Figure 1.
MDL-12,330A and SQ 22536 have similar effects on cAMP, but differ in insulin secretion.
A. effects of MDL-12,330A (MDL, 10 µM, n = 6) and SQ 22536 (SQ, 10 µM, n = 6) on intracellular cAMP in the absence or presence of forskolin (FSK, 10 µM ) at 2.8 mM glucose (2.8 G) or 8.3 mM glucose (8.3 G) conditions. B. effects of MDL-12,330A (10 µM, n = 6) and SQ 22536 (10 µM, n = 8) on insulin secretion at 8.3 mM glucose condition. C. effects of H89 (1 µM, n = 8; 5 µM, n = 7; 10 µM, n = 8) and ESI-09 (ESI, 1 µM, n = 6; 5 µM, n = 6; 10 µM, n = 5) on insulin secretion at 8.3 mM glucose condition. **P<0.01, ***P<0.001, †P<0.05.
Figure 2.
MDL-12,330A, but not SQ 22536, produces prolonged APDs.
Pancreatic beta cells were seeded on glass coverslips and cultured in culture dish for 24-clamp experiments. Artificial APs were elicited by applying 4 ms, 150 pA current injections in perforated whole-cell configuration and were recorded before and 2 min after application of reagents in the same cell. Representative action potential waveforms and summary of mean APDs were shown for cells treated with MDL-12,330A (MDL, 10 µM, n = 6), SQ 22536 (SQ, 10 µM, n = 7) or H89 (1 µM n = 7) at 11.1 mM glucose (Ctrl) conditions. **p<0.01.
Figure 3.
MDL-12,330A inhibits KV channels in pancreatic beta cells.
KV currents were recorded in conventional whole-cell configuration from a holding potential of −70 mV to various depolarizing voltages (−70 to 80 mV) as the protocol shown in inset. A. representative current traces recorded under the different treatments as indicated. B. current-voltage relationship curves of KV channels from rat beta cell. C. summary of the mean current density of KV channels recorded at 70 mV depolarization. Control (Ctrl, n = 12); MDL-12,330A (MDL, 10 µM, n = 6); SQ 22536 (SQ, 10 µM, n = 7); H89 (1 µM, n = 7). ***P<0.001.
Figure 4.
MDL-12,330A inhibits KV currents in a dose-dependent manner in rat beta cells.
A. representative time-response curve of KV currents in response to 400 ms voltage steps to +70 mV in the absence and presence of 1, 5, 10, 15 and 30 µM MDL-12,330A (MDL) in the same cell. The holding potential was −70 mV and voltage steps were applied every 3 s in perforated whole-cell configuration. The addition of MDL-12,330A is indicated by the bar with the specific concentrations. B. plot of the fraction of control current blocked (Fraction Blocked) versus MDL-12,330A concentration for all cells tested (n = 5). The continuous line is a fit of the four-parameter logistic equation to the data. Parameters of the fit are: IC50 = 7.22 µM. C and D. representative time-response scatterplot of KV currents in the absence and presence of H89 (n = 7) and ESI-09 (ESI, n = 7).
Figure 5.
MDL-12,330A does not influence currents through voltage-dependent Ca2+ channels.
We employed extracellular Ba2+ to replace Ca2+ as the charge carrier to amplify current responses through Ca2+ channels during the stimulus of depolarization. Ba2+ currents were recorded in conventional whole-cell configuration from a holding potential of −70 mV to various depolarizing voltages (−50 to 20 mV). A. representative current traces recorded under the treatments as indicated. B. current-voltage relationship curves of Ca2+ channels from rat beta cell. C. summary of the mean current density of Ba2+ currents recorded at 0 mV depolarization. Control (Ctrl, n = 6); MDL-12,330A (MDL, 10 µM, n = 6).
Figure 6.
MDL-12,330A influences insulin secretion via KV channels rather than KATP channels in rat pancreatic islets.
All islets were preliminary incubated for 30(2.8 G), then incubated in KRB solution containing 2.8 G or 8.3 G in the presence or absence of the compounds as indicated. Insulin release from these islets was subsequently assayed and the values were normalized to basal secretion at 2.8 G. A. effects of MDL-12,330A on insulin secretion at 2.8 G (MDL 0 µM, n = 6; MDL 5 µM, n = 6; MDL 10 µM, n = 5) and at 8.3 G condition (MDL 0 µM, n = 5; MDL 5 µM, n = 6; MDL 10 µM, n = 5). B. effects of TEA (20 mM) on insulin secretion in the absence or presence of MDL-12,330A (10 µM) at 2.8 G (Ctrl, n = 6; TEA, n = 5; TEA+MDL, n = 5) and at 8.3 G condition (Ctrl, n = 6; TEA, n = 5; TEA+MDL, n = 5). *P<0.05, †P<0.05.
Figure 7.
MDL-12,330A increases [Ca2+]i level.
[Ca2+]i was measured from beta cells loaded with Fluo 4-AM using a laser scanning confocal microscope at room temperature. The ratio of fluorescence change F/F0 was plotted to represent the change in [Ca2+]i levels. A, B. cells were treated with 10 µM MDL-12,330A (MDL, n = 10) in the presence of 8.3 mM glucose (8.3G). C, D. Cells were treated with 10 µM SQ 22536 (SQ, n = 9) in the presence of 8.3 G. E,F. cells were treated with 1 µM H89 (n = 8) in the presence of 8.3 G. As a positive control, Tolbutamide (Tol, 300 µM) was applied after SQ 22536 or H89 treatments.