Dynamic regulation of [Ca2+](i) by plasma membrane Ca2+-ATPase and Na+/Ca2+ exchange during capacitative Ca2+ entry in bovine vascular endothelial cells

The dynamic regulation of Ca2+ extrusion by the plasma membrane Ca2+-ATPase (PMCA) and Na+/Ca2+ exchange (NCX) was investigated in single cultured cal f pulmonary artery endothelial (CPAE) cells using indo-1 microfluorimetry t o measure cytoplasmic Ca2+ concentration ([Ca2+](i)). The quantitative anal ysis of the recovery from an increase of [Ca2+](i) elicited by activation o f capacitative Ca2+ entry (CCE) served to characterize kinetic parameters o f these Ca2+ extrusion systems in the intact cell. In CPAE cells the PMCA i s activated in a Ca2+- and time-dependent manner. Full activation of the pu mp occurs only after [Ca2+], has been elevated for at least 1 min which res ults in an increase of the affinity of the pump for Ca2+ and an increase in the apparent maximal extrusion rate (V-max). Application of calmodulin ant agonists W-7 and calmidazolium chloride (compound R 24571) revealed that ca lmodulin is a major regulator of PMCA activity in vivo. Sequential and simu ltaneous inhibition of PMCA and NCX suggested that both contribute to Ca2extrusion in a non-additive fashion. The activity of one system is dynamica lly adjusted to compensate for changes in the extrusion rate by the alterna tive transporter. It was concluded that in vascular endothelial cells, the PMCA functions as a calmodulin-regulated, high-affinity Ca2+ removal system . The contribution by the low-affinity NCX to Ca2+ clearance became apparen t at [Ca2+](i) >similar to 150 nM under conditions of submaximal activation of the PMCA.