Physiological effects of Na+/Ca2+ exchanger knockdown by antisense oligodeoxynucleotides in arterial myocytes.

Antisense oligodeoxynucleotides (AS-oligos) targeted to the Na+/Ca2+ exchanger (NCX) inhibit NCX-mediated Ca2+ influx in mesenteric artery (MA) myocytes [Am. J. Physiol. 269 (Cell Physiol. 38): C1340-C1345, 1995]. Here, we show AS-oligo knockdown of NCX-mediated Ca2+ efflux. In initial experiments, the cytosolic free Ca2+ concentration ([Ca2+]cyt) was raised, and sarcoplasmic reticulum (SR) Ca2+ sequestration was blocked with caffeine and cyclopiazonic acid; the extracellular Na+-dependent (NCX) component of Ca2+ efflux was then selectively inhibited in AS-oligo-treated cells but not in controls (no oligos or nonsense oligos). In contrast, the La3+-sensitive (plasmalemma Ca2+ pump) component of Ca2+ efflux was unaffected in AS-oligo-treated cells. Knockdown of NCX activity was reversed by incubating AS-oligo-treated cells in normal media for 5 days. Transient [Ca2+]cyt elevations evoked by serotonin (5-HT) at 15-min intervals in AS-oligo-treated cells were indistinguishable from those in controls. When cells were stimulated every 3 min, however, the peak amplitudes of the second and third responses were larger, and [Ca2+]cyt returned to baseline more slowly, in AS-oligo-treated cells than in controls. Peak 5-HT-evoked responses in the controls, but not AS-oligo-treated cells, were augmented more than twofold in Na+-free media. This implies that NCX is involved in Na+ gradient modulation of SR Ca2+ stores and cell responsiveness. The repetitive stimulation data suggest that the NCX may be important during tonic activation of arterial myocytes.

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.

Antisense inhibition of Na+/Ca2+ exchange in primary cultured arterial myocytes.

The effects of chimeric phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted against the Na+/Ca2+ exchanger (NCX) were tested in primary cultured rat mesenteric artery myocytes. In parallel cultures, myocytes proliferated and were morphologically normal in the presence of scrambled nonsense (NS-) or AS-oligos or no oligos (controls). NCX function was examined with digital imaging, using fura 2 to estimate the cytosolic free Ca2+ concentration ([Ca2+]cyt). Resting [Ca2+]cyt was higher (145 +/- 4 nM; P < 0.05) in AS-oligo-treated cells than in controls (125 +/- 5 nM) or NS-oligo-treated cells (131 +/- 4 nM). Lowering external Na+, to promote Ca2+ entry via NCX, increased [Ca2+]cyt transiently in controls and NS-oligo-treated cells but not in AS-oligo-treated cells. Raising the cytosolic free Na+ concentration with ouabain augmented the low-Na(+)-induced rise in [Ca2+]cyt in controls and NS-oligo-treated cells, but AS-oligo-treated cells still did not respond. Nevertheless, serotonin (5-HT) increased [Ca2+]cyt in all three groups. Thus AS-oligos selectively blocked NCX activity but not the 5-HT response. To determine the effect of NCX knockdown on the modulation of stored Ca2+, the 5-HT response was tested immediately after removal of external Ca2+. Ouabain augmented the 5-HT-induced rise in [Ca2+]cyt in control and NS-oligo-treated cells but not AS-oligo-treated cells. This indicates that the NCX can modulate intracellular Ca2+ stores. We conclude that AS-oligos are useful for investigating the physiological role of NCX in vascular smooth muscle.