CALCIUM-RELEASE-ACTIVATED CALCIUM INFLUX IN ENDOTHELIUM

Signaling pathways activated by the tachykinin substance P (SP) were i nvestigated in pig coronary artery endothelial cells (PCAECs). Single cells ere obtained after enzymatic digestion of coronary arteries. Int racellular Ca2+ ([Ca2+](i)) was measured from fura-2 fluorescence whil e membrane potential or ionic current was measured using patch-clamp t echniques. In physiological saline solution, SP induced hyperpolarizat ions or outward currents which coincided with biphasic [Ca2+](i) incre ases representing store release of Ca2+ and Ca2+ influx. Single channe l recording protocols showed that both sources of Ca2+ activated a sma ll conductance K+ channel, resulting in cell hyperpolarization. When o utward currents were blocked by d-tubocurare. Cs+, or BAP-TA, an inwar d 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 La-3, and (3) inactivated by high extracellular [Ca2+]. Tyrosine kinase inhibitors also blocked the inward current. The same c urrent was acti- ated 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-activa ted Ca2+ channel. The characteristics of this channel are compared to those of Ca2+ channels found in other nonexcitable cells.