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.