In non-excitable cells, many agonists increase the intracellular Ca2+ concentration ([Ca2+]i) by inducing an inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the intracellular stores. Ca2+ influx from the extracellular medium may then sustain the Ca2+ signal. [Ca2+]i recovers its resting level as a consequence of Ca2+-removing mechanisms, i.e. plasma-membrane Ca2+-ATPase (PMCA) pump, Na+/Ca2+ exchanger (NCX) and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump. In a study performed in pancreatic acinar cells, evidence has been provided suggesting that, during the decay phase of the agonist-evoked Ca2+ transients, the Ca2+ concentration within the intracellular stores remains essentially constant [Mogami, Tepikin and Petersen (1998) EMBO J. 17, 435–442]. It was therefore hypothesized that, in such a situation, intracellular Ca2+ is not only picked up by the SERCA pump, but is also newly released through IP3-sensitive Ca2+ channels, with the balance between these two processes being approximately null. The main aim of the present work was to test this hypothesis by a different experimental approach. Using cardiac microvascular endothelial cells, we found that inhibition of the SERCA pump has no effect on the time course of agonist-evoked Ca2+ transients. This result was not due to a low capacity of the SERCA pump since, after agonist removal, this pump proved to be very powerful in clearing the excess of intracellular Ca2+. We showed further that: (i) in order to avoid a rapid removal of Ca2+ by the SERCA pump, continuous IP3 production appears to be required throughout all of the decay phase of the Ca2+ transient; and (ii) Ca2+ picked up by the SERCA pump can be fully and immediately released by agonist application. All these results support the model of Mogami, Tepikin and Petersen [(1998) EMBO J. 17, 435–442]. Since the SERCA pump did not appear to be involved in shaping the decay phase of the agonist-evoked Ca2+ transient, we inhibited the PMCA pump with carboxyeosin, and NCX with benzamil and by removing extracellular Na+. The results indicate that, during the decay phase of the agonist-evoked Ca2+ transient, the intracellular Ca2+ is removed by both the PMCA pump and NCX. Finally, we provide evidence indicating that mitochondria have no role in clearing intracellular Ca2+ during agonist-evoked Ca2+ transients.
Abbreviations used: [Ca2+]i, intracellular Ca2+ concentration; IP3, inositol 1,4,5-trisphosphate; PMCA, plasma-membrane Ca2+-ATPase; NCX, Na+/Ca2+ exchanger; SERCA, sarco-endoplasmic reticulum Ca2+-ATPase; EC, endothelial cell; CMEC, cardiac microvascular EC; DMEM, Dulbecco's modified Eagle's medium; HBSS, Hanks balanced salt solution; PSS, physiological salt solution; CPA, cyclopiazonic acid; PLC, phospholipase C; PKC, protein kinase C; CCCP, carbonyl cyanide m-chlorophenylhydrazone; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.