Antisense inhibition of Na+/Ca2+ exchange in primary cultured arterial myocytes
Slodzinski, M.K.; Juhaszova, M.; Blaustein, M.P.
American Journal of Physiology 269(5 Pt 1): C1340-C1345
1995
ISSN/ISBN: 0002-9513 PMID: 7491927 Document Number: 453177
The effects of chimeric phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted against the Na+/Ca-2+ 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 Ca-2+ concentration ((Ca-2+)-cyt). Resting (Ca-2+)-cyt was higher (145 +- 4 nM; P lt 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 Ca-2+ entry via NCX, increased (Ca-2+)-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 (Ca-2+)-cyt in controls and NS-oligo-treated cells, but AS-oligo-treated cells still did not respond. Nevertheless, serotonin (5-HT) increased (Ca-2+)-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 Ca-2+, the 5-HT response was tested immediately after removal of external Ca-2+. Ouabain augmented the 5-HT-induced rise in (Ca-2+)-cyt in control and NS-oligo-treated cells but not AS-oligo-treated cells. This indicates that the NCX can modulate intracellular Ca-2+ stores. We conclude that AS-oligos are useful for investigating the physiological role of NCX in vascular smooth muscle.