Modulation of Endothelial Cell K _Ca 3.1 Channels During Endothelium-Derived Hyperpolarizing Factor Signaling in Mesenteric Resistance Arteries

Abstract

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 ...

Recycling of the Ca2+-activated K+ Channel, KCa2.3, Is Dependent upon RME-1, Rab35/EPI64C, and an N-terminal Domain

“…However, mounting evidence indicates that the hyperpolarization induced in the endothelial cells by Ca 2ϩ -mediated agonists, and hence activation of KCa channels, is directly communicated to the smooth muscle cells via myoendothelial gap junctions (10 -12). Indeed, uncoupling of the gap junctions attenuated the acetylcholine-induced hyperpolarization of the underlying smooth muscle, consistent with a direct current flow between cells (10). Recent evidence, using double KCa2.3/ KCa3.1 knock-out mice, demonstrates that KCa3.1 deficiency attenuates the acetylcholine-induced, EDHF-mediated vasodilation, whereas KCa2.3 knock-out impairs the NO-mediated dilation induced by acetylcholine (9).…”

“…3 and KCa3.1 are perfectly positioned to play a role in the EDHF response as outlined above, i.e. KCa3.1 has been shown to be localized primarily to the myoendothelial gap junctions, whereas KCa2.3 is more uniformly distributed across the endothelial cell surface, including at the myoendothelial gap junctions (10,14).…”

“…Additionally, KCa3.1 appears to play a critical role in the propagation of the acetylcholine-induced dilation along the arterioles (13). A second, perhaps parallel role for KCa channels is to conduct K ϩ ions into the intracellular space resulting in an activation of the Na ϩ /K ϩ -ATPase and ultimately vascular relaxation (10). Recent immunolocalization studies have suggested that KCa2.…”

Low intravascular pressure activates endothelial cell TRPV4 channels, local Ca 2+ events, and IK Ca channels, reducing arteriolar tone

“…Further, this control mechanism is part of a membrane-signaling microdomain located within MEJs. These EC projections are a focus for IP 3 R-mediated Ca 2+ events, and this Ca 2+ will generate SMC hyperpolarization predominantly by activation of the EC IK Ca channels densely expressed within this microdomain (1,5,7).…”

“…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 )-mediated Ca 2+ events, discovered in unpressurized arteries and termed Ca 2+ pulsars (7).…”