Effects of sulphonylureas and diazoxide on insulin secretion and nucleotide‐sensitive channels in an insulin‐secreting cell line

Abstract

1 The effects of various sulphonylureas and diazoxide on insulin secretion and the activity of various channels have been studied using tissue culture and patch-clamp methods in an insulinsecreting cell line derived from a rat islet cell tumour. 2 Tolbutamide, glibenclamide and HB699 increased the rate of insulin release by 2-5 fold. The concentrations of tolbutamide and glibenclamide giving half-maximum effects on insulin secretion were approximately 40pM and 0.2pm, respectively. 3 Diazoxide (0.6-1.0mM) per se, had either no effect or produced a small increase in insulin secretion, whereas when secretion was maximally stimulated by the combination of glucose (3 mM) and leucine (20mM), it produced inhibition. Tolbutamide-induced release was also inhibited by diazoxide. 4 Tolbutamide, glibenclamide, HB699 and HB985 reduced the open-state probability of the ATP-K+ channel in a dose-dependent manner. Tolbutamide and glibenclamide were shown to be effective regardless of which side of the membrane they were applied. 5 In whole cell recording, in which the total ATP-sensitive K+ conductance of the cell could be measured, dose-inhibition curves for tolbutamide and glibenclamide were constructed, resulting in Ki values of 17pM and 27nm, respectively. The value of Ki for tolbutamide was unchanged when ATP (0.1 mM) was present in the electrode. 6 Diazoxide (0.6mM) activated the ATP-K+ channels only when they had first been inhibited by intracellular ATP (0.1 mM) or bath applied tolbutamide (3-30 pM). The inhibition produced by glibenclamide could not be reversed by diazoxide. 7 Neither tolbutamide (1.0mM) nor glibenclamide (10 M) altered the open-state probability of the Ca2 + -activated K+ channel or the Ca2 + -activated non-selective cation channel which are present in this cell line. 8 It is concluded that the sulphonylureas and related hypoglycaemic drugs and diazoxide regulate insulin secretion by direct effects on the ATP-K+ channel or a protein closely associated with this channel.

Ligustrazine selectively blocks ATP‐sensitive K+ channels in mouse pancreatic β cells

“…Fig.2 shows that 0.74 mM ligustrazine inhibited K-ATP currents (by 34.2 -t 1.3070, mean f S.E.M., n = 8 cells). The response to ligustrazine was much slower than that to tolbutamide, but similar to that of the more potent sulphonylurea glibenclamide [7]. Doubling the dose of ligustrazine to 1.48 mM did not significantly change the percentage inhibition (32.4 + 1.3%; n = 6; P>O.d, unpaired Student's t-test) suggesting that the lower dose is maximal.…”

“…This effect was observed both during the continued presence of ligustrazine ( fig.3A) and also after its removal when the recovery of K-ATP currents was incomplete ( fig.3B). This latter finding suggests that the failure of K-ATP currents to recover after ligustrazine was not due to their running-down, as d&oxide does not reactivate channels subject to rundown [7].…”

“…This variability appeared to be independent of the duration of its application. D&oxide is a potent activator of K-ATP channels which reverses the blocking effect of tolbutamide [2,7]. Fig.3 demonstrates that bath application of 0.4 mM diazoxide rapidly reversed the effect of ligustrazine, evoking a current which was in many cases greater than the control level.…”

“…We measured K-ATP currents as the response to 10 mV depolarizing voltage steps (200 ms duration, 0.5 Hz) from a holding potential of -70 mV: an experimental protocol which measures only current through K-ATP channels [2,7]. Characteristic features of this current were: (i) On first obtaining the whole-cell configuration the currents were small but then increased in amplitude (run-up) as intracellular ATP was dialysed from the cell, reflecting the relief of ATP inhibition of K-ATP channels (fig,lA).…”

“…A reduction of the negative holding current accompanied this run-up. (ii) The application to the bath of 0.2 mM tolbutamide, a specific and potent blocker of K-ATP channels [2,7], resulted in rapid, reversible inhibition of this conductance and an increase in the holding current ( fig.lB). The rate at which tolbutamide inhibited K-ATP currents reflected the rate of exchange of the bath solutions.…”