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.
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000819:agonists / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008638:Mesenteric Arteries / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology*; D051381:Rats; D019831:Sodium-Calcium Exchanger
D002352:Carrier Proteins; D016376:Oligonucleotides, Antisense; D019831:Sodium-Calcium Exchanger; D002118:Calcium