The ATP- and tolbutamide-sensitivity of the ATP-sensitive K-channel from human pancreatic B cells

Abstract

Summary. The ATP-and sulphonylurea-sensitivity of the ATP-sensitive K-channel was measured in human pancreatic B cells. In inside-out patches, half-maximal inhibition of channel activity was produced by 10 p~mol/l ATP (with 2 mM Mg 2+) and ATP-inhibition was partially antagonised by ADP. A significantly lower sensitivity to ATP was found in whole-cell recordings. Tolbutamide inhibited whole-cell ATPsensitive K-currents half-maximally at 18 ~tmol/l; the sensitivity to tolbutamide ~vas somewhat less in the inside-out patch.Ca-activated K-channels were unaffected by tolbutamide (10 mmol/1). These results resemble those found for rodent B cells and suggest that sulphonylureas exert their therapeutic effects in Type 2 (non-insulin dependent) diabetes by inhibition of the ATP-sensitive K-channel.Key words: ATP-sensitive K-channel, pancreatic B cell, human islet, sulphonylurea, tolbutamide, insulin secretion.The activity of the ATP-sensitive K-channel is thought to play a central role in the physiological regulation of insulin secretion from the pancreatic B-cell [1,2]. Studies of rodent B cells and B cell lines have led to the proposal that an increase in the cytosolic ATP/ADP ratio, resulting from glucose metabolism, inhibits channel activity. This produces membrane depolarization and precipitates a chain of events that culminates in insulin secretion.The channel is also the target for the hypoglycaemic sulphonylureas used clinically in the treatment of Type 2 (non-insulin-dependent) diabetes mellitus. These drugs inhibit channel activity in rodent B cells [3][4][5][6][7] and thereby stimulate electrical activity and insulin release [8]. It has therefore been suggested that a defect in the regulation of the ATP-sensitive K-channel may be involved in the initial pathology of Type 2 diabetes [9].Information about the properties of this channel in man is scarce. We have shown previously that the normal human B-cell possesses a K-channel inhibited by both ATP and the sulphonylurea, tolbutamide [9]. A similar channel is also found in human insulinoma ceils [10]. In this paper we present a detailed analysis of the properties of the ATP-sensitive K-channel of the human B cell. Our results indicate that the rodent B cell constitutes a good model for that of the human and provide support for the idea that the therapeutic action of tolbutamide in man is mediated via inhibition of the ATP-sensitive K-channel. Materials and methods PreparationHuman pancreata were removed (with permission) from nondiabetic, heart-beating cadaw.~r organ donors and islets of Langerhans isolated under aseptic conditions using a method similar to that described previously [11]. "[here were 2 male donors aged 27 and 37, and 3 female donors aged 19, 55 and 58. In brief, the pancreatic duct was cannulated and the pancreas distended with 2 ml/g prewarmed collagenase (6 mg/ml) in Iq[ank's solution. The pancreas was incubated in a water bath at 39~ for 25 rain then cooled rapidly in ice cold Hank's containing 20 mmol/1 HEPES and 5 g/1 bovine serum albumin....

Nutrient Control of Insulin Secretion in Isolated Normal Human Islets

“…Basal, peak first-phase (5-8 min), and second-phase (21-30 min) insulin secretion averaged 0.008 Ϯ 0.001, 0.147 Ϯ 0.020 (ϳ18-fold increase), and 0.078 Ϯ 0.013% per min (ϳ10-fold increase). The addition of diazoxide, an effective opener of K ATP channels in human ␤-cells (29), suppressed glucose-induced insulin secretion, whereas subsequent closure of the channels by tolbutamide (29,30) completely reversed the inhibition. The addition of forskolin augmented the secretory rate within 5 min.…”

“…Figure 3 shows that tolbutamide, purportedly used at the high concentration of 500 mol/l to block all K ATP channels (29,30), and 30 mmol/l KCl, used to depo- larize ␤-cells despite opening of K ATP channels by diazoxide (31), rapidly increased insulin secretion in 1 mmol/l glucose. Subsequent stimulation with 15 mmol/l glucose further increased the secretory rate.…”

“…First, the critical role of K ATP channels is convincingly demonstrated by the abrogation of insulin secretion with diazoxide and its reversible restoration with tolbutamide. Patch-clamp studies have shown that K ATP channels in human ␤-cells are similar to those in rodent ␤-cells (29,30). Second, the K ATP channel-independent amplifying pathway is also functional.…”

Tolbutamide excites rat glucoreceptive ventromedial hypothallamic neurones by indirect inhibition of ATP‐K+ channels

“…A reduction in the effectiveness of tolbutamide at inhibiting ATP-K' channel activity in inside-out patches compared to whole-cell currents has been demonstrated for mouse (Zunkler et al, 1988) and human (Ashcroft et al, 1989) pancreatic fl-cells. It appears likely that this discrepancy is due to the absence of cytosolic ADP in the excised patch, as Zunkler et al (1988) have shown that this nucleotide enhances the channel sensitivity to tolbutamide.…”

P2Y-Purinoceptor Mediated Inhibition of L-type Ca2+ Channels in Rat Pancreatic .BETA.-Cells.

“…In cell-attached experiments for recording of action currents, the pipettes were filled with an extracellular solution composed of (mM): KCl 140; CaCl2 2; and HEPES 5.0 (pH was adjusted to 7.4 with KOH). In experiments on whole-cell configuration for recording of K + channel currents, the pipette solution contained (mM): KCl 50; K2SO4 35; MgCl2 3.0; EGTA 11; CaCl2 1; HEPES 11 (pH 7.2 adjusted with KOH) and 0.1 mM ATP plus 0.1 mM ADP were added to record the ATP-sensitive K + channel currents (Ashcroft et al, 1989). In experiments on the whole-cell configuration for recording of Ca 2+ channel currents under the condition of inhibition of both K + and Na + currents, the bath solution was (mM): NaCl 125; KCl 4.8; CaCl2 10; MgCl2 1.2; tetraethylammonium chloride (TEA-Cl) 10; HEPES 5.0; tetrodotoxin (TTX) 0.3 mM (pH was adjusted to 7.4 with NaOH) and the pipette solution contained (mM): MgCl2 4; EGTA 10; ATP 3; GTP 0.1; HEPES 5 and CsCl 125 (pH was adjusted to 7.2 with CsOH).…”