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...

In rat mesenteric artery, endothelium-derived hyperpolarizing factor (EDHF) is blocked by a combination of apamin and charybdotoxin (ChTX). The site of action of these toxins has not been established. We compared the effects of ChTX and apamin applied selectively to the endothelium and to the smooth muscle. In isometrically mounted arteries, ACh (0.01–10 μm), in the presence of indomethacin (2.8 μM) and N ω-nitro-l-arginine methyl ester (l-NAME) (100 μM), concentration dependently relaxed phenylephrine (PE)-stimulated tone (EC50 50 nM; n = 10). Apamin (50 nM) and ChTX (50 nM) abolished this relaxation ( n = 5). In pressurized arteries, ACh (10 μM), applied intraluminally in the presence of indomethacin (2.8 μM) andl-NAME (100 μM), dilated both PE-stimulated (0.3–0.5 μM; n = 5) and myogenic tone ( n = 3). Apamin (50 nM ) and ChTX (50 nM) applied intraluminally abolished ACh-induced dilatations. Bath superperfusion of apamin and ChTX did not affect ACh-induced dilatations of either PE-stimulated ( n = 5) or myogenic tone ( n = 3). This is the first demonstration that ChTX and apamin act selectively on the endothelium to block EDHF-mediated relaxation.

Abstract-Inhibition of vascular smooth muscle (VSM) delayed rectifier K ϩ channels (K DR ) by 4-aminopyridine (4-AP; 200 mol/L) or correolide (1 mol/L), a selective inhibitor of Kv1 channels, enhanced myogenic contraction of rat mesenteric arteries (RMAs) in response to increases in intraluminal pressure. The molecular identity of K DR of RMA myocytes was characterized using RT-PCR, real-time PCR, and immunocytochemistry. Transcripts encoding the pore-forming Kv␣ subunits, Kv1.2, Kv1.4, Kv1.5, and Kv1.6, were identified and confirmed at the protein level with subunit-specific antibodies. Kv␤ transcript (␤1.1, ␤1.2, ␤1.3, and ␤2.1) expression was also identified. Kv1.5 message was Ϸ2-fold more abundant than that for Kv1.2 and Kv1.6. Transcripts encoding these three Kv1␣ subunits were Ϸ2-fold more abundant in 1st/2nd order conduit compared with 4th order resistance RMAs, and Kv␤1 was 8-fold higher than Kv␤2 message. RMA K DR activated positive to Ϫ50 mV, exhibited incomplete inactivation, and were inhibited by 4-AP and correolide. However, neither ␣-dendrotoxin or -dendrotoxin affected RMA K DR , implicating the presence of Kv1.5 in all channels and the absence of Kv1.1, respectively. Currents mediated by channels because of coexpression of Kv1.2, Kv1.5, Kv1.6, and Kv␤1.2 in human embryonic kidney 293 cells had biophysical and pharmacological properties similar to those of RMA K DR . It is concluded that K DR channels composed of heteromultimers of Kv1 subunits play a critical role in myogenic control of arterial diameter. Key Words: delayed rectifier potassium channel Ⅲ KCNA Ⅲ vascular smooth muscle Ⅲ myogenic contraction Ⅲ arterial diameter T he ability of small resistance arteries to develop myogenic tone in response to elevations in intraluminal (or transmural) pressure is an essential autoregulatory mechanism and an important determinant of peripheral vascular resistance, regional blood flow, and blood pressure. Pressureinduced depolarization of vascular smooth muscle (VSM) leading to increased intracellular Ca 2ϩ ([Ca 2ϩ ] i ) via voltagedependent activation of Ca 2ϩ channels is required for myogenic tone development. 1,2 However, the depolarization does not evoke regenerative action potentials, rather incremental changes in diameter are achieved by graded, steady-state depolarizations. 3 Our understanding of the ionic basis of this precise control of myogenic depolarization is incomplete.Increased intraluminal pressure is thought to activate nonselective cation or Cl Ϫ channels of VSM cells, 2 with the level of depolarization attributable to these channels precisely controlled by an activation of VSM K ϩ channels. 3 Compelling evidence for a contribution of large Ca 2ϩ -activated K ϩ channels (BK Ca ) to this feedback control comes from studies using specific inhibitors (eg, iberiotoxin) and transgenic mice lacking the BK Ca modulatory ␤1 subunit. 4 However, whether other channels are also involved in controlling myogenic depolarization in VSM is not clear.Voltage-gated delayed rectifier channels (K DR ) ...

The endogenous cannabinoid, anandamide, has been suggested as an endothelium-derived hyperpolarizing factor (EDHF). We found that anandamide-evoked relaxation in isolated segments of rat mesenteric artery was associated with smooth muscle hyperpolarization. However, although anandamide-evoked relaxation was inhibited by either charybdotoxin (ChTX) or iberiotoxin, inhibition of the relaxation to EDHF required a combination of ChTX and apamin. The relaxations induced by either anandamide or EDHF were not inhibited by the cannabinoid receptor (CB 1 ) antagonist SRI41716A, or mimicked by selective CB 1 agonists. Thus, anandamide appears to cause smooth muscle relaxation via a CB 1 receptorindependent mechanism and cannabinoid receptor activation apparently does not contribute to EDHFmediated relaxation in this resistance artery.

1 The effect of native low-density lipoproteins (LDL) and oxidized LDL (OXLDL) on the relaxations to endothelium-derived relaxing factor (EDRF) in isolated, intact aortic rings of the rabbit were investigated. 2 Native LDL induced a concentration-dependent reversible inhibition of the relaxations elicited by acetylcholine (ACh) or A23187, in rings pre-contracted by noradrenaline (NA), adrenaline (Ad) and 5-hydroxytryptamine (5-HT), but not phenylephrine (PE), which was not influenced by indomethacin. 3 The inhibition was surmountable in the rings pre-contracted with NA and Ad and only partially in those pre-contracted with 5-HT. 4 OXLDL induced an inhibition of the relaxations elicited by ACh and A23187 which was independent of the contractile agonist. The extent of inhibition and its reversibility varied with the LDL from individual donors, but was unaffected by indomethacin. 5 Native and oxidized LDL inhibited relaxations evoked by exogenous nitric oxide (NO) to the same extent. Higher concentrations of NO overcame the inhibition. The inhibition was independent of the contractile agonist and the preparation of LDL from individual donors. 6 Only OXLDL inhibited reversibly relaxations evoked by glyceryl trinitrate (GTN) and the inhibition was independent of the LDL preparation from individual donors. 7 This study demonstrates that native and OXLDL influence the response to EDRF in isolated aorta. We suggest that these lipoproteins may contribute to the attenuation of responses to EDRF found in isolated arteries from hypercholesterolaemic and atherosclerotic animals.