Modulation of Substance P-Induced K⁺Current in Coronary Endothelium

Substance P (SP) activates an outward K+ current, membrane hyperpolarization and increases cytosolic Ca2+ ([Ca2+],) in porcine coronary artery endothelial cells (PCAECs). We tested the importance of Ca2+, CAMP, cGMP, arachidonic acid and G protein-linked pathways in modulating whole-cell K+ currents activated by SP. Current or membrane potential and [Ca2+], were measured simultaneously in single cells in response to SP (10 nM), using the perforated patch technique and fura-2 microfluorimetry. Partial block of Kcachannels by D-tubocurarine resulted in a significant reduction in SP-induced membrane hyperpolarization and the plateau phase of the cell [Ca2+]i response. In PCAECs preloaded with BAPTA, SP failed to elevate [Ca2+]i or activate outward current. In Ca2+-free bath solution, SP induced transient elevations in [Ca2+]i and outward current. With SP still present, addition of 1 mM Ca2+ to the bath after [Ca2+]i returned to resting levels elevated [Ca2+]i and elicited outward current. Acute application of forskolin (20 pM), a CAMP activator, 8-bromo-cGMP (100 pM), a membrane-permeant analog of cGMP or arachidonic acid (200 pM) did not significantly affect the magnitude or time-course of SP-induced K+ current. Open probability (P,) of single K+ channels was measured in inside-out patches as a function of bath [Ca2+]. P0 increased in a sigmoidal fashion with [Calf] but the relation was not significantly altered by GTP (200 pM), GDPPS (500 pM) or GTPyS (200 1M). These results suggest that SP-induced K+ current and subsequent hyperpolarization in PCAECs are important for regulation of Ca2+ influx and can be triggered by Ca2+ release from stores or by extracellular Ca2+ entry. In addition, K+ channel activity in PCAECs is primarily modulated by changes in [Ca2+], but not by CAMP, cGMP or arachidonic acid pathways and is not directly linked to a G protein.