Calcitonin gene-related peptide hyperpolarizes mouse pulmonary artery endothelial tubes through KATP channel activation

The sensory neurotransmitter calcitonin gene-related peptide (CGRP) is associated with vasodilation of systemic arteries through activation of ATP-sensitive K+ (K-ATP) channels in smooth muscle cells (SMCs); however, its effects on endothelial cell (EC) membrane potential (V-m) are unresolved. In pulmonary arteries (PAs) of C57BL/6J mice, we questioned whether CGRP would hyperpolarize ECs as well as SMCs. Intact PAs were isolated and immunostained for CGRP to confirm sensory innervation; vessel segments (1-2 mm long, similar to 150 mu m diameter) with intact or denuded endothelium were cannulated and pressurized to 16 cmH(2)O at 37 degrees C. Increasing concentrations (10(-10)-10(-6) M) of CGRP progressively dilated PAs preconstricted with UTP (10(-5) M); SMCs hyperpolarized similarly (Delta Vm similar to 20 mV) before and after endothelial denudation. To study native intact PA ECs, SMCs were dissociated to isolate endothelial tubes, and their integrity was confirmed by vital dye uptake, nuclear staining, and reproducible electrical and intracellular Ca2+ responses to acetylcholine (10(-5) M) over 2 h. Increasing [CGRP] hyperpolarized ECs in a manner similar to SMCs, with each cell layer demonstrating robust immunostaining for CGRP receptor proteins. Increasing concentrations (10(-10)-10(-6) M) of pinacidil, a KATP channel agonist, resulted in progressive hyperpolarization of SMCs of intact PAs (Delta Vm similar to 30 mV), which was blocked by glibenclamide (10(-6) M), as was hyperpolarization of ECs and SMCs to CGRP. Inhibition of protein kinase A with protein kinase inhibitor (10(-5) M) also inhibited hyperpolarization to CGRP. We demonstrate [CGRP]-dependent hyperpolarization of ECs for the first time while validating freshly isolated PA endothelial tubes as an experimental model. Redundant electrical signaling to CGRP in ECs and SMCs implies an integral role for K-ATP channels in PA dilation.