Desensitization of insulin secretory response to imidazolines, tolbutamide, and quinine. I. Secretory and morphological studies.

The desensitization of pancreatic B-cells against stimulation by insulin secretagogues that inhibit ATP-dependent K(+) channels (K(ATP) channels) was investigated by measuring insulin secretion of perifused pancreatic islets. Additionally, the islet insulin content and the number of secretory granules per B-cell were determined. Prior to the measurement of secretion, islets were cultured for 18 h in the presence or absence of the test agents in a cell-culture medium containing 5 mM glucose. The effects of three imidazolines, phentolamine, alinidine, and idazoxan (100 microM each) were compared with those of the well-characterized sulfonylurea, tolbutamide (500 microM), and those of the ion channel-blocking alkaloid, quinine (100 microM). Insulin secretion was strongly reduced upon re-exposure to phentolamine, alinidine, tolbutamide, and quinine, whereas idazoxan, which stimulated secretion only weakly, had no significant effect. The imidazoline secretagogues phentolamine and alinidine induced a cross-desensitization against the stimulatory effect of tolbutamide and quinine. A long-term depolarization with 40 mM KCl was also able to induce a significant reduction of the secretory response to all of the above secretagogues. The insulin content of cultured islets was moderately, but significantly reduced by alinidine, whereas the reduction by phentolamine, tolbutamide, and quinine was not significant. In contrast to these observations, the ultrastructural examination revealed that tolbutamide-treated B-cells had a high degree of degranulation, whereas the other test agents and 40 mM KCl produced only a partial degranulation, except for phentolamine, which produced no significant degranulation at all. These results suggest that the desensitization of insulin secretion is a common property of all agents that stimulate insulin secretion by depolarisation of the plasma membrane. Depending on the specific secretagogue, additional mechanisms, proximal and distal to Ca(2+) influx, appear to contribute to the desensitization (see Rustenbeck et al., pages 1695-1703, this issue).     

Desensitization of insulin secretory response to imidazolines, tolbutamide, and quinine. II. Electrophysiological and fluorimetric studies.

Prolonged in vitro exposure (18 h) of pancreatic islets to insulin secretagogues that block ATP-dependent K(+) channels (K(ATP) channels), such as sulfonylureas, imidazolines, and quinine, induced a desensitization of insulin secretion (Rustenbeck et al., pages 1685-1694, this issue). To elucidate the underlying mechanisms, K(ATP) channel activity, plasma membrane potential and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) were measured in mouse single B-cells. In B-cells desensitized by phentolamine or quinine (100 microM each) K(ATP) channel activity was virtually absent and could not be elicited by diazoxide. Desensitization by alinidine (100 microM) induced a marked reduction of K(ATP) channel activity, which could be reversed by diazoxide, whereas exposure to idazoxan (100 microM) or tolbutamide (500 microM) had no lasting effect on K(ATP) channel activity. Correspondingly, phentolamine-, alinidine-, and quinine-desensitized B-cells were markedly depolarized, whereas B-cells that had been exposed to tolbutamide or idazoxan had an unchanged resting membrane potential. The increase in [Ca(2+)](i) normally elicited by phentolamine and alinidine was suppressed after desensitization by these compounds, whereas the [Ca(2+)](i) increase by re-exposure to quinine was markedly reduced and that by tolbutamide only minimally affected as compared with control-cultured B-cells. The increase in [Ca(2+)](i) elicited by a K(+) depolarization was diminished in secretagogue-pretreated B-cells, the extent depending on the secretagogue. This effect was closely correlated with the degree of depolarization after pretreatment with the respective secretagogue. In conclusion, the apparently uniform desensitization of secretion by K(ATP) channel blockers is due to different effects at two stages located distally in the stimulus-secretion coupling: either at the stage of [Ca(2+)](i) regulation, where the increase is depressed as a consequence of a persistent depolarization (e.g. in the case of phentolamine or alinidine) and/or at the stage of exocytosis, which responds only weakly to substantial increases in [Ca(2+)](i) (in the case of tolbutamide).     

Antagonism of the insulinotropic action of first generation imidazolines by openers of K(ATP) channels

The antagonism between K(ATP) channel-blocking insulinotropic imidazolines - phentolamine, alinidine, idazoxan and efaroxan - and K(ATP) channel openers, diazoxide and nucleoside diphosphates, was studied in mouse pancreatic islets and B-cells. In inside-out patches from B-cells, 500muM MgGDP abolished the inhibitory effect of the imidazolines. 300muM diazoxide further increased channel activity. The depolarizing effect of all imidazolines (100muM) on the B-cell membrane potential was practically completely antagonized by 300muM diazoxide. In contrast, diazoxide was unable to decrease the cytosolic Ca(2+) concentration ([Ca(2+)](i)) which was elevated by phentolamine, whereas the [Ca(2+)](i) increases induced by the other imidazolines were promptly antagonized. The effects on [Ca(2+)](i) were reflected by the secretory activity in that the stimulatory effects of alinidine, idazoxan and efaroxan, but not that of phentolamine were antagonized by diazoxide. Metabolic inhibition of intact B-cells by 250muM NaCN, most likely by a decrease of the ATP/ADP ratio, significantly diminished the K(ATP) channel-blocking effect of a low concentration of alinidine (10muM), whereas efaroxan proved to be susceptible even at a highly effective concentration (100muM). This may explain the oscillatory pattern of the [Ca(2+)](i) increase typically produced by efaroxan in pancreatic B-cells. In conclusion, the inhibitory effect of imidazolines on K(ATP) channels, which is exerted at the pore-forming subunit, Kir6.2, is susceptible to the action of endogenous and exogenous K(ATP) channel openers acting at the regulatory subunit SUR, which confers tissue specificity. With intact cells this antagonism can be obscured, possibly by intracellular accumulation of some imidazolines.     

Heterogeneous characteristics of imidazoline-induced insulin secretion

Imidazolines are regarded as a pharmacological group of insulin secretagogues with one uniform mechanism of action, namely closure of ATP-dependent K⁺ channels (K_ATP channels) and, in consequence, depolarization of the plasma membrane, Ca²⁺ influx and stimulation of secretion. This assumption was investigated by measuring insulin secretion from perifused pancreatic islets in response to three imidazoline compounds and comparing the characteristics of secretion with changes in membrane potential and cytosolic Ca²⁺ concentration [Ca²⁺]_i of single β-cells. Phentolamine (32 μM) stimulated insulin secretion from perifused mouse islets in the presence of stimulatory (10 mM and 30 mM) and substimulatory (5 mM) glucose concentrations and even in the absence of glucose. Idazoxan in concentrations up to 500 μM was virtually ineffective in the presence of 5 mM glucose. At 10 mM glucose, there was a moderate but significant increase of secretion by idazoxan, 20 μM being nearly as effective as 100 μM. The effect of phentolamine was of slow onset and irreversible in the time frame of the experiments, while the effect of idazoxan was of fast onset and reversible. Alinidine also stimulated secretion in the presence of 10 mM glucose with fast and reversible kinetics, but in contrast to idazoxan, 100 μM was clearly more effective than 20 μM. These heterogeneous characteristics of secretion were reflected by changes of [Ca²⁺]_i: the increase of [Ca²⁺]_i by phentolamine was slow and only partially reversible, whereas idazoxan led to a smaller, but faster and reversible response. The increase of [Ca²⁺]_i by phentolamine and idazoxan was abolished by the Ca²⁺ channel blocker D 600. Surprisingly, all three compounds depolarized the β-cell plasma membrane from a resting potential of –71 mV to about –36 mV. Again, the effect of phentolamine was slow and that of idazoxan and alinidine fast. Thus, the characteristics of phentolamine-induced secretion appear to be attributable to the consequences of K_ATP channel closure. It is unclear, however, why all three test compounds achieved the same degree of depolarization in spite of their known different efficiency to close K_ATP channels. Apparently, there are additional mechanisms involved in the action of idazoxan and alinidine, which may contribute to the obvious differences in the characteristics of secretion. Received: 2 October 1998 / Accepted: 21 December 1998     

Effects of imidazoline binding site ligands on the growth and viability of clonal pancreatic β-cells

Pancreatic beta-cells express imidazoline binding sites which play a role in the regulation of insulin secretion, but it is not known whether ligands for these sites also affect other aspects of beta-cell physiology. In the present study, we have investigated the effects of a range of imidazoline reagents on the growth and viability of clonal pancreatic beta-cells (RINm5F and HIT-T15). Three imidazoline compounds (idazoxan, phentolamine and antazoline) were found to cause marked inhibition of beta-cell growth in a time and concentration dependent manner. Idazoxan was the most potent of these with as little as 0.5 microM causing a significant decrease in beta-cell viability (EC50 approximately 10 microM). All three imidazolines also decreased the viability of clonal beta-cells in parallel with their inhibitory effects on cell growth. These effects were not reproduced by any of a wide-range of other imidazoline compounds, including effective insulin secretagogues such as efaroxan and RX821002. The effects of the three ligands did not correlate with their relative potencies for binding to any of the well-characterised imidazoline binding sites nor to alpha2-adrenoceptors. In addition, the inhibitory responses were not antagonised by other imidazoline binding site ligands. The inhibitory effects of idazoxan on the growth of RINm5F and HIT-T15 beta-cells required as little as 3-h exposure to the imidazoline and were not readily reversible when the reagent was removed. Reductions in growth rate were accompanied by marked alterations in the morphology of the cells, which could be detected before loss of viability. Cells exposed to phentolamine showed the characteristic features of apoptosis in that the nuclei were condensed (as judged by acridine orange staining) and electrophoresis of DNA revealed the presence of oligonucleosomal fragmentation. These changes could not be detected in cells exposed to idazoxan despite the more profound reduction in viability induced by this agent. We conclude that a sub-group of imidazoline compounds can exert profoundly detrimental effects on the growth and viability of clonal beta-cells but that these effects do not correlate with their binding affinity at imidazoline binding sites or alpha2-adrenoceptors.     

Pulses of external ATP aid to the synchronization of pancreatic beta-cells by generating premature Ca(2+) oscillations

Pancreatic beta-cells respond to glucose stimulation with increase of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), manifested as membrane-derived slow oscillations sometimes superimposed with transients of intracellular origin. The effect of external ATP on the oscillatory Ca(2+) signal for pulsatile insulin release was studied by digital imaging of fura-2 loaded beta-cells and small aggregates isolated from islets of ob/ob-mice. Addition of ATP (0.01-100 microM) to media containing 20mM glucose temporarily synchronized the [Ca(2+)](i) rhythmicity in the absence of cell contact by eliciting premature oscillations. External ATP triggered premature [Ca(2+)](i) oscillations also when the sarcoendoplasmic reticulum Ca(2+)-ATPase was inhibited with 50 microM cyclopiazonic acid and phospholipase C inhibited with 10 microM U-73122. The effect of ATP was mimicked by other activators of cytoplasmic phospholipase A(2) (10nM acetylcholine, 0.1-1 micro M of the C-terminal octapeptide of cholecystokinin and 2 microg/ml melittin) and suppressed by an inhibitor of the enzyme (50 microM p-amylcinnamoylanthranilic acid). Premature oscillations generated by pulses of ATP sometimes triggered subsequent oscillations. However, prolonged exposure to high concentrations of the nucleotide (10-100 microM) had a suppressive action on the beta-cell rhythmicity. The early effects of ATP included generation of transients induced by inositol (1,4,5) trisphosphate and superimposed on the premature oscillation or on an ordinary oscillation induced by glucose. The results support the idea that purinergic activation of phospholipase A(2) has a co-ordinating effect on the beta-cell rhythmicity by triggering premature [Ca(2+)](i) oscillations mediated by closure of ATP-sensitive K(+) channels.     

Elevation of cytosolic calcium by imidazolines in mouse islets of Langerhans: implications for stimulus-response coupling of insulin release.

1. Microfluorimetry techniques with fura-2 were used to characterize the effects of efaroxan (200 microM), phenotolamine (200-500 microM) and idazoxan (200-500 microM) on the intracellular free Ca2+ concentration ([Ca2+]i) in mouse isolated islets of Langerhans. 2. The imidazoline receptor agonists efaroxan and phentolamine consistently elevated cytosolic Ca2+ by mechanisms that were dependent upon Ca2+ influx across the plasma membrane; there was no rise in [Ca2+]i when Ca2+ was removed from outside of the islets and diazoxide (100-250 microM) attenuated the responses. 3. Modulation of cytosolic [Ca2+]i by efaroxan and phentolamine was augmented by glucose (5-10 mM) which both potentiated the magnitude of the response and reduced the onset time of imidazoline-induced rises in [Ca2+]i. 4. Efaroxan- and phentolamine-evoked increases in [Ca2+]i were unaffected by overnight pretreatment of islets with the imidazolines. Idazoxan failed to increase [Ca2+]i under any experimental condition tested. 5. The putative endogenous ligand of imidazoline receptors, agmatine (1 microM-1 mM), blocked KATP channels in isolated patches of beta-cell membrane, but effects upon [Ca2+]i could not be further investigated since agmatine disrupts fura-2 fluorescence. 6. In conclusion, the present study shows that imidazolines will evoke rises in [Ca2+]i in intact islets, and this provides an explanation to account for the previously described effects of imidazolines on KATP channels, the cell membrane potential and insulin secretion in pancreatic beta-cells.     

Inhibition of ATP-sensitive K+ channels by taurine through a benzamido-binding site on sulfonylurea receptor 1

ATP-sensitive potassium (K(ATP)) channels in pancreatic beta-cells comprise sulfonylurea receptor (SUR) 1 and inwardly-rectifying potassium channel (Kir) 6.2 subunits. We have evaluated the effect of intracellular taurine on K(ATP) channel activity in rat pancreatic beta-cells using the patch-clamp technique. The mechanism of taurine action was also examined using recombinant K(ATP) channels. The islets and single beta-cells from male Sprague-Dawley rats were collected by collagenase digestion technique. Single K(ATP) channel currents were recorded by the inside-out mode at a membrane potential of -60mV. Cytosolic free-Ca(2+) concentration ([Ca(2+)](c)) and insulin secretory capacity were measured by the dual-excitation fluorimetry and radioimmunoassay, respectively. The native beta-cell K(ATP) channel was directly inhibited by taurine in a dose-dependent manner. Taurine did not influence ATP-mediated inhibition or MgADP-induced activation of the channel activity. The sensitivity of the K(ATP) channel to glybenclamide, but not gliclazide, was enhanced by taurine. Glybenclamide elicited a greater increase in [Ca(2+)](c) and increased insulin secretion in the beta-cells when pretreated with taurine. Taurine did not inhibit Kir6.2DeltaC36 currents, a truncated form of Kir6.2, expressed in Xenopus oocytes without SUR. These results demonstrate that taurine inhibits the K(ATP) channel activity in the beta-cells, interacting with a benzamido-binding site on SUR1, but not Kir6.2.     

Desensitization of insulin secretion

Desensitization of insulin secretion describes a reversible state of decreased secretory responsiveness of the pancreatic beta-cell, induced by a prolonged exposure to a multitude of stimuli. These include the main physiological stimulator, glucose, but also other nutrients like free fatty acids and practically all pharmacological stimulators acting by depolarization and Ca2+ influx into the beta-cell. Desensitization of insulin secretion appears to be an important step in the manifestation of type 2 diabetes and in the secondary failure of oral antidiabetic treatment. In this commentary, the basic concepts and the controversial issues in the field will be outlined. With regard to glucose-induced desensitization, two fundamentally opposing concepts have emerged. The first is that desensitization is the consequence of functional changes in the beta-cell that impair glucose-recognition. The second is that long-term increased secretory activity leads to a depletion of releasable insulin, often in spite of increased insulin synthesis. The latter concept is more appropriately termed beta-cell exhaustion. The same dichotomy applies to the desensitization evoked by pharmacological stimuli: again the relative contributions of a decreased insulin content versus alterations in signal transduction are in dispute. The action of tolbutamide on beta-cells may be an example of desensitization caused by a lack of releasable insulin since the signaling mechanisms are nearly unchanged, whereas the action of phentolamine, an imidazoline, induces a strong desensitization without reducing insulin content or secretory granules, apparently by abolishing Ca2+ influx. With pharmacological agents it seems that both, alterations in signal transduction and decreased availability of releasable insulin, can contribute to the desensitized state of the beta-cell, the relative contribution being variable depending upon the exact nature of the secretory stimulus.     

Stimulation of insulin secretion by glucose in the absence of diminished potassium (86Rb+) permeability.

Two inhibitors of the nucleotide-sensitive K+ (KATP) channel, tolbutamide and quinine, were utilized in order to assess the role of this channel in glucose-stimulated insulin release from perifused rat islets. In the absence of these drugs, the addition of 15 mM glucose elicited a marked biphasic stimulation of insulin secretion concomitant with a reduction in the rate of 86Rb+ efflux. In the presence of either 500 microM tolbutamide or 100 microM quinine, a reduced rate of efflux of 86Rb+ was observed together with an elevated rate of insulin release. Under such conditions, the addition of 15 mM glucose retained the ability to stimulate insulin secretion though this was associated with a marked increase in 86Rb+ efflux. It is concluded that a net reduction in beta-cell K+ permeability is not an obligatory step in glucose-stimulated insulin release. Thus, glucose is likely to exert depolarizing actions on the beta-cell in addition to the closure of K+ channels.     

Inhibition of ATP-sensitive K(+)-channels by a sulfonylurea analogue with a phosphate group

Hypoglycaemic sulfonylureas initiate insulin secretion by direct inhibition of ATP-sensitive K(+)-channels in the pancreatic beta-cells. These channels are composed of two proteins, a pore-forming subunit (K(IR)6.2 in the case of beta-cells) and a regulatory subunit, the sulfonylurea receptor (SUR). In the present study we characterised the interaction with SURs of the new sulfonylurea analogues 5-chloro-N-[2-(4-hydroxyphenyl)ethyl]-2-methoxybenzamide (compound I) and (4-[2-(5-chloro-2-methoxybenzamido)ethyl]phenyl)phosphate (compound II). Compounds I and II differ from the sulfonylurea analogue meglitinide only in so far as the carboxylic group of meglitinide is replaced by a hydroxyl group or a phosphate group, respectively. The binding affinities of compound II for the SUR subtypes SUR1 (identified in beta-cells) and SUR2A (identified in heart and skeletal muscle) were higher by 55 or 21-fold, respectively, than the corresponding affinities for compound I. In inside-out patch-clamp experiments compound II inhibited ATP-sensitive K(+)-channels of the SUR1/K(IR)6.2-type (characteristic of beta-cells) with an IC(50) value of 0.16 microM which is 6-fold lower than the corresponding value for meglitinide. These findings strongly support the conclusion that the interaction of sulfonylureas and acidic analogues with SURs is favoured by the anionic group of these drugs and that a phosphate group allows more efficient ligand interaction with SUR1 than a carboxylic group.     

Fluorescence microscopy studies with a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic beta-cell ATP-sensitive K+ currents

Hypoglycemic sulfonylureas (e.g. tolbutamide, glibenclamide) exert their stimulatory effects on pancreatic beta-cells by closure of ATP-sensitive K(+) (K(ATP)) channels. Pancreatic K(ATP) channels are composed of two subunits, a pore-forming inwardly rectifying K(+) channel (Kir6.2) subunit and a regulatory subunit (the sulfonylurea receptor of subtype 1 (SUR1)) in a (SUR1/Kir6.2)(4) stoichiometry. The aim of the present study was to characterize the interaction of green-fluorescent 3-[3-(4,4 difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacen-3-yl)propanamido] glibenclamide (Bodipy-glibenclamide) with pancreatic beta-cell K(ATP) channels using patch-clamp and fluorescence microscopy techniques. Bodipy-glibenclamide inhibited K(ATP) currents from the clonal insulinoma cell line RINm5F half-maximally at a concentration of 0.6nM. Using laser-scanning confocal microscopy Bodipy-glibenclamide was shown to induce a diffuse fluorescence across the RINm5F cell, but only about 17% of total Bodipy-glibenclamide-induced fluorescence intensity in RINm5F cells was due to specific binding to SUR1. Using fluorescence correlation spectroscopy, it could be demonstrated that the fluorescence label contributes to the protein binding and, therefore, possibly also to the non-specific binding of Bodipy-glibenclamide observed in RINm5F cells. Specific binding of Bodipy-glibenclamide to SUR1 in RINm5F cells might be localized to different intracellular structures (nuclear envelope, endoplasmic reticulum, Golgi compartment, insulin secretory granules) as well as to the plasma membrane. In conclusion, Bodipy-glibenclamide is a high-affinity blocker of pancreatic beta-cell K(ATP) currents and can be used for visualizing SUR1 in intact pancreatic beta-cells, although non-specific binding must be taken into account in confocal microscopy experiments on intact beta-cells.     

Effects of the beta-carbolines, harmane and pinoline, on insulin secretion from isolated human islets of Langerhans

It is well known that certain imidazoline compounds can stimulate insulin secretion and this has been attributed to the activation of imidazoline I(3) binding sites in the pancreatic beta-cell. Recently, it has been proposed that beta-carbolines may be endogenous ligands having activity at imidazoline sites and we have, therefore, studied the effects of beta-carbolines on insulin secretion. The beta-carbolines harmane, norharmane and pinoline increased insulin secretion two- to threefold from isolated human islets of Langerhans. The effects of harmane and pinoline were dose-dependent (EC(50): 5 and 25 microM, respectively) and these agents also blocked the inhibitory effects of the potassium channel agonist, diazoxide, on glucose-induced insulin release. Stimulation of insulin secretion by harmane was glucose-dependent but, unlike the imidazoline I(3) receptor agonist efaroxan, it increased the rate of insulin release beyond that elicited by 20 mM glucose (20 mM glucose alone: 253+/-34% vs. basal; 20 mM glucose plus 100 microM harmane: 327+/-15%; P<0.01). Stimulation of insulin secretion by harmane was attenuated by the imidazoline I(3) receptor antagonist KU14R (2 (2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole) and was reduced when islets were treated with efaroxan for 18 h, prior to the addition of harmane. The results reveal that beta-carbolines can potentiate the rate of insulin secretion from human islets and suggest that these agents may be useful prototypes for the development of novel insulin secretagogues.     

Activation of BKCa channels via cyclic AMP- and cyclic GMP-dependent protein kinases by eugenosedin-A in rat basilar artery myocytes

BACKGROUND AND PURPOSE: The study investigated whether eugenosedin-A, a 5-hydroxytryptamine and alpha/beta adrenoceptor antagonist, enhanced delayed-rectifier potassium (K(DR))- or large-conductance Ca(2+)-activated potassium (BK(Ca))-channel activity in basilar artery myocytes through cyclic AMP/GMP-dependent and -independent protein kinases. EXPERIMENTAL APPROACH: Cerebral smooth muscle cells (SMCs) were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K(+)- and Ca(2+)-channel activities. KEY RESULTS: Eugenosedin-A (1 microM) did not affect the K(DR) current but dramatically augmented BK(Ca) channel activity in a concentration-dependent manner. Increased BK(Ca) current was abolished by charybdotoxin (ChTX, 0.1 microM) or iberiotoxin (IbTX, 0.1 microM), but not affected by a small-conductance K(Ca) blocker (apamin, 100 microM). BK(Ca) current activation by eugenosedin-A was significantly inhibited by an adenylate cyclase inhibitor (SQ 22536, 10 microM), a soluble guanylate cyclase inhibitor (ODQ, 10 microM), competitive antagonists of cAMP and cGMP (Rp-cAMP, 100 microM and Rp-cGMP, 100 microM), and cAMP- and cGMP-dependent protein kinase inhibitors (KT5720, 0.3 microM and KT5823, 0.3 microM). Eugenosedin-A reversed the inhibition of BK(Ca) current induced by the protein kinase C activator, phorbol myristyl acetate (PMA, 0.1 microM). Eugenosedin-A also prevented BK(Ca) current inhibition induced by adding PMA, KT5720 and KT5823. Moreover, eugenosedin-A reduced the amplitude of voltage-dependent L-type Ca(2+) current (I(Ca,L)), but without modifying the voltage-dependence of the current. CONCLUSIONS AND IMPLICATIONS: Eugenosedin-A enhanced BK(Ca) currents by stimulating the activity of cyclic nucleotide-dependent protein kinases. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and thereby relax cerebral SMCs.     

Effects of COX-2 inhibition on spinal nociception: the role of endocannabinoids

Background and purpose:

ATP-sensitive potassium channels (K_ATP channels) in beta cells are a major target for insulinotropic drugs. Here, we studied the effects of selected stimulatory and inhibitory pharmacological agents in islets lacking K_ATP channels.

Experimental approach:

We compared insulin secretion (IS) and cytosolic calcium ([Ca²⁺]_c) changes in islets isolated from control mice and mice lacking sulphonylurea receptor1 (SUR1), and thus K_ATP channels in their beta cells (Sur1KO).

Key results:

While similarly increasing [Ca²⁺]_c and IS in controls, agents binding to site A (tolbutamide) or site B (meglitinide) of SUR1 were ineffective in Sur1KO islets. Of two non-selective blockers of potassium channels, quinine was inactive, whereas tetraethylammonium was more active in Sur1KO compared with control islets. Phentolamine, efaroxan and alinidine, three imidazolines binding to K_IR6.2 (pore of K_ATP channels), stimulated control islets, but only phentolamine retained weaker stimulatory effects on [Ca²⁺]_c and IS in Sur1KO islets. Neither K_ATP channel opener (diazoxide, pinacidil) inhibited Sur1KO islets. Calcium channel blockers (nimodipine, verapamil) or diphenylhydantoin decreased [Ca²⁺]_c and IS in both types of islets, verapamil and diphenylhydantoin being more efficient in Sur1KO islets. Activation of α₂-adrenoceptors or dopamine receptors strongly inhibited IS while partially (clonidine > dopamine) lowering [Ca²⁺]_c (control > Sur1KO islets).

Conclusions and implications:

Those drugs retaining effects on IS in islets lacking K_ATP channels, also affected [Ca²⁺]_c, indicating actions on other ionic channels. The greater effects of some inhibitors in Sur1KO than in control islets might be relevant to medical treatment of congenital hyperinsulinism caused by inactivating mutations of K_ATP channels.     

The imidazoline site involved in control of insulin secretion: characteristics that distinguish it from I1- and I2-sites.

1. The nature of the binding site mediating the insulin secretagogue activity of certain imidazoline compounds remains unclear and the pharmacology of the I1- and I2-imidazoline sites, described in many tissues, does not correlate with the observed responses to imidazolines in islets. In the present paper, we describe further results which support the concept that the islet imidazoline site may represent a novel subtype of imidazoline receptor. 2. Culture of rat isolated islets in the presence of imidazoline secretagogues (either efaroxan or phentolamine) resulted in loss of responsiveness on subsequent re-exposure to these agents. However, culture of islets with either idazoxan or UK14,304 (imidazoline ligands that do not stimulate insulin secretion) did not lead to any loss of response when the islets were subsequently exposed to efaroxan. By contrast, islets cultured with UK14,304 (a potent alpha 2-adrenoceptor agonist), displayed loss of sensitivity to noradrenaline, consistent with down-regulation of alpha 2-adrenoceptors. 3. In order to characterize the imidazoline site further, radioligand binding studies were performed in membranes from RINm5F insulinoma cells using [3H]-RX821002, an imidazoline insulin secretagogue that does not interact significantly with imidazoline sites in other tissues. [3H]-RX821002 labelled alpha 2-adrenoceptors with high affinity (2.01 +/- 0.7 nM) but also labelled a second, non-adrenoceptor site with much lower affinity. 4. Under conditions of alpha 2-adrenoceptor blockade (in the presence of adrenaline), efaroxan displaced [3H]-RX821002 binding to the low affinity site, in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple effects and stimulation of insulin secretion by the tyrosine kinase inhibitor genistein in normal mouse islets.

1. Islets from normal mice were used to test the acute effects of genistein, a potent tyrosine kinase inhibitor, on stimulus-secretion coupling in pancreatic beta-cells. 2. Genistein produced a concentration-dependent (10-100 microM), reversible, increase of insulin release. This effect was marginal on basal release or in the presence of non-metabolized secretagogues, and much larger in the presence of glucose or other nutrients. The increase in insulin release caused by 100 microM genistein was abolished by adrenaline or omission of extracellular Ca2+. It was not accompanied by any rise of cyclic AMP, inositol phosphate or adenine nucleotide levels. 3. Although genistein slightly inhibited ATP-sensitive K+ channels, as shown by 86Rb efflux and patch-clamp experiments, this effect could not explain the action of the drug on insulin release because the latter persisted when ATP-sensitive K+ channels were all blocked by maximally effective concentrations of glucose and tolbutamide. Genistein was also effective when ATP-sensitive K+ channels were opened by diazoxide and the beta-cell membrane depolarized by 30 mM K, but ineffective in the presence of diazoxide and normal extracellular K. 4. Genistein paradoxically decreased Ca2+ influx in beta-cells, as shown by the inhibition of glucose-induced electrical activity, by the inhibition of Ca2+ currents (perforated patches) and by the lowering of cytosolic [Ca2+]i (fura-2 technique). Genistein thus increases insulin release in spite of a lowering of [Ca2+]i in beta-cells. 5. Daidzein, an analogue of genistein reported not to affect tyrosine kinases, was slightly less potent than genistein on K+ and Ca2+ channels, but increased insulin secretion in a similar way.(ABSTRACT TRUNCATED AT 250 WORDS)     

Y-26763: ATP-sensitive K+ channel activation and the inhibition of insulin release from human pancreatic beta-cells

The effect of Y-26763 [(-)-(3S,4R)-4-(N-acetyl-N-hydroxyamino)-6-cyano-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-3-ol], a novel ATP-sensitive K(+) (K(ATP)) channel activator, was tested on insulin secretion from human pancreatic islets in vitro. Y-26763 was able to inhibit both glucose- and tolbutamide-induced insulin secretion from islets as assessed by radioimmunoassay. The mechanism for inhibition of insulin secretion was characterised using patch clamp electrophysiology on dispersed human pancreatic beta-cells which express K(ATP) channels comprised of Kir6.2 and SUR1, and the NES2Y human beta-cell line, transfected with Kir6.2DeltaC26. Y-26763 activated K(ATP) channels in a reversible manner with a similar activity to diazoxide. This required the presence of hydrolysable nucleotides and appeared to be mediated by interaction of Y-26763 with SUR1 since: (a) tolbutamide was able to reverse the actions of Y-26763 and (b) Y-26763 failed to activate Kir6.2DeltaC26 in the absence of SUR1. We conclude that Y-26763-induced inhibition of insulin release is dependent upon the activation of K(ATP) channels in human beta-cells.     

Alterations of insulin secretion following long-term manipulation of ATP-sensitive potassium channels by diazoxide and nateglinide

Previous studies have shown that prolonged exposure to drugs, which act via blocking KATP channels, can desensitize the insulinotropic effects of drugs and nutrients acting via KATP channels. In this study, effects of prolonged exposure to diazoxide, a KATP channel opener, on beta cell function were examined using clonal BRIN-BD11 cells. The findings were compared to the long-term effects of KATP channel blockers nateglinide and tolbutamide. Following 18 h exposure to 200 microM diazoxide, the amounts of insulin secreted in response to glucose, amino acids and insulinotropic drugs were increased. Secretory responsiveness to a variety of agents acting via KATP channels was retained following prolonged diazoxide exposure. In contrast, 18 h exposure to 100 microM nateglinide significantly attenuated the insulin secretory responses to tolbutamide, nateglinide and BTS 67 582. Glucose- and L-alanine-stimulated insulin release were unaffected by prolonged nateglinide exposure, however responsiveness to L-leucine and L-arginine was diminished. Prolonged exposure to nateglinide had no effect on forskolin- and PMA-stimulated insulin release, and the overall pattern of desensitization was similar to that induced by 100 microM tolbutamide. We conclude that in contrast to chronic long-term KATP channel blockade, long-term diazoxide treatment is not harmful to KATP channel mediated insulin secretion and may have beneficial protective effects on beta cell function.