Modulation of Endothelial Cell KCa3.1 Channels During Endothelium-Derived Hyperpolarizing Factor Signaling in Mesenteric Resistance Arteries

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 2+ ] o but blocked either with forskolin to activate protein kinase A or by limiting smooth muscle [Ca 2+ ] i increases stimulated by phenylephrine depolarization. Imaging [Ca 2+ ] i within the endothelial cell projections forming myoendothelial gap junctions revealed increases in cytoplasmic [Ca 2+ ] i during endothelial stimulation with ACh that were unaffected by simultaneous increases in muscle [Ca 2+ ] 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 + /K + -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 + /K + -ATPase α 2 /α 3 subunits were highly concentrated in endothelial cell projections and adjacent to myoendothelial gap junctions. Thus, extracellular [Ca 2+ ] o appears to modify K Ca 3.1 channel activity through a protein kinase A–dependent mechanism independent of changes in endothelial [Ca 2+ ] i . The resulting hyperpolarization links to arterial relaxation largely through Na + /K + -ATPase, possibly reflecting K + 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 + and myoendothelial coupling evoke EDHF-mediated relaxation through distinct, definable pathways.