The cardiac NCX protein half-life and the effects of chimeric phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted against the cardiac and arterial Na - Ca2+ exchanger (NCX) were studied in rat myocytes. Low concentrations (0.5 muM) of chimeric, phosphorothioated AS-oligos targeted to the NCX1 transcript were used to knock down NCX protein and activity. Control cardiac myocytes incubated without oligos or with scrambled (NS-) oligos exhibited spontaneous Ca2+ transients (measured with fura-2). The cytosolic free Ca2+ concentration ([Ca2+]i) rose in control cells when external Na (Nao) was removed. In contrast, 60% of cells treated with AS-oligos did not exhibit spontaneous Ca2+ transients or respond to Na-free medium. In all cells, 50 mM K increased [Ca2+]i. NCX protein was reduced by 50% in cells treated with AS-oligos for 7 days, but was not reduced after only 2 days. The biochemical and protein half-life are consistent with the physiological evidence of about 60% knock down of cardiac NCX activity after 4 days. Arterial myocytes proliferated normally in the presence of NS-, AS-, or no oligos, without detectable quantitative or morphological differences. Reduction of Nao to 5 mM, which promotes the Nai-dependent component of Ca2+ influx caused [Ca2+]i to rise in controls and in NS-oligo treated cells, but not in AS-oligos treated cells. Incubation with 1 mM ouabain to raise Nai augmented Nai-dependent component of Ca2+ influx in controls and NS-oligo treated cells, but the AS-oligo treated cells still did not respond with a rise in [Ca2+]i. Nevertheless, all three groups of cells exhibited increases in [Ca2+]i in response to 10 muM serotonin (5-HT). With La3+ inhibition of the PMCA pump and Na-free media (to inhibit NCX), CAF and CPA elevated [Ca2+]i; the Nao-dependent component of Ca2+ efflux was selectively inhibited by AS-oligos, but not in sense (S-), or NS-oligos treated cells or controls. The knock down of NCX activity in arterial myocytes was reversible. The La3+-sensitive (PMCA pump) component of Ca2+ efflux was unaffected in AS-oligos treated cells. The transient [Ca2+]i elevations by 5-HT (10 muM, 30 s) at low frequency (15 min) in AS-oligo treated cells were no different from those in controls and NS-oligos treated cells in terms of [Ca2+]i amplitude, and rates of rise and recovery. [Ca2+]i recovery in AS-oligo treated cells was markedly slowed when the PMCA pump was inhibited by La3+. When cells were stimulated at higher frequency (3 min), however, the peak amplitudes of the 2nd and 3rd responses were larger, and [Ca2+]cyt returned to baseline more slowly in AS-oligo treated cells than controls. Peak 5-HT evoked responses in controls, but not AS-oligo treated cells, were augmented >2-fold in Na-free media. The response to 5-HT was tested immediately after removal of external Ca2+ before and after 10 min incubation with 1 muM ouabain. Ouabain significantly augmented the 5-HT induced rise in [Ca2+]cyt in controls, but had no effect on AS-oligos treated cells. The latter observation implies that the arterial myocytes NCX is involved in Na+ gradient modulation of SR Ca2+ stores and cell responsiveness, while the results of the repetitive stimulation experiments suggest that the NCX may be important during tonic activation.