Calcitonin gene-related peptide hyperpolarizes mouse pulmonary artery endothelial tubes through K_ATP channel...

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, ∼150 µm diameter) with intact or denuded endothelium were cannulated and pressurized to 16 cmH₂O at 37°C. Increasing concentrations (10^-10-10^-6 M) of CGRP progressively dilated PAs preconstricted with UTP (10^-5 M); SMCs hyperpolarized similarly (Δ V_m ∼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 K_ATP channel agonist, resulted in progressive hyperpolarization of SMCs of intact PAs (Δ V_m ∼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.