Na+/Ca2+ exchange in neonatal rat heart cells: antisense inhibition and protein half-life.
Cardiac Na+/Ca2+ exchanger (NCX) protein half-life (t1/2) and antisense knockdown were studied in primary cultured neonatal rat cardiomyocytes. Protein t1/2 was determined using [35S]methionine with a pulse-chase protocol. The 35S signal in NCX was identified by immunoprecipitation and Western blotting. The t1/2 of NCX protein was 33 h. Low concentrations (0.5 microM) of chimeric, phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted to the region around the start codon of NCX1 transcript were used to knock down NCX protein and activity. Control myocytes (no oligos or scrambled oligos for at least 4 days) exhibited spontaneous Ca2+ transients (measured with fura 2). The sustained ("diastolic") Ca2+ concentration in the cytosol ([Ca2+]cyt) of control cells was unaffected by cyclopiazonic acid (CPA) plus caffeine (Caf), which promote depletion of sarcoplasmic reticular Ca2+ stores, but [Ca2+]cyt rose in control cells when external Na+ was removed. In contrast, approximately 60% of cells treated with AS-oligos for at least 4 days did not exhibit spontaneous Ca2+ transients or respond to Na+-free medium; however, CPA + Caf did induce a prolonged elevation in [Ca2+]cyt in these cells. In all cells, 50 mM K+ increased [Ca2+]cyt. NCX protein was reduced by approximately 50% in cells treated with AS-oligos for 7 days but was not reduced after only 2 days. These biochemical data are consistent with the physiological evidence of NCX knockdown in approximately 60% of cells.
Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
M K
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Slodzinski
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Blaustein
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003062:Codon; D003593:Cytoplasm / Q000378:metabolism; D006352:Heart Ventricles; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors
D003062:Codon; D016376:Oligonucleotides, Antisense; D019831:Sodium-Calcium Exchanger; D013873:Thionucleotides; C110374:sodium-calcium exchanger 1; D012964:Sodium; D002118:Calcium