Modulation of endothelial cell KCa3.1-channels during EDHF signaling in mesenteric resistance arteries

摘要

Arterial hyperpolarization to ACh reflects co-activation of KCa3.1 (IKCa)-channels and KCa2.3 (SKCa)-channels in the endothelium, that transfers through myoendothelial gap junctions (MEGJs) and diffusible factor(s) to affect smooth muscle relaxation (EDHF response). However, ACh can differentially activate KCa3.1 and KCa2.3-channels and we investigated the mechanisms responsible in rat mesenteric arteries. KCa3.1-channel input to EDHF-hyperpolarization was enhanced by reducing external [Ca²⁺]o, but blocked either with forskolin to activate PKA or by limiting smooth muscle [Ca²⁺]i increases stimulated by phenylephrine (PE) depolarization. Imaging [Ca²⁺]i within the endothelial cell projections forming MEGJs, revealed increases in cytoplasmic [Ca²⁺]i during endothelial stimulation with ACh that were unaffected by simultaneous increases in muscle [Ca²⁺]i evoked by PE. If gap junctions were uncoupled, KCa3.1-channels became the predominant input to EDHF-hyperpolarization, and relaxation was inhibited with ouabain implicating a crucial link through Na⁺/K⁺-ATPase. There was no evidence for an equivalent link through KCa2.3-channels, nor between these channels and the putative EDHF-pathway involving natriuretic peptide receptor-C (NPR-C). Reconstruction of confocal z-stack images from pressurized arteries revealed KCa2.3-immunostain at endothelial cell borders, including endothelial cell projections, while KCa3.1-channels and Na⁺/K⁺-ATPase α₂/α₃-subunits were highly concentrated in endothelial cell projections and adjacent to MEGJs. Thus, extracellular [Ca²⁺]_o appears to modify K_Ca3.1-channel activity through a PKA-dependent mechanism independent of changes in endothelial [Ca²⁺]_i. The resulting hyperpolarization links to arterial relaxation largely through Na⁺/K⁺-ATPase, possibly reflecting K⁺ acting as an EDHF. In contrast, K_Ca2.3-hyperpolarization appears mainly to affect relaxation through MEGJs. Overall, these data suggest that K⁺ and myoendothelial coupling evoke EDHF-mediated relaxation through distinct, definable pathways.