Na(+)/Ca(2+) exchanger in porcine oocytes.
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.
Macháty
Zoltán
Z
Alexion Pharmaceuticals, Inc., Cheshire, Connecticut 06410, USA. machatyz@columbusfarming.com
Ramsoondar
Jagdeece J
JJ
Bonk
Aaron J
AJ
Prather
Randall S
RS
Bondioli
Kenneth R
KR
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D015764:Bepridil / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes; D016257:Fura-2; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009865:Oocytes / Q000737:chemistry* / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D010868:Pimozide / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017385:Sequence Homology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D013552:Swine; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D019831:Sodium-Calcium Exchanger; D010868:Pimozide; D010042:Ouabain; D015764:Bepridil; D000584:Amiloride; D012964:Sodium; D014700:Verapamil; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium; D016257:Fura-2