Dual-phase response of bovine pulmonary artery endothelial cells to agonists which increase free cytoplasmic calcium concentration

The regulation of free cytoplasmic calcium concentration (Ca2+]i) was studied in bovine pulmonary artery endothelial cells (BPAEC). The cells were seeded on the inner surface of glass cuvettes, grown to confluency and loaded with INDO-1. Using a multiwavelength method for estimation of Ca2+]i it was shown that in Ca2+ containing medium a rapid rise of Ca2+]i occurs in response to bradykinin, ATP or thrombin followed by a much slower decrease in free cytoplasmic calcium. Binding of extracellular Ca2+ by EGTA lowered basal Ca2+]i but had no effect on the rate of agonist-induced Ca2+]i increase or its absolute amount. In contrast, the kinetics of Ca2+]i decrease were entirely different. A rapid (less than 0.5 min) decrease in Ca2+]i to the basal level was observed immediately after the maximum had been achieved. If excess Ca2+ was added to the medium after EGTA, a second Ca2+]i rise in response to the agonists occurred. The decrease in Ca2+]i after the second peak was several times slower than the decrease in Ca2+ free medium. It is concluded that Ca2+ entry from the external medium had no effect on the maximal increase in Ca2+]i but provides a severalfold increase in the duration the endothelial cell responses to the agonists.