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Na⁺/Ca²⁺ Exchanger in Porcine Oocytes¹

^a Alexion Pharmaceuticals, Inc., Cheshire, Connecticut 06410 ^b Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211-5300

The presence of the Na⁺/Ca²⁺ exchange mechanism was investigated in porcine oocytes. Immature and in vitro-matured oocytes were loaded with the Ca²⁺-sensitive fluorescent dye fura 2 and changes in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) were monitored after altering the Na⁺ concentration gradient across the plasma membrane. Decreasing the extracellular Na⁺ concentration induced an increase in [Ca²⁺]_i possibly by a Ca²⁺ influx via the Na⁺/Ca²⁺ exchanger. A similar Ca²⁺ 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 [Ca²⁺]_i was due to Ca²⁺ influx since it was abolished in the absence of extracellular Ca²⁺, and the increase was mediated by the Na⁺/Ca²⁺ exchanger since it was blocked by the application of amiloride or bepridil, inhibitors of Na⁺/Ca²⁺ exchange. Verapamil (50 µM) and pimozide (50 µM), inhibitors of L- and T-type voltage-gated Ca²⁺ channels, respectively, could not block the Ca²⁺ influx. The Ca²⁺ entry via the Na⁺/Ca²⁺ exchanger could not induce the release of cortical granules and did not stimulate the resumption of meiosis. This was unexpected because Ca²⁺ is thought to be a universal trigger for activation. Using antibodies raised against the exchanger, it was demonstrated that the Na⁺/Ca²⁺ 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⁺/Ca²⁺ exchanger.

¹ This material is based on work supported by the Cooperative State Research, Education and Extension Service, U.S. Department of Agriculture, under agreement 99-35203-7675.

² Correspondence: Zoltán Macháty, Columbus Farming Corporation, P.O. Box 1160, Sherburne, NY 13460. FAX: 607 674 6309; machatyz{at}columbusfarming.com

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