Na(+)/Ca(2+) exchanger in porcine oocytes.

Abstract

The presence of the Na(+)/Ca(2+) exchange mechanism was investigated in porcine oocytes. Immature and in vitro-matured oocytes were loaded with the Ca(2+)-sensitive fluorescent dye fura 2 and changes in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) were monitored after altering the Na(+) concentration gradient across the plasma membrane. Decreasing the extracellular Na(+) concentration induced an increase in [Ca(2+)](i) possibly by a Ca(2+) influx via the Na(+)/Ca(2+) exchanger. A similar Ca(2+) influx could also be triggered after increasing the intracellular Na(+) concentration by incubation in the presence of ouabain (0.4 mM), a Na(+)/K(+)-ATPase inhibitor. The increase in the [Ca(2+)](i) was due to Ca(2+) influx since it was abolished in the absence of extracellular Ca(2+), and the increase was mediated by the Na(+)/Ca(2+) exchanger since it was blocked by the application of amiloride or bepridil, inhibitors of Na(+)/Ca(2+) exchange. Verapamil (50 micro M) and pimozide (50 micro M), inhibitors of L- and T-type voltage-gated Ca(2+) channels, respectively, could not block the Ca(2+) influx. The Ca(2+) entry via the Na(+)/Ca(2+) exchanger could not induce the release of cortical granules and did not stimulate the resumption of meiosis. This was unexpected because Ca(2+) is thought to be a universal trigger for activation. Using antibodies raised against the exchanger, it was demonstrated that the Na(+)/Ca(2+) exchanger was localized predominantly in the plasma membrane. Reverse transcription-polymerase chain reaction revealed that porcine oocytes contain a transcript that shows 98.1% homology to the NACA3 isoform of the porcine Na(+)/Ca(2+) exchanger.