Na+/Ca2+ exchange in neonatal rat heart cells: Antisense inhibition and protein half-life

Martin K. Slodzinski, Mordecai P. Blaustein

Research output: Contribution to journalArticlepeer-review

Abstract

Cardiac Na+/Ca2+ exchanger (NCX) protein half-life (t( 1/4 )) and antisense knockdown were studied in primary cultured neonatal rat cardiomyocytes. Protein t( 1/4 ) was determined using [35S]methionine with a pulse-chase protocol. The 35S signal in NCX was identified by immunoprecipitation and Western blotting. The t( 1/4 ) of NCX protein was 33 h. Low concentrations (0.5 μM) 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, ~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 ~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 ~60% of cells.

Original languageEnglish (US)
Pages (from-to)C459-C467
JournalAmerican Journal of Physiology - Cell Physiology
Volume275
Issue number2 44-2
DOIs
StatePublished - Aug 1998

Keywords

  • Caffeine
  • Cyclopiazonic acid
  • Oligodeoxynucleotides
  • Potassium depolarization
  • Sarcoplasmic reticulum

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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