Role of high-voltage-activated calcium channels in glucose-regulated beta-cell calcium homeostasis and insulin release.

High-voltage-activated (HVA) calcium channels are known to be the primary source of calcium for glucose-stimulated insulin secretion. However, few studies have investigated how these channels can be regulated by chronically elevated levels of glucose. In the present study, we determined the level of expression of the four major HVA calcium channels (N-type, P/Q-type, L(C)-type, and L(D)-type) in rat pancreatic beta-cells. Using quantitative real-time PCR (QRT-PCR), we found the expression of all four HVA genes in rat insulinoma cells (INS-1) and in primary isolated rat islet cells. We then determined the role of each channel in insulin secretion by using channel-selective antagonists. Insulin secretion analysis revealed that N- and L-type channels are both involved in immediate glucose-induced insulin secretion. However, L-type was preferentially coupled to secretion at later time points. P/Q-type channels were not found to play a role in insulin secretion at any stage. It was also found that long-term exposure to elevated glucose increases basal calcium in these cells. Interestingly, chronically elevated glucose decreased the mRNA expression of the channels involved with insulin secretion and diminished the level of stimulated calcium influx in these cells. Using whole cell patch clamp, we found that N- and L-type channel currents increase gradually subsequent to lower intracellular calcium perfusion, suggesting that these channels may be regulated by glucose-induced changes in calcium.

Characterization of the inhibitory effects of erythromycin and clarithromycin on the HERG potassium channel.

Both erythromycin and clarithromycin have been reported to cause QT prolongation and the cardiac arrhythmia torsade de pointes in humans, however direct evidence documenting that these drugs produce this effect by blocking human cardiac ion channels is lacking. The goal of this study was to test the hypothesis that these macrolide antibiotics significantly block the delayed rectifier current (IKr) encoded by HERG (the human ether-a-go-go-related gene) at drug concentrations, temperature and ionic conditions mimicking those occurring in human subjects. Potassium currents in HEK 293 cells stably transfected with HERG were recorded using a whole cell voltage clamp method. Exposure of cells to erythromycin reduced the HERG encoded potassium current in a concentration dependent manner with an IC50 of 38.9 +/- 1.2 microM and Hill Slope factor of 0.4 +/- 0.1. Clarithromycin produced a similar concentration-dependent block with an IC50 of 45.7 +/- 1.1 microM and Hill Slope factor of 1.0 +/- 0.1. Erythromycin (25-250 microM) and clarithromycin (5 or 25 microM) also produced a significant decrease in the integral of the current evoked by an action potential shaped voltage clamp protocol. The results of this study document that both erythromycin and clarithromycin significantly inhibit the HERG potassium current at clinically relevant concentrations.