No abstract

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP(3)) flux activates a discrete pool of IP(3) receptors (IP(3)Rs), elicits localized endothelial Ca(2+) transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca(2+)-activated K(+) channels (IK) and Ca(2+) mobilization from the endoplasmic reticulum (ER) enhanced the contractile/electrical responsiveness of feed arteries to phenylephrine. Next, structural analysis confirmed that endothelial projections make contact with the overlying smooth muscle. These projections retained membranous ER networks, and IP(3)Rs and IK channels localized in or near this structure. Finally, Ca(2+) imaging revealed that phenylephrine induced discrete endothelial Ca(2+) events through IP(3)R activation. These events were termed recruitable Ca(2+) wavelets on the basis of their spatiotemporal characteristics. From these findings, we conclude that IP(3) flux across myoendothelial gap junctions is sufficient to induce focal Ca(2+) release from IP(3)Rs and activate a discrete pool of IK channels within or near endothelial projections. The resulting hyperpolarization feeds back on smooth muscle to moderate agonist-induced depolarization and constriction.

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.

It is controversial whether the endothelial cell release of nitric oxide (NO) or a different factor(s) accounts for endothelium-dependent hyperpolarization, because in many arteries endothelium-dependent relaxation and hyperpolarization resists inhibitors of NO synthase. The contribution of NO to acetylcholine-induced endotheliumdependent hyperpolarization and relaxation of the rabbit carotid artery was determined by measuring NO with electrochemical and chemiluminescence techniques. In the presence of phenylephrine to depolarize and contract the smooth muscle cells, acetylcholine caused concentration-dependent hyperpolarization and relaxation which were closely correlated to the release of NO. N -nitro-L-arginine methyl ester (30 M) partially reduced the release of NO and caused a similar reduction in smooth muscle cell relaxation and hyperpolarization. To determine if the residual responses were mediated by another endothelium-derived mediator or NO released despite treatment with N -nitro-L-arginine methyl ester, N -nitro-L-arginine (300 M) was added. The combined inhibitors further reduced, but did not eliminate, NO release, smooth muscle relaxation, and hyperpolarization. Hyperpolarization and relaxation to acetylcholine remained closely correlated with the release of NO in the presence of the inhibitors. In addition, the NO donor, SIN-1, caused hyperpolarization and relaxation which correlated with the concentrations of NO that it released. These studies indicate that (i) the release of NO by acetylcholine is only partially inhibited by these inhibitors of NO synthase when used even at high concentrations, and (ii) NO rather than another factor accounts fully for endothelium-dependent responses of the rabbit carotid artery.Nitric oxide (NO) is now accepted to be the major mediator of endothelium-dependent, arterial smooth muscle relaxation (1). However, whether or not NO accounts for endotheliumdependent hyperpolarization is controversial (2, 3). Authentic NO and NO donors hyperpolarize vascular smooth muscle cells (4); however, in some studies, concentrations of NO that were too high to be considered physiological were required, or the hyperpolarization observed was smaller than that caused by an endothelial cell agonist (2, 3). In addition, in many in vitro and in vivo studies of human and animal arteries, endotheliumdependent relaxation, vasodilatation, and hyperpolarization persist in the presence of L-arginine analogues that are inhibitors of NO synthase (NOS) (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). This has led to the supposition that factors other than NO are important mediators of endothelium-dependent responses including hyperpolarization (2, 3). Cytochrome P450 metabolites of arachidonic acid (21,22), and carbon monoxide (23) have been proposed as alternative mediators. Neither the release from the endothelium of sufficient quantities of these mediators to account for the responses has been measured, nor has NO release been measured directly to exclude its contributio...