In addition, they were significantly more common in the distal compared with the proximal regions of this arterial bed. Pentalaminar gap junctions between adjacent endothelial cells were much larger and were common in both proximal and distal mesenteric arteries. These latter junctions were frequently found near the myoendothelial gap junctions. These results provide the first evidence for the presence of sites for electrical communication between endothelial cells and smooth muscle cells in the mesenteric vascular bed. Furthermore, the relative incidence of these sites suggests that there may be a relationship between the activity of EDHF and the presence of myoendothelial gap junctions.

Abstract-The nature of the vasodilator endothelium-derived hyperpolarizing factor (EDHF) is controversial, putatively involving diffusible factors and/or electrotonic spread of hyperpolarization generated in the endothelium via myoendothelial gap junctions (MEGJs). In this study, we investigated the relationship between the existence of MEGJs, endothelial cell (EC) hyperpolarization, and EDHF-attributed smooth muscle cell (SMC) hyperpolarization in two different arteries: the rat mesenteric artery, where EDHF-mediated vasodilation is prominent, and the femoral artery, where there is no EDHF-dependent relaxation. In the rat mesenteric artery, stimulation of the endothelium with acetylcholine (ACh) evoked hyperpolarization of both ECs and SMCs, and characteristic pentalaminar MEGJs were found connecting the two cell layers. Key Words: endothelium-derived hyperpolarizing factor Ⅲ myoendothelial gap junctions Ⅲ endothelium Ⅲ smooth muscle Ⅲ electrical coupling T he endothelium plays a central role in the regulation of vascular tone. 1 The endothelium is capable of exerting a profound relaxing influence on the underlying smooth muscle, mediated by at least three different factors, depending on the vascular bed. These include nitric oxide (NO) and prostacyclin, both diffusible factors. [2][3][4][5][6] In addition, after blockade of NO and prostacyclin synthesis, stimulation of the endothelium is capable of evoking vascular smooth muscle relaxation that has been attributed to a third factor, endothelium-derived hyperpolarizing factor (EDHF). [7][8][9][10][11][12][13][14][15] The hallmark of the vasorelaxation attributed to EDHF is that it is accompanied by membrane hyperpolarization. Consensus regarding the nature of EDHF is lacking, with evidence suggesting the involvement of a diffusible factor(s) released from the endothelium in some vascular beds. 11,12,16,17 Others propose that the hyperpolarization generated in endothelial cells (ECs) is capable of spreading electrotonically to the underlying smooth muscle cells (SMCs), 18,19,21,23,25 most likely via myoendothelial gap junctions (MEGJs). 9,18,19,21,[23][24][25][26] Evidence to support the transfer of a small molecule (eg, cAMP) via MEGJs has also been presented. 20,22 Support for the MEGJ hypothesis has come from studies in which gap junctions have been pharmacologically blocked, either with peptide mimetics of connexin 43 (Cx43) [27][28][29][30] or with glycyrrhetinic acid derivatives. 13,26,[31][32][33] Although the former have been demonstrated to indeed reduce dye coupling between Cx-transfected cells, 34 the latter seem to have some nonspecific actions. [31][32][33]35 An alternative and complementary approach would be to study an artery that lacked an EDHF-dependent relaxation and to test for the existence of agonist-induced hyperpolarization in the ECs and for the presence of MEGJs. The femoral artery represents such a tissue because EDHF does not contribute to endothelium-dependent SMC relaxation in this vessel. 36,37 In the present study, this artery was ...

1. Modulation of vascular cell calcium is critical for the control of vascular tone, blood flow and pressure. 2. Specialized microdomain signalling sites associated with calcium modulation are present in vascular smooth muscle cells, where spatially localized channels and calcium store receptors interact functionally. Anatomical studies suggest that such sites are also present in endothelial cells. 3. The characteristics of these sites near heterocellular myoendothelial gap junctions (MEGJs) are described, focusing on rat mesenteric artery. The MEGJs enable current and small molecule transfer to coordinate arterial function and are thus critical for endothelium-derived hyperpolarization, regulation of smooth muscle cell diameter in response to contractile stimuli and vasomotor conduction over distance. 4. Although MEGJs occur on endothelial cell projections within internal elastic lamina (IEL) holes, not all IEL holes have MEGJ-related projections (approximately 0-50% of such holes have MEGJ-related projections, with variations occurring within and between vessels, species, strains and disease). 5. In rat mesenteric, saphenous and caudal cerebellar artery and hamster cheek pouch arteriole, but not rat middle cerebral artery or cremaster arteriole, intermediate conductance calcium-activated potassium channels (IK(Ca)) localize to endothelial cell projections. 6. Rat mesenteric artery MEGJ connexins and IK(Ca) are in close spatial association with endothelial cell inositol 1,4,5-trisphosphate receptors and endoplasmic reticulum. 7. Data suggest a relationship between spatially associated endothelial cell ion channels and calcium stores in modulation of calcium release and action. Differences in spatial relationships between ion channels and calcium stores in different vessels reflect heterogeneity in vasomotor function, representing a selective target for the control of endothelial and vascular function.

Intracellular recordings were made from short segments of the muscular wall of the guinea-pig gastric antrum. Preparations were impaled using two independent microelectrodes, one positioned in the circular layer and the other either in the longitudinal layer, in the network of myenteric interstitial cells of Cajal (ICC MY ) or in the circular layer. Cells in each layer displayed characteristic patterns of rhythmical activity, with the largest signals being generated by ICC MY . Current pulses injected into the circular muscle layer produced electrotonic potentials in each cell layer, indicating that the layers are electrically interconnected. The amplitudes of these electrotonic potentials were largest in the circular layer and smallest in the longitudinal layer. An analysis of electrical coupling between the three layers suggests that although the cells in each layer are well coupled to neighbouring cells, the coupling between either muscle layer and the network of ICC MY is relatively poor. The electrical connections between ICC MY and the circular layer did not rectify. In parallel immunohistochemical studies, the distribution of the connexins Cx40, Cx43 and Cx45 within the antral wall was determined. Only Cx43 was detected; it was widely distributed on ICC MY and throughout the circular smooth muscle layer, being concentrated around ICC IM , but was less abundant in the circular muscle layer immediately adjacent to ICC MY . Although the electrophysiological studies indicate that smooth muscle cells in the longitudinal muscle layer are electrically coupled to each other, none of the connexins examined were detected in this layer.

Many arteries and arterioles exhibit rhythmical contractions which are synchronous over considerable distances. This vasomotion is likely to assist in tissue perfusion especially during periods of altered metabolism or perfusion pressure. While the mechanism underlying vascular rhythmicity has been investigated for many years, it has only been recently, with the advent of imaging techniques for visualizing intracellular calcium release, that significant advances have been made. These methods, when combined with mechanical and electrophysiological recordings, have demonstrated that the rhythm depends critically on calcium released from intracellular stores within the smooth muscle cells and on cell coupling via gap junctions to synchronize oscillations in calcium release amongst adjacent cells. While these factors are common to all vessels studied to date, the contribution of voltage-dependent channels and the endothelium varies amongst different vessels. The basic mechanism for rhythmical activity in arteries thus differs from its counterpart in non-vascular smooth muscle, where specific networks of pacemaker cells generate electrical potentials which drive activity within the otherwise quiescent muscle cells. Spontaneous, rhythmical contractions are generated in many different types of smooth muscle, from the gastrointestinal tract, urinary tract and lymphatic vessels through to arteries and veins (Tomita, 1981;Van Helden, 1993; Hashitani et al. 1996). In blood vessels, this activity, known as vasomotion, occurs in small resistance vessels of the microcirculation, as well as in larger arteries both in vivo and in vitro (see Shimamura et al. 1999; Nilsson & Aalkjaer, 2003 for details). While rhythmicity in non-vascular smooth muscles is often propagated, serving to actively move intraluminal contents in a peristaltic fashion, rhythmicity in vascular smooth muscle is apparently synchronous over considerable lengths of arteries. Vasomotion is thus expected to increase flow as its amplitude increases, in turn resulting in a decrease in vascular resistance (Funk et al. 1983;Meyer et al. 2002). In this case vasomotion may be seen to be beneficial and its up-regulation during pathological conditions, such as hypertension, may be considered to be protective. However the effect of vasomotion on vascular resistance is currently controversial (Gratton et al. 1998;Meyer et al. 2002) and hence its physiological significance is yet to be clearly defined.Vasomotion occurs in arteries in vitro either spontaneously or in response to pressure, stretch, application of vasoconstrictor agonists or increases in extracellular potassium concentration (Duling et al. 1981; Hayashida et al. 1986; Katusic et al. 1988;Chemtob et al. 1992;Gustafsson, 1993; Lee & Earm, 1994; Stork & Cocks, 1994;Porret et al. 1995;Eddinger & Ratz, 1997; Hill et al. 1999). Since many studies have described a critical role for voltage-dependent calcium channels (VDCCs;Colantuoni et al. 1984; Hayashida et al. 1986; Hundley et al. 1988;Fujii et al. 1990;Chem...

Although dihydropyridines are widely used for the treatment of vasospasm, their effectiveness is questionable, suggesting that other voltage-dependent calcium channels (VDCCs) contribute to control of cerebrovascular tone. This study therefore investigated the role of dihydropyridine-insensitive VDCCs in cerebrovascular function. Using quantitative PCR and immunohistochemistry, we found mRNA and protein for L-type (Ca(V)1.2) and T-type (Ca(V)3.1 and Ca(V)3.2) channels in adult rat basilar and middle cerebral arteries and their branches. Immunoelectron microscopy revealed both L- and T-type channels in smooth muscle cell (SMC) membranes. Using patch clamp electrophysiology, we found that a high-voltage-activated calcium current, showing T-type channel kinetics and insensitivity to nifedipine and nimodipine, comprised approximately 20% of current in SMCs of the main arteries and approximately 45% of current in SMCs from branches. Both components were abolished by the T-type antagonists mibefradil, NNC 55-0396, and efonidipine. Although nifedipine completely blocked vasoconstriction in pressurized basilar arteries, a nifedipine-insensitive constriction was found in branches and this increased in magnitude as vessel size decreased. We conclude that a heterogeneous population of VDCCs contributes to cerebrovascular function, with dihydropyridine-insensitive channels having a larger role in smaller vessels. Sensitivity of these currents to nonselective T-type channel antagonists suggests that these drugs may provide a more effective treatment for therapy-refractory cerebrovascular constriction.

The renal vasculature and mesangial cells are well coupled on the preglomerular side but there is little evidence that the coupling extends into the efferent arteriole. This pattern of cell coupling is accentuated during diabetes.

Control of cerebral vasculature differs from that of systemic vessels outside the blood-brain barrier. The hypothesis that the endothelium modulates vasomotion via direct myoendothelial coupling was investigated in a small vessel of the cerebral circulation. In the primary branch of the rat basilar artery, membrane potential, diameter, and calcium dynamics associated with vasomotion were examined using selective inhibitors of endothelial function in intact and endothelium-denuded arteries. Vessel anatomy, protein, and mRNA expression were studied using conventional electron microscopy high-resolution ultrastructural and confocal immunohistochemistry and quantitative PCR. Membrane potential oscillations were present in both endothelial cells and smooth muscle cells (SMCs), and these preceded rhythmical contractions during which adjacent SMC intracellular calcium concentration ([Ca 2ϩ ]i) waves were synchronized. Endothelium removal abolished vasomotion and desynchronized adjacent smooth muscle cell [Ca 2ϩ ]i waves. N G -nitro-L-arginine methyl ester (10 M) did not mimic this effect, and dibutyryl cGMP (300 M) failed to resynchronize [Ca 2ϩ ]i waves in endothelium-denuded arteries. Combined charybdotoxin and apamin abolished vasomotion and depolarized and constricted vessels, even in absence of endothelium. Separately, 37,43 Gap27 and 40 Gap27 abolished vasomotion. Extensive myoendothelial gap junctions (3 per endothelial cell) composed of connexins 37 and 40 connected the endothelial cell and SMC layers. Synchronized vasomotion in rat basilar artery is endothelium dependent, with [Ca 2ϩ ]i waves generated within SMCs being coordinated by electrical coupling via myoendothelial gap junctions.connexin; electron microscopy; endothelial function; potassium channel; membrane potential VASOMOTION is an intrinsic feature of many arteries and arterioles under normal and pathological conditions and may contribute to regulation of blood flow and pressure (1,14). Both spontaneous and agonist-induced vasomotion occur when oscillations in the concentration of intracellular calcium ([Ca 2ϩ ] i ) become synchronized among adjacent smooth muscle cells, giving rise to global oscillations in [Ca 2ϩ ] i across the vessel wall (13,15,26,27,33,41,(47)(48)(49).There is clear heterogeneity in the mechanisms that underlie vasomotion, and specifically whether the endothelium plays a mandatory or modulatory role. In the rat iris arteriole, rabbit ear, and superior mesenteric artery, vasomotion is reported to be endothelium independent (3, 18, 39), whereas in the rat mesenteric, rabbit femoral artery, and hamster aorta, vasomotion is suggested to be endothelium dependent, as shown by its abolition with endothelium denudation (12,21,34,38,40).Endothelium-dependent effects on vasomotion have been attributed to the vasodilatory factors nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). In some studies, endothelial cells play an essential role in synchronizing the activity of smooth muscle cells through the rel...