Calcium-Release-Activated Calcium Influx in Endothelium

Abstract

Signaling pathways activated by the tachykinin substance P (SP) were investigated in pig coronary artery endothelial cells (PCAECs). Single cells were obtained after enzymatic digestion of coronary arteries. Intracellular Ca²⁺ ([Ca²⁺]i) was measured from fura-2 fluorescence while membrane potential or ionic current was measured using patch-clamp techniques. In physiological saline solution, SP induced hyperpolarizations or outward currents which coincided with biphasic [Ca²⁺]i increases representing store release of Ca²⁺ and Ca²⁺ influx. Single channel recording protocols showed that both sources of Ca²⁺ activated a small conductance K⁺ channel, resulting in cell hyperpolarization. When outward currents were blocked by d-tubocurare, Cs⁺, or BAP-TA, an inward current was unmasked. Ion substitution protocols showed that the SP-induced inward current was (1) carried by a mixture of Ca²⁺ and Na⁺, (2) blocked by La³⁺, and (3) inactivated by high extracellular [Ca²⁺]. Tyrosine kinase inhibitors also blocked the inward current. The same current was activated by bath application of BHQ, an inhibitor of the endoplasmic reticulum Ca²⁺ ATPase, or by cell dialysis with IP3. These results suggest that the plateau phase of the agonist-activated [Ca²⁺]i increase in PCAECs reflects Ca²⁺ entry through a depletion-activated Ca²⁺ channel. The characteristics of this channel are compared to those of Ca²⁺ channels found in other nonexcitable cells.