In arteries, muscarinic agonists such as acetylcholine release an unidentified, endothelium-derived hyperpolarizing factor (EDHF) which is neither prostacyclin nor nitric oxide. Here we show that EDHF-induced hyperpolarization of smooth muscle and relaxation of small resistance arteries are inhibited by ouabain plus Ba2+; ouabain is a blocker of Na+/K+ ATPase and Ba2+ blocks inwardly rectifying K+ channels. Small increases in the amount of extracellular K+ mimic these effects of EDHF in a ouabain- and Ba2+-sensitive, but endothelium-independent, manner. Acetylcholine hyperpolarizes endothelial cells and increases the K+ concentration in the myoendothelial space; these effects are abolished by charbdotoxin plus apamin. Hyperpolarization of smooth muscle by EDHF is also abolished by this toxin combination, but these toxins do not affect the hyperpolarizaiton of smooth muscle by added K+. These data show that EDHF is K+ that effluxes through charybdotoxin- and apamin-sensitive K+ channels on endothelial cells. The resulting increase in myoendothelial K+ concentration hyperpolarizes and relaxes adjacent smooth-muscle cells by activating Ba2+-sensitive K+ channels and Na+/K+ ATPase. These results show that fluctuations in K+ levels originating within the blood vessel itself are important in regulating mammalian blood pressure and flow.

It is well known that vascular smooth muscle tone can be modulated by signals arising in the endothelium (e.g., endothelium-derived relaxing factor, endothelium-derived hyperpolarizing factor, and prostaglandins). Here we show that during vasoconstriction a signal can originate in smooth muscle cells and act on the endothelium to cause synthesis of endothelium-derived relaxing factor. We studied responses to two vasoconstrictors (phenylephrine and KCl) that act by initiating a rise in smooth muscle cell intracellular Ca2+ concentration ([Ca2+]i) and exert little or no direct effect on the endothelium. Fluo-3 was used as a Ca2+ indicator in either smooth muscle or endothelial cells of arterioles from the hamster cheek pouch. Phenylephrine and KCl caused the expected rise in smooth muscle cell [Ca2+]i that was accompanied by an elevation in endothelial cell [Ca2+]i. The rise in endothelial cell [Ca2+]i was followed by increased synthesis of NO, as evidenced by an enhancement of the vasoconstriction induced by both agents after blockade of NO synthesis. The molecule involved in signal transmission from smooth muscle to endothelium is as yet unknown. However, given that myoendothelial cell junctions are frequent in these vessels, we hypothesize that the rise in smooth muscle cell Ca2+ generates a diffusion gradient that drives Ca2+ through myoendothelial cell junctions and into the endothelial cells, thereby initiating the synthesis of NO.

Abstract-Arterial hyperpolarization to acetylcholine (ACh) reflects coactivation of K Ca 3.1 (IK Ca ) channels and K Ca 2.3 (SK Ca ) channels in the endothelium that transfers through myoendothelial gap junctions and diffusible factor(s) to affect smooth muscle relaxation (endothelium-derived hyperpolarizing factor [EDHF] response). However, ACh can differentially activate K Ca 3.1 and K Ca 2.3 channels, and we investigated the mechanisms responsible in rat mesenteric arteries. K Ca 3.1 channel input to EDHF hyperpolarization was enhanced by reducing external [Ca 2ϩ ] o but blocked either with forskolin to activate protein kinase A or by limiting smooth muscle [Ca 2ϩ ] i increases stimulated by phenylephrine depolarization. Imaging [Ca 2ϩ ] i within the endothelial cell projections forming myoendothelial gap junctions revealed increases in cytoplasmic [Ca 2ϩ ] i during endothelial stimulation with ACh that were unaffected by simultaneous increases in muscle [Ca 2ϩ ] i evoked by phenylephrine. If gap junctions were uncoupled, K Ca 3.1 channels became the predominant input to EDHF hyperpolarization, and relaxation was inhibited with ouabain, implicating a crucial link through Na ϩ /K ϩ -ATPase. There was no evidence for an equivalent link through K Ca 2.3 channels nor between these channels and the putative EDHF pathway involving natriuretic peptide receptor-C. Reconstruction of confocal z-stack images from pressurized arteries revealed K Ca 2.3 immunostain at endothelial cell borders, including endothelial cell projections, whereas K Ca 3.1 channels and Na ϩ /K ϩ -ATPase ␣ 2 /␣ 3 subunits were highly concentrated in endothelial cell projections and adjacent to myoendothelial gap junctions. Thus, extracellular [Ca 2ϩ ] o appears to modify K Ca 3.1 channel activity through a protein kinase A-dependent mechanism independent of changes in endothelial [Ca 2ϩ ] i . The resulting hyperpolarization links to arterial relaxation largely through Na ϩ /K ϩ -ATPase, possibly reflecting K ϩ acting as an EDHF. In contrast, K Ca 2.3 hyperpolarization appears mainly to affect relaxation through myoendothelial gap junctions. Overall, these data suggest that K ϩ and myoendothelial coupling evoke EDHF-mediated relaxation through distinct, definable pathways. (Circ Res. 2008;102:1247-1255.) Key Words: potassium channel Ⅲ endothelial cells Ⅲ hyperpolarization Ⅲ membrane potential Ⅲ electrophysiology Ⅲ vasodilation T he importance of the arterial endothelium for relaxation of the subjacent smooth muscle is well established. Whatever the final endothelium-derived effector, a key event is an initial increase in endothelial cell [Ca 2ϩ ] i . In the case of endothelium-derived hyperpolarizing factor (EDHF) (the NO-and prostanoid-independent pathway), this increase crucially activates endothelial K Ca 2.3 and K Ca 3.1 channels. Activation of these K Ca channels leads on to arterial hyperpolarization and dilation (see elsewhere for review 1 ), and a changing role for each subtype has been implicated in pathological responses ...

Endothelial cell (EC) Ca 2+ -activated K channels (SK Ca and IK Ca channels) generate hyperpolarization that passes to the adjacent smooth muscle cells causing vasodilation. IK Ca channels focused within EC projections toward the smooth muscle cells are activated by spontaneous Ca 2+ events (Ca 2+ puffs/pulsars). We now show that transient receptor potential, vanilloid 4 channels (TRPV4 channels) also cluster within this microdomain and are selectively activated at low intravascular pressure. In arterioles pressurized to 80 mmHg, ECs generated low-frequency (∼2 min −1 ) inositol 1,4,5-trisphosphate receptor-based Ca 2+ events. Decreasing intraluminal pressure below 50 mmHg increased the frequency of EC Ca 2+ events twofold to threefold, an effect blocked with the TRPV4 antagonist RN1734. These discrete events represent both TRPV4-sparklet-and nonsparklet-evoked Ca 2+ increases, which on occasion led to intracellular Ca 2+ waves. The concurrent vasodilation associated with increases in Ca 2+ event frequency was inhibited, and basal myogenic tone was increased, by either RN1734 or TRAM-34 (IK Ca channel blocker), but not by apamin (SK Ca channel blocker). These data show that intraluminal pressure influences an endothelial microdomain inversely to alter Ca 2+ event frequency; at low pressures the consequence is activation of EC IK Ca channels and vasodilation, reducing the myogenic tone that underpins tissue blood-flow autoregulation.endothelial cell calcium | cremaster arterioles | mesenteric arteries C a 2+ -activated K + (K Ca ) channels in arteriolar endothelial cells (ECs) are activated by intrinsic spontaneous or receptormediated Ca 2+ events, each leading to hyperpolarization of smooth muscle cells (SMCs) and vasodilation independent of nitric oxide or prostacyclin-the endothelium-dependent hyperpolarization (EDH) response. This hyperpolarization spreads both radially and longitudinally through the vascular wall via patent gap junctions to evoke local and conducted dilation, and it is central to cardiovascular function (1, 2).EDH is the predominant endothelium-dependent mechanism in smaller "resistance" arteries and arterioles. The underlying hyperpolarization is generated by two subtypes of K Ca channels found in the EC, but not SMC, membrane, the small (SK Ca ,K Ca 2.3) and intermediate (IK Ca, K Ca 3.1) conductance forms that may be activated independently of each other (3). The physiological importance of independent activation is apparent from studies with K Ca 3.1-deficient mice in which the mean blood pressure is raised by ∼7 mmHg, but further elevated by disrupting both K Ca channels (4). In mesenteric resistance arteries, IK Ca channels are focused within EC projections through the internal elastic lamina (IEL) termed myoendothelial junctions (MEJs). MEJs can contain gap junctions (MEGJs) coupling ECs to SMCs, and EDH can spread by direct electrical coupling and/or a diffusible factor (5, 6). The IK Ca channels enriched within MEJs can be activated by spontaneous inositol 1,4,5-trisphosphate (IP 3 ...

Abstract-In resistance arteries, spread of hyperpolarization from the endothelium to the adjacent smooth muscle is suggested to be a crucial component of dilation resulting from endothelium-derived hyperpolarizing factor (EDHF). To probe the role of endothelial gap junctions in EDHF-mediated dilation, we developed a method, which was originally used to load membrane impermeant molecules into cells in culture, to load connexin (Cx)-specific inhibitory molecules rapidly (Ϸ15 minutes) into endothelial cells within isolated, pressurized mesenteric arteries of the rat. Validation was achieved by luminally loading cell-impermeant fluorescent dyes selectively into virtually all the arterial endothelial cells, without affecting either tissue morphology or function. The endothelial monolayer served as an effective barrier, preventing macromolecules from entering the underlying smooth muscle cells. Using this technique, endothelial cell loading either with antibodies to the intracellular carboxyl-terminal region of Cx40 (residues 340 to 358) or mimetic peptide for the cytoplasmic loop (Cx40; residues 130 to 140) each markedly depressed EDHF-mediated dilation. In contrast, multiple antibodies directed against different intracellular regions of Cx37 and Cx43, and mimetic peptide for the intracellular loop region of Cx37, were each without effect. Furthermore, simultaneous intra-and extraluminal incubation of pressurized arteries with inhibitory peptides targeted against extracellular regions of endothelial cell Cxs ( 43 Gap 26, 40 Gap 27, and 37,43 Gap 27; 300 mol/L each) for 2 hours also failed to modify the EDHF response. High-resolution immunohistochemistry localized Cx40 to the end of endothelial cell projections at myoendothelial gap junctions. These data directly demonstrate a critical role for Cx40 in EDHF-mediated dilation of rat mesenteric arteries. Key Words: endothelium-derived hyperpolarizing factor Ⅲ myoendothelial gap junctions Ⅲ endothelium-dependent dilation Ⅲ acetylcholine Ⅲ connexin 40 I n arterioles and some arteries, gap junctions between endothelial and smooth muscle cells (myoendothelial gap junctions [MEGJs]) enable changes in membrane potential to spread over considerable distances and, as a consequence, regulate blood flow by coordinating diameter change through the microcirculation. 1-3 For example, injection of hyperpolarizing current into a single endothelial cell can evoke extensive relaxation involving many smooth muscle cells throughout an isolated arteriole. 4,5 An important aspect of this response is that MEGJs may provide a crucial route for endothelial cell hyperpolarization to spread radially to the adjacent smooth muscle and evoke the dilation attributed to endothelium-derived hyperpolarizing factor (EDHF). 6 -8 In many arteries, there is more than 1 underlying mechanism for endothelium-dependent hyperpolarization of smooth muscle cells. In the rat mesenteric artery, during submaximal contraction to phenylephrine (PE), EDHF dilation appears to reflect hyperpolarizing current spread thro...