G. Trube

Opposite effects of tolbutamide and diazoxide on the ATP-dependent K+ channel in mouse pancreatic beta-cells.

The influence of the antidiabetic sulphonylurea tolbutamide on K+ channels of mouse pancreatic β-cells was investigated using different configurations of the patch clamp technique. The dominant channel in resting cells is a K+ channel with a single-channel conductance of 60 pS that is inhibited by intracellular ATP or, in intact cells, by stimulation with glucose. In isolated patches of β-cells membrane, this channel was blocked by tolbutamide (0.1 mM) when applied to either the intracellular or extracellular side of the membrane. The dose-dependence of the tolbutamide-induced block was obtained from whole-cell experiments and revealed that 50% inhibition was attained at approximately 7 μM. In cell-attached patches low concentrations of glucose augmented the action of tolbutamide. Thus, the simultaneous presence of 5 mM glucose and 0.1 mM tolbutamide abolished channel activity and induced action potentials. These were not produced when either of these substances was added alone at these concentrations. The inhibitory action of tolbutamide or glucose on the K+ channel was counteracted by the hyperglycaemic sulphonamide diazoxide (0.4 mM). Tolbutamide (1 mM) did not affect Ca2+-dependent K+ channels. It is concluded that the hypo- and hyperglycaemic properties of tolbutamide and diazoxide reflect their ability to induce the closure or opening, respectively, of ATP-regulated K+ channels.

Glucose dependent K+-channels in pancreaticβ-cells are regulated by intracellular ATP

The resting conductance of culturedβ-cells from murine pancreases was investigated using the whole-cell, cell-attached and isolated patch modes of the patch-clamp technique. Whole-cell experiments revealed a high input resistance of the cells (>20 GΩ per cell or>100 kΩ·cm2), if the medium dialysing the cell interior contained 3 mM ATP. The absence of ATP evoked a large additional K+ conductance. In cell-attached patches single K+-channels were observed in the absence of glucose. Adition of glucose (20 mM) to the bath suppressed the channel activity and initiated action potentials. Similar single-channel currents were recorded from isolated patches. In this case the channels were reversibly blocked by adding ATP (3 mM) to the solution at the intracellular side of the membrane. The conductances (51 pS and 56 pS for [K+]0=145 mM, T=21° C) and kinetics (at −70 mV: τopen=2.2 ms and τclosed=0.38 ms and 0.33 ms) of the glucose- and ATP-dependent channels were found to be very similar. It is concluded that both channels are identical. The result suggests that glucose could depolarize theβ-cell by increasing the cytoplasmic concentration of ATP.

Concentration-dependent effects of tolbutamide, meglitinide, glipizide, glibenclamide and diazoxide on ATP-regulated K+ currents in pancreatic B-cells

SummaryThe influence of the hypoglycemic drugs tolbutamide, meglitinide, glipizide and glibenclamide on ATP-dependent K+ currents of mouse pancreatic B-cells was studied using the whole-cell configuration of the patch-clamp technique. In the absence of albumin, tolbutamide blocked the currents half maximally at 4.1 μmol/l. In the presence of 2 mg/ml albumin half maximal inhibition of the currents was observed at 2.1 μmol/l meglitinide, 6.4 nmol/l glipizide and 4.0 nmol/1 glibenclamide. The hyperglycemic sulfonamide diazoxide opened ATP-dependent K+channels. Half maximally effective concentrations of diazoxide were 20 μmol/l with 0.3 mmol/1 ATPand102 μmol/l with 1 mmol/1 ATP in the recording pipette. Thus, the action of diazoxide was dependent on the presence of ATP in the recording pipette. The free concentrations of the drugs which influenced ATP-dependent K+ currents were comparable with the free plasma concentrations in humans and the free concentrations which affected insulin secretion in vitro. The results support the view that the target for the actions of sulfonylureas and of diazoxide is the ATP-dependent K+ channel of the pancreatic B-cell or a structure closely related to this channel.

Dual effects of ATP on K+ currents of mouse pancreatic β-cells

K+ currents through ATP-dependent channels were recorded from inside-out patches of β-cell membrane as previously described (Rorsman and Trube 1985). Channels were opened by removing ATP from the intracellular side of the membrane. The open probability and/or the number of active channels declined spontaneously (“run-down”) when ATP was absent for periods longer than about 30 s. Channels subject to the run-down could be activated again after applying a blocking concentration (>0.1 mM) of ATP in presence of 1 mM MgCl2 for at least 2 min. ATP in absence of Mg and the ATP-analogues AMP-PNP, AMP-PCP and ATPγS were ineffective in reactivating the channels. This suggests that phosphorylation of the channels or associated proteins of hydrolysis of ATP may be necessary for keeping the channels available. In contrast to the differential effects on the run-down, ATP in presence and absence of Mg and the ATP analogues were similarly effective in blocking the channels at concentrations above 0.1 mM. Using an experimental protocol avoiding the run-down ghe dose-inhibition curve for ATP was found to reach 50% at 18 μM.

Changes of membrane currents in cardiac cells induced by long whole-cell recordings and tolbutamide

Single isolated myocytes were obtained from the ventricles of adult guinea pig hearts. The whole-cell recording configuration of the patch-clamp technique was used to measure membrane currents. A decrease (run-down) of the Ca2+ inward current and an increase of a time-independent K+ outward current were observed during long lasting (1–3 h) recordings. The time at which the outward current developed depended on the intracellular ATP concentration in the pipette, suggesting that this current is identical to the ATP-dependent K+ current described by Noma and Shibasaki (1985). However, the maximum outward current reached in the experiments was independent of the ATP concentration indicating a limited diffusion of ATP in the cell interior. In single-channel experiments on isolated patches of cell membrane and in whole-cell recordings the ATP-dependent K+ current could be blocked by the hypoglycaemic sulphonylurea tolbutamide. The IC50 of 0.38 mM was about 50 times higher than that reported for pancreatic β-cells (Trube et al. 1986). The Ca2+ inward current and the inwardly retifying K+ current were not affected by tolbutamide (3 mM).

Single Ca channel currents in mouse pancreatic B-cells.

Barium currents flowing through single Ca2+ channels were recorded from outside-out patches isolated from mouse pancreatic B-cells. Only one type of Ca2+ channel was observed. In 110 mM Ba2+, the single channel conductance was 24 pS (at negative membrane potentials) and the current amplitude at 0 mV was−0.7 pA. Channel openings were activated by depolarisations more positive than −30 mV and showed little inactivation during 200 ms pulses. Open times were increased by BAY K 8644 an decreased by micromolar Cd2+. Channel activity was subject to rundown in excised patches and little activity remained after 10 min. These properties resemble those of L-type Ca2+ channels in other tissues. It is suggested that this Ca2+ channel participates in the generation of the B-cell action potential and mediates the increase in Ca2+ influx required for insulin secretion.

Forskolin-induced block of delayed rectifying K+ channels in pancreaticβ-cells is not mediated by cAMP

K+ channels in the membrane of murine pancreatic β-cells were studied using the patch-clamp technique. The delayed outward current was activated in whole-cell experiments by depolarizing voltage pulses to potentials between −30 mV and 0 mV. Forskolin blocked the current rapidly (<5 s) and reversibly with 50% inhibition at 13 μM. The inhibition did not depend on a stimulation of the adenylate cyclase since it occurred even in presence of 1 mM cAMP in the pipette solution which replaced the cytoplasm. Membrane permeant cAMP analogues and phosphodiesterase inhibitors did not influence the delayed outward current. In experiments on outside-out patches forskolin (100 μM) shortened the openings of a channel of about 10 pS conductance at 0 mV and a time course of activation and inactivation similar to the whole-cell current. Another smaller, slowly activating channel and the Ca2+- and ATP-dependent K+ channels were influenced only weakly or not at all. It is therefore concluded that the 10-pS channel generates most of the delayed outward K+ current in murine pancreatic β-cells. The Ca2+-independent part of the delayed outward current in bovine adrenal chromaffin cells was also blocked by forskolin (100 μM).

Low resting potentials in single isolated heart cells due to membrane damage by the recording microelectrode

Single myocytes from adult rat hearts were prepared following the method of Powell and co-workers (9, 10, 11). Low resting potentials (Em) could be improved by three techniques. (i) Elevation of Cao to 7.2 mM which, however, mostly resulted in spontaneity and irreversible contracture. (ii) Pre-incubation in a “KB medium” (6). (iii) Use of suction pipettes instead of tapered microelectrodes for intracellular recordings (2). It is concluded that low Em measured previously (11) were due to membrane damage upon microelectrode impalement accopanied by insfufficient healing of the membrane around the electrode insertion.