To characterize the role played by Na/Ca exchange in the pancreatic beta-cell, phosphorothioated antisense oligonucleotides (AS-oligos) were used to knock down the exchanger in rat pancreatic beta-cells. Na/Ca exchange activity was evaluated by measuring cytosolic free Ca2+ concentration ([Ca2+]i) in single cells using fura-2. Exposure of beta-cells to 500 nmol/l of the AS-oligos for 24 h inhibited Na/Ca exchange activity by approximately 77%. In contrast, control oligonucleotides (scrambled and mismatched) did not affect Na/Ca exchange activity. In AS-oligo-treated cells, the increase in [Ca2+]i induced by membrane depolarization (K+ or the hypoglycemic sulfonylurea, tolbutamide) was reduced by 28 or 40%, respectively. Likewise, the rate of [Ca2+]i decrease after K+ or tolbutamide removal was reduced by 72 or 40%, respectively. AS-oligos treatment also abolished the nifedipine-resistant increase in [Ca2+]i induced by K+ and profoundly altered the oscillatory or sustained increases in [Ca2+]i induced by 11.1 mmol/l glucose. The present study shows that AS-oligos may specifically inhibit Na/Ca exchange in rat pancreatic beta-cells. In those cells, Na/Ca exchange appears to mediate Ca2+ entry in response to membrane depolarization and to be responsible for up to 70% of Ca2+ removal from the cytoplasm upon membrane repolarization.

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