Contribution of Na/Ca exchange to Ca2+ outflow and entry in the rat pancreatic beta-cell: studies with antisense oligonucleotides.

Abstract

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.

Inhibition of Mitochondrial Na+-Ca2+ Exchanger Increases Mitochondrial Metabolism and Potentiates Glucose-Stimulated Insulin Secretion in Rat Pancreatic Islets

“…To avoid cholinergic effects, the Na ϩ -free buffer contained atropine (10 mol/l). Cytosolic calcium ([Ca 2ϩ ] i ) was calculated as described previously (39). KB-R7943 (Tocris, Ballwin, MO), an inhibitor of the NCX, was used as a positive control.…”

Plasma Membrane Ca2+-ATPase Overexpression Reduces Ca2+ Oscillations and Increases Insulin Release Induced by Glucose in Insulin-Secreting BRIN-BD11 Cells

“…Fura-2 fluorescence of single cells was measured by dual-excitation fluorimetry using a camera-based image analysis system (Magical; Applied Imaging, Sunderland, U.K.). The excitation and emission wavelengths were set at 340/380 and 510 nm, respectively, and a pair of ratioable images (at the excitations of 340 and 380 nm, 30-ms interval) were taken every 2.5 s. [Ca 2ϩ ] i was calculated from the ratios of the 340-and 380-nm signals as previously described (28).…”

The suppressor of cytokine signalling 2 (SOCS2) is a key repressor of insulin secretion

“…Fura-2 fluorescence of single islets was measured by dual excitation fluorimetry using a camerabased image analysis system (Metafluor, Universal Imaging). Data are presented as the ratios of the 340 and 380 nm signals as described [34]. …”