AJP - Cell Physiology, Vol 269, Issue 5 C1340-C1345, Copyright © 1995 by American Physiological Society

ARTICLES

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

M. K. Slodzinski, M. Juhaszova and M. P. Blaustein
Department of Physiology, University of Maryland School of Medicine, Baltimore 21201, USA.

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.

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