Endothelial cells respond to bradykinin and other agonists with an increase in cytoplasmic calcium. The calcium comes from two sources. One of these is a release from an internal storage site. The other component is influx from the extracellular fluid through the plasma membrane. In spite of its importance in regulation of intracellular calcium the identity and the characteristics of the influx pathway remain unknown. Recently we have taken advantage of the electrical coupling among endothelial cells to develop a unique multicellular preparation to characterize the ionic pathway underlying the inward calcium current evoked in response to bradykinin. We have used appropriate blocking agents identified in our previous studies to eliminate other ionic currents that might obscure the inward calcium current in whole cell current recordings. Presently, this is the only reported endothelial cell preparation that provides consistent and reproducible electrophysiological responses to agonist. The influx pathway is being characterized for voltage dependency, ionic selectivity, sensitivity to blocking agents and bradykinin concentration. The dependence of the current on second messenger systems including components of the phosphoinositol pathway, the guanine nucleotides, and internal calcium are being examined. The hypothesis that the calcium influx is dependent upon depletion of an intracellular calcium store is being examined. The hypothesis that the calcium influx is dependent upon depletion of an intracellular calcium store is being examined. When the characterization of the pathway is complete its relationship to the bradykinin-induced rise in intracellular calcium will be confirmed using simultaneous measurement of the whole cell current and the intracellular calcium using fura-2. We will proceed to a set of experiments designed to determine, at the single channel level, whether any of the several calcium permeable ion channels that have been previously identified in endothelial cells is responsible for the BK-activated calcium influx. These studies will provide important information on the mechanisms that regulate agonist- induced changes in intracellular calcium and subsequent release of endothelial derived mediators of vascular resistance.
