Sulfonylurea-Mediated Stimulation of Insulin Exocytosis via an ATP-Sensitive K+ Channel–Independent Action
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Figures
- FIG. 1.
Photoaffinity labeling by [3H]-glibenclamide in the granule membrane fraction (GR; black curve) and plasma membrane fraction (PM; gray curve) of normal mouse islets. In the granule membrane fraction, a 65-kd protein is labeled, whereas in the plasma membrane fraction, a 140-kd protein with the expected molecular weight (Mr) for SUR-1 was detected. Radioactivity is plotted as fraction of peak radioactivity (599 cpm for PM and 1,109 cpm for GR). Granule protein (60 μg) and plasma membrane protein (73 μg) were equilibrated with 100 nmol/l [3H]-glibenclamide for 1 h and ultraviolet irradiated (3 min, 312 nm). Membrane protein from granule or plasma membrane fractions were separated by SDS-PAGE on 7.5% acrylamide gels, and radioactivity measured in a liquid scintillation counter. Using a low concentration of [3H]-glibenclamide (20 nmol/l), the granular 65-kd sulfonylurea receptor was inconsistently labeled, suggesting that it has a lower affinity than that of SUR-1. These experiments were performed using methods described in Braun et al. (19).
- FIG. 2.
A: Deprotonation of the granule interior estimated as the relative decrease (in percentage terms) of the initial LysoSensor-fluorescence intensity [(F0-F)/F0] after establishment of the standard whole-cell configuration. A standard Ca2+- and ATP-containing intracellular solution was used and supplemented with carbonyl cyanide m-chlorophenylhydrazone (CCCP) (ctrl; black, n = 14); ADP and CCCP (ADP; gray, n = 9); tolbutamide, ADP, and CCCP (tolb; gray, n = 10); diazoxide and CCCP (dz; black, n = 7); 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and CCCP (DIDS; black, n = 5); and anti-ClC-3, a functional antibody directed against the ClC-3 channel, and CCCP (anti-ClC-3; gray, n = 9). The arrow indicates the establishment of the standard whole-cell configuration. B: Average decrease (percentage of initial) in Lysosensor fluorescence after 60 s recording [(F0-F)/F0]. Data are means ± SE. Statistical significances were evaluated comparing the responses in the respective groups with the responses obtained with the control solution including CCCP. C: Increases in cell capacitance (ΔC) under control conditions (ctrl; black, n = 26), after addition of Mg-ADP (ADP; gray, n = 12), tolbutamide and Mg-ADP (tolb; gray, n = 12), diazoxide (dz; black, n = 19), or DIDS (black, n = 8). D: Average rate of changes in cell capacitance (ΔC/Δt) ± SE. **P < 0.01; ***P < 0.001. Data are modified from Barg et al. (14), where the methodology is described in detail.
- FIG. 3.
Model for the sulfonylurea-mediated stimulation of Ca2+-dependent insulin secretion. Sulfonylurea binding to a 65-kd receptor in the granule membrane (g-SUR) activates granular ClC-3 Cl− channels (ClC-3). The ClC-3 channels act in concert with the v-type H+-ATPase in the granule membrane to promote acidification of the granule interior, which is essential for the insulin granule to gain release competence. The exact nature of the interaction between g-SUR and the ClC-3 channel remains to be established. This putative g-SUR/ClC-3 channel complex is, however, under metabolic control and is activated when the ATP/ADP ratio is high. By analogy to the KATP channel, it seems likely that the inhibitory actions of ADP and diazoxide are mediated via g-SUR, whereas 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) acts directly on the ClC-3 channel. The sulfonylurea-mediated stimulation of granule acidification, and in turn exocytosis, then result from perturbation of the ADP-mediated actions.
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