Life Sciences, Vol. Printed in the U S A
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Pergamon
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MINIREVIEW K + C H A N N E L OPENERS A N D INSULIN RELEASE P. Lebrun, M.-H. Antoine and A. Herchuelz. Laboratory of Pharmacology - Brussels Free University - School of Medicine, 808 route de Lennik, B-1070 Brussels, Belgium. (Received
in final
form July 6, 1992)
Summary Recent in vivo and in vitro experiments suggested that the smooth muscle relaxation mediated by diverse pharmacologic agents resulted from K + channel opening. Pinacidil, cromakalim, nicorandil, RP 4 9 3 5 6 , minoxidil sulfate and diazoxide belong to this n e w group of smooth muscle relaxants : the "K + channel openers". Because modifications in the K + permeability are k n o w n to represent a critical event in the insulin-releasing process, numerous studies have been performed in order to examine the putative effects of K + channel openers on B-cell function. The aim of the present review is to summarize these experimental data which are sometimes divergent. Under physiological conditions, the maintenance of homeostatic blood glucose concentrations is mainly dependent on an appropriate secretion of insulin from the pancreatic B-cell. The primary physiological stimulant and metabolic substrate of the B-cell is the molecule of glucose. Today, it is generally believed that the metabolism of the nutrient in the pancreatic B-cell leads to an e x t e n s i v e remodeling of ionic fluxes across the plasma membrane (1). Radioisotopic and electrophysiological experiments revealed that a rise in the extracellular glucose concentration initially provokes a decrease in membrane permeability to K + (2,3). This reduction in B-cell membrane K + permeability appears to reflect the closure of ATP-sensitive K + channels resulting from the increase in the ATP/ADP ratio mediated by glucose metabolism (4). Notably, the activity of the ATP-regulated K + channels equipping the B-cell plasma membrane is also reduced by hypoglycemic sulfonylureas (4-6). Because the resting potential of the pancreatic B-cell is mainly determined by a high permeability to K + ions, the decrease in K + channels activity leads to membrane depolarization.
Correspondence address : P. Lebrun, Laboratory of Pharmacology, Brussels Free University, School of Medicine, Route de Lennik, 808, B-1070 Brussels Belgium, Fax : 3 2 / 2 / 5 5 5 . 6 3 . 7 0 .
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The reduction of the electrical potential difference across the plasma membrane provokes, in turn, the opening of voltage-sensitive Ca 2 + channels with subsequent Ca 2+ inflow. The enhanced Ca 2+ entry would then raise the cytosolic concentration of ionized Ca 2 + and, hence, triggers exocytosis by affecting enzyme activities, electrostatic membrane charges and/or the effector system responsible for the translocation of secretory granules (7). Theoretical studies on the electrical activity of pancreatic B-cells also predicted this sequence of events leading to insulin release (8,9). In the last few years, great interest has been focused on new pharmacological agents exhibiting a n t i h y p e r t e n s i v e a c t i v i t y "in v i v o " and vasorelaxant properties "in vitro". Several studies have suggested that such compounds could activate membrane K + channels thereby controlling the membrane potential and the excitability of smooth muscle cells (10,11). Cromakalim (BRL 34915), pinacidil, nicorandil, minoxidil sulfate, RP 49356 and diazoxide belong to this new group of smooth muscle relaxants : the K + channel openers (10,11). These drugs present several potential clinical applications including the treatment of hypertension, angina pectoris, asthma, irritable bladder syndrome, cerebral ischemia and peripheral vascular diseases (10,11 ). Because modifications in K + permeability represent a crucial step in the sequence of events leading to insulin release (2,3,7), numerous radioisotopic, fluorimetric, electrophysiological and pharmacological studies have been performed in order to examine the putative effects of K + channel openers on pancreatic B-cell function. In these investigations, whole islets as well as isolated cells from different insulin secreting cell lines have been used. The experimental data emerging from these studies are sometimes divergent and the purpose of the present commentary is to review them. Moreover, an attempt is made to show that the excitable pancreatic B-cell is an excellent model system which may provide new clues to the understanding of drug action. "in v i t r o " effects o f diazoxide
Diazoxide ( 3 - m e t h y l - 7 - c h l o r o - l , 2 , 4 - b e n z o t h i a d i a z i n e known to have vasodilator and hyperglycemic properties.
1,1-dioxide)is
The hyperglycemic activity of diazoxide is primarily attributed to its ability to inhibit insulin secretion from pancreatic B-cells. From measurements of 86Rb outflow, used as a tracer for K +, it was concluded about ten years ago that the sulfonamide increased the K + permeability of the B-cell membrane (12,13). The resultant hyperpolarisation was proposed to close the voltageresponsive Ca 2 + channels, to reduce Ca 2 + influx and ultimately to inhibit the secretory process (1 2,13). Patch clamp studies conducted on single B-cells confirmed the capacity of diazoxide to increase K + currents and suggested that the drug activated the ATP-sensitive K + channels equiping the B-cell plasma membrane (14-22). The
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stimulatory effect of diazoxide on the KAT P channels appeared to be specific. Indeed, the drug was shown neither to affect the delayed rectifier-type K + channel (19) nor the calcium and voltage-activated K + channel (16,19). Although the diazoxide-induced inhibition of insulin release is currently ascribed to the activation of a class of K + channels characterized by their sensitivity to ATP, the interaction of the sulfonamide derivative with its target site is not fully understood. The drug appears to have an internal site of action and analysis of single channel data showed that diazoxide provoked an increase in both the individual open state probability and the number of functional KAT P channels (20). A more detailed analysis of the kinetic behavior of the KAT P channels revealed that diazoxide increased channel burst duration and reduced the interburst closed intervals (19). Patch clamp experiments also indicated that the presence of ATP at the intracellular membrane surface was required in order for diazoxide to activate the KAT P channels (14-17, 19,20). On the other hand, experiments conducted on RINm5F cells suggested that Mg 2+ ion was not a necessary cofactor (15) whilst studies performed on CRIG1 cells concluded that the presence of Mg 2+ was essential to detect a stimulatory effect of diazoxide on KAT P channels (20). The latter finding and the failure of diazoxide to increase KAT P channel activity in the presence of a non-hydrolysable ATP analogue, whether Mg 2 + was present or not, led to the proposal that a phosphorylation process was involved in the diazoxide-induced KAT P channel activation (20-22). In cells from the rat pancreatic cell line CRIG1, diazoxide (600 pM) was also shown to progressively inhibit KAT P currents when the solution bathing the cytosolic membrane surface was devoid of both ATP and Mg 2 + (20). "in vitro" effects of pinacidil Pinacidil ( N " - c y a n o - N - 4 - p y r i d y I - N ' - 1 , 2 , 2 - t r i m e t h y l p r o p y l g u a n i d i n e monohydrate) is a cyanoguanidine derivative belonging to the new group of smooth muscle relaxants : the K + channel openers (10,11). The first observation of an effect of pinacidil on B-cell function came from experiments conducted on whole rat pancreatic islets (23). It was shown that, whether in the presence or absence of extracellular Ca 2+, pinacidil increased 86Rb outflow from islets perifused in the presence of glucose, 2-ketoisocaproate or tolbutamide. Moreover, the drug inhibited 45Ca outflow (reflecting an inhibition of 4°Ca entry into the islet cells) and the glucoseinduced insulin release. These findings suggested that, in endocrine islet cells like in smooth muscle cells, the primary effect of ~inacidil was to increase the K + permeability. Because pinacidil increased 86Rb outflow only under experimental conditions where the activity of KAT P channels was inhibited (4), it was proposed that the drug activated such K * channels (23). A more detailed study indicated that, in whole islets, pinacidil and diazoxide similarly affected the ionic (45Ca outflow, 86Rb outflow, 86Rb inflow, [Ca2+]i ) and secretory responses to glucose or tolbutamide (24). It should also be stressed that pinacidil such as diazoxide failed to affect the increase in 45Ca efflux, insulin
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release and cytosolic free Ca 2 + concentration resulting from KCI depolarization (24). Taken together, these observations substantiated the hypothesis that the primary effect of pinacidil was to increase the B-cell K + permeability through the activation of KAT P channels. The hy•erpolarisation resulting from K + channel activation is likely to impede Ca z+ inflow through voltagesensitive Ca 2+ channels and consequently inhibit the secretory process (23,24). The recording of bioelectrical activity in intact mouse islets indicated that pinacidil, like diazoxide, abolished the regular membrane potential fluctuations elicited by intermediate concentrations of glucose (18). On the other hand, whole cell ATP-sensitive K + currents were enhanced by pinacidil in a way similar to diazoxide (18,25). The cyanoguanidine derivative was also shown to activate single KAT P currents from excised outside-out membrane patches (25).
Thus, the electrophysiological data confirm that the pinacidil-induced inhibition of insulin release is due to the opening of B-cell ATP-sensitive K + channels with subsequent membrane hyperpolarisation and reduction of Ca 2+ influx. Incidentally, in RINm5F cells, the application of pinacidil to the cytosolic side of the membrane in the absence of ATP unexpectedly provoked a reduction in the KATP channel activity (25). This finding was taken as evidence that pinacidil-induced channel activation required protein phosphorylation (25).
Pinacidil/Diazoxide I_Cro___makali______m_ ~//~ / ~
KATP i h anne' ~on ~ Membrane Polarizati
I RP 49356 I
,.-
Ca
channel
[Nicorandil] Minoxidilsulfate intracellular Ca 2+ redistribution
Ca 2+ influx CystolicCa 2+ concentration Insulinrelease Fig.1 : "In vitro" effects of putative "K + channel openers" on ionic and secretory events in pancreatic B-cells.
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"in vitro" effects of cromakalim Cromakalim [BRL 34915; 6-cyano-3,4-dihydro-2,2-dimethyl- trans-4-(2oxo-l-pyrollidyl)-2H-benzo(b)pyran-3-ol] appears to be the most potent smooth muscle relaxant belonging to the "K + channel openers". However, the effects of cromakalim recorded on normal pancreatic B cells and on tumoral cell lines are conflicting. In rat and/or mouse pancreatic islets, the drug unexpectedly decreased rather than increased 86Rb outflow (18,26). The cromakalim-induced inhibition of 86Rb outflow was a concentration-dependent phenomenon and several findings suggested that this inhibitory effect could reflect a reduction in the activity of the B-cell ATP-sensitive K + channels (26). First, cromakalim decreased 86Rb outflow in islets exposed to a glucose-free medium; an experimental condition where 86Rb extrusion is mainly mediated by ATPdependent K + channels. Second, the inhibitory effect of cromakalim was attenuated by glibenclamide, a hypoglycemic sulfonylurea known to selectively block the ATP-sensitive K + channels in islet cells (4-6). Third, patch clamp studies on mouse pancreatic B-cells revealed that cromakalim reduced the current through KAT P channels (18). The experiments conducted on whole islets further indicated that cromakalim also inhibited a Ca2+-sensitive modality of 86Rb extrusion (26). Indeed, when the B-cell KAT P channels were almost fully inhibited by the presence of high glucose concentrations in the medium (4), the drug still decreased 86Rb outflow. Moreover, the cromakalim-induced-reduction in 86Rb outflow was markedly reduced in islets exposed to a Ca 2 +-free medium. If the target site of cromakalim was restricted to the K + channels, the drug-induced decrease in 86Rb outflow, which mimics the effect of most insulin secretagogs, should cause facilitation of insulin release. Cromakalim, however, elicited a concentration-dependent inhibition of the glucose-induced insulin release (18,26). Radioisotopic data further showed that cromakalim provoked a dose-dependent reduction in 45Ca outflow (reflecting the isotopic exchange between influent 40Ca and effluent 45Ca) from islets perifused in the presence of glucose and extracellular Ca 2+ (26). In islets exposed to a glucose free or a Ca 2+ free medium, the drug was ineffective. Moreover, cromakalim inhibited the increased 45Ca outflow in response to K + depolarisation, an effect reminiscent of that evoked by Ca 2 + channel blockers (26, 27). These observations were interpreted as being due to an inhibitory effect of cromakalim on the B-cell Ca 2+ channels. A decrease in Ca 2+ entry as mediated by cromakalim may explain the effect of the drug on the insulin releasing process as well as its action on the Ca2+-sensitive modality of 86Rb extrusion. In summary, the experiments conducted on normal rat and/or mouse B cells revealed that cromakalim had mixed effects. On the one hand, the drug reduces the activity of the KAT P channels and indirectly inhibit the Ca 2+activated K + channels. On the other hand, cromakalim inhibits Ca 2 + channels
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and subsequently reduces insulin secretion. The results of electrophysiological experiments carried out on insulin secreting cells isolated from tumoral cell lines contrast with the above mentioned findings. At 100 pM, a concentration reported to affect 86Rb outflow, 45Ca outflow and insulin secretion from rat pancreatic islets, cromakalim was found to have no effects on the whole cell or single channel ATP-K + currents recorded in CRI-G1 cells (20). In whole cell patch clamp experiments performed on the clonal insulin-secreting cell line RINm5F, cromakalim (100-200 #M) was found to hyperpolarise the cell membrane and to inhibit the spiking activity generated by low glucose concentrations (21). The concomitant measurement of single cell intracellular calcium concentration demonstrated that the drug reduced the glucose-induced rise in [Ca2+]i (21). Cromakalim, in the same range of concentrations as those used in normal rat and/or mouse B cells, also enhanced the whole cell KAT P current (21). In excised outside-out membrane patches of RINm5F, the drug activated single KAT P channels; an effect counteracted by tolbutamide (21). Lastly, using the open-cell variation of the patch clamp technique, it was shown that application of cromakalim (200800 #M) to the inside of the RINm5F membrane in the complete absence of internal ATP either did not affect or reduced the activity of K ATP channels (28). "in v i t r o " effects o f R P 4 9 3 5 6
RP49356 (N-methyl-2-(3-pyridil)-tetrahydrothiopyran-2-carbothiamide- 1oxide) has been reported to provoke relaxation of K + depolarized rat aortic rings (29), to relax airway smooth muscle (30) and to activate KAT P channels in isolated cardiac cells (31). The drug was recently shown to counteract the depolarizing effect of glucose in RINm5F cells and to enhance the KAT P currents recorded in the same clonal insulinoma cell line (25). When open cell experiments were conducted in the absence of ATP on the inside of the plasma membrane of RINm5F cells, RP49356 usually provoked an inhibition of the KAT P channel activity characterized by a decrease in the single channel current amplitude (25). Radioisotopic experiments conducted on whole rat pancreatic islets challenge the view that RP49356 could increase the membrane permeability to K +. Indeed, the drug did not stimulate but rather reduced 86Rb outflow from prelabeled and perifused pancreatic islets (32). The drug-induced reduction in 86Rb outflow was recorded in islets exposed to a high glucose concentration, when most ATP-sensitive K + channels are closed and Ca2+-activated K + channels are stimulated (4). Furthermore, glibenclamide, a specific blocker of ATP-modulated K + channels (4-6), did not reverse the inhibitory effect of RP49356 whereas the absence of extracellular Ca 2 + did. These findings were taken as indication that RP49356 inhibited a Ca2+-sensitive modality of 86Rb extrusion. Because the drug also inhibited insulin output and 45Ca outflow
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from islets perifused in the presence but not the absence of extracellular Ca 2+, it was proposed that RP49356 could reduce the activity of voltagedependent Ca 2+ channels (32). A decrease in Ca 2+ entry as mediated by RP49356 would, in turn, reduce the insulin output and the Ca2+-dependent 86Rb outflow. "in v i t r o " effects o f nicorandil
Few studies have been devoted to the effect of nicorandil (N-(nitroxy-2 ethyl)pyridinecarboxamide-3) on B-cell function. In RINm5F cells, nicorandil behaved like pinacidil and RP49356 (25). The drug provoked a membrane hyperpolarisation which was inhibited by tolbutamide. Nicorandil was also shown to enhance the current flowing through the KAT P channels (25). The stimulatory effect of nicorandil on KAT P channel activity appeared to require the presence of ATP at the cytosolic aspect of the membrane. Indeed, when added to the inside of the membrane in the absence of ATP, the drug exhibited the capacity to inhibit the K + channels (25). Patch clamp experiments in B-cells from mouse pancreatic islets failed to confirm the above results (18). Whole cell recordings indicated that nicorandil was without effect on the ATP-sensitive K + current (18). The same study revealed that high concentrations of nicorandil slightly reduced the glucoseinduced insulin release and marginally accelerated 86Rb outflow from whole mouse islets perifused in the presence of glucose (18). The same concentrations of nicorandil also inhibited 45Ca outflow from glucose-stimulated rat pancreatic islets (Lebrun et al., unpublished observations). Thus, these results suggested that the inhibition of insulin output mediated by nicorandil could be dependent, in part only, on an increase in membrane K + permeability with subsequent reduction in 40Ca entry. However, the experiments conducted on normal B-cells do not confirm that the modifications in membrane K + permeability reflect alterations in KAT P channel activity (18). "in v i t r o " effects o f m i n o x i d i l sulfate
Minoxidil sulfate, the active metabolite of minoxidil (2,4-diamino-6piperidinyl-pyrimidine 3-oxide), is known to provoke arterial dilatation by activating membrane K + channels (33). The drug was unexpectedly found to increase the release of insulin from glucose-stimulated rat and/or mouse pancreatic islets (18,34). These observations contrast with the effects of other K + channel openers on B-cell function. Minoxidil sulfate was also shown to provoke a concentration-dependent reduction in 86Rb outflow (18,34). The magnitude of this inhibitory effect was reduced in a concentration-dependent manner by glucose, tolbutamide and glibenclamide (34). Such insulin secretagogs are known to reduce the activity of the KAT P channels (4-6). Further patch clamp studies conducted on single mouse B-cells also revealed an inhibitory effect of the drug on the ATP-K + cur-
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rents (18). These combined data were taken as an indication that minoxidil sulfate potentiated insulin secretion by primarily decreasing the activity of the ATP-sensitive K + channels (18, 34). However, measurements of 45Ca outflow and cytosolic free Ca 2+ concentrations clearly indicated that, in addition to its effect on the K + permeability, minoxidil sulfate promoted an intracellular calcium redistribution (34). It is thus conceivable that this calcium translocating effect of minoxidil sulfate may account, at least in part, for the stimulatory effect of the drug on the insulin-releasing process. "in v i v o " e f f e c t s o f K + c h a n n e l ooeners
Diazoxide, at doses producing a marked reduction in blood pressure, is known to cause a decrease in plasma insulin levels with subsequent hyperglycemia (35-37). Because of such properties, the drug has been used in clinical practice to treat both hypertensive emergencies and various forms of hypoglycemia (38). Regarding the other K + channel openers, the concentrations required to affect insulin release "in vitro" were far superior to that necessary to relax a large variety of smooth muscles. Hence, it is unlikely that such compounds may cause or favor hyperglycemia, at least in patients with normal glucose tolerance and receiving recommended cardiovascular therapeutic doses of the drugs. The effects of cromakalim, nicorandil and RP52891 (the active enantiomer of the racemate RP 49356) on glucose and insulin plasma concentrations have been investigated in rats. In vivo, cromakalim was found to have minimal effects on plasma glucose and insulin levels at concentrations producing large falls in blood pressure (35). Moreover, at concentrations exerting hypotensive effects, cromakalim, nicorandil and RP 52891 were found not to affect the plasma insulin concentration in hyperglycemic rats (37). The effects of pinacidil administered during two weeks at doses recommended in clinical antihypertensive therapy (25-50 mg once a day) was investigated in six healthy humans (39). The vasodilator pinacidil, which in healthy subjects failed to affect heart rate and blood pressure, did not modify glucosestimulated insulin secretion or impair the oral glucose tolerance test. This short term treatment with pinacidil also failed to change the fasting blood levels of glucose or insulin. Although most drugs reviewed here (except diazoxide), appear unlikely to perturb glucose homeostasis, no studies are available in diabetic patients. Discussion Pinacidil, diazoxide, cromakalim, nicorandil and RP 4 9 3 5 6 were reported to inhibit whereas minoxidil sulfate was found to potentiate the release of insulin from glucose-stimulated B-cells. Among the different drugs, only diazoxide and pinacidil appeared to behave, in the B-cell, as pure K + channel openers. Indeed, these two
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compounds only have been shown, under physiological conditions, to specifically activate the KAT P channels equiping the B-cell plasma membrane. The resulting hyperpolarization may in turn restrict the opening of voltage-sensitive Ca 2+ channels, reduce Ca 2+ inflow, decrease the cytosolic free Ca 2+ concentration and ultimately inhibit the secretory process. Cromakalim and minoxidil sulfate were found to reduce rather than to increase the activity of the ATP-sensitive K + channels in normal B cells. Nicorandil was reported to have no effect on the ATP-sensitive K + current in mouse B-cells whereas RP 4 9 3 5 6 was shown to inhibit a sulfonylureainsensitive modality of 86Rb outflow from pancreatic rat islets. Incidentally, radioisotopic and fluorimetric studies conducted in whole islets further revealed that cromakalim as well as RP 49356 might exhibit antagonistic actions on B-cell Ca 2+ channels whilst minoxidil sulfate could promote an intracellular translocation of Ca 2 +. By contrast, experiments conducted on insulin secreting cells isolated from tumoral cell lines indicated that cromakalim had either no effect or activated single KAT P channels. Furthermore, RP 4 9 3 5 6 and nicorandil were shown to enhance the KAT P current recorded in the clonal insulinoma cell line RINm5F. At present, the origin of the different reactions of normal and tumoral cells to "K + channel openers" appears unclear. It has long been known that t h e islets of Langerhans represent highly organized functional units consisting of a central core of B-cells surrounded by at least three different cell types. This structural organization allows islets hormones to exert paracrine and/or autocrine effects (40). Moreover, there is now ample evidence that the cells within an islet act in concert and display close contacts with homologous and heterologous cells (3,40). The gap junctions interconnecting B and non-B islet cells represent an intracellular pathway for metabolic and ionic signals. Such a functional organization of the islets appears to be a prerequisite for an appropriate glucose-induced insulin release (41). Thus, the use of isolated islets has the potential advantage to respect the intraislet arrangement of pancreatic B-cells and represents a useful approach to the study of B cell physiology and pharmacology. The perifusion of intact islets has been found to be an appropriate method to characterize the dynamics of insulin secretion and parallel changes in ionic fluxes. When radioisotopic and conventional bioelectric data (intracellular impalements in whole islets) collected under close-to-identical experimental conditions are combined, a fine understanding of changes in ionic fluxes evoked by physiological and/or pharmacological agents can be reached. This experimental approach can still be improved by the use of the patch clamp method which allows the study of channel properties for individual ion channels. Although the technique fails to respect the integrity of the pancreatic islets, whole cell recordings performed on multicellular clusters of cells initially dispersed from normal islets of Langerhans can help to characterize the type of ionic channel involved in the response to physiological and/or pharmacological stimuli.
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Such a combination of techniques has been used to characterize the effects of putative "K + channel openers" on normal pancreatic B-cells. The results of the different investigations led to identical conclusions regarding the different drugs. In contrast, electrophysiological data obtained on clonal B-cells are often different from those recorded in B-cells isolated from pancreatic islets. Cell lines established from islet cell tumour have been developed to provide a easy supply of insulin secreting cells. However, tumoral islet cells present defects of signal transduction and differ from normal islet cells in several distinct respects (42). In RINm5F cells, the most studied line of tumoral islet cells, anomalies in hexose transport, in the generation and channeling of hexose phosphates, in phosphofructokinase activation, in mitochondrial, oxidative events, in the anomeric specificity of glucose metabolism, in insulin biosynthesis, storage and release have been described (42). Radioisotopic data also revealed that the cationic response of RINm5F cells to nutrient secretagogs was impaired (43,44). Moreover, cell lines may change their properties during culture (45). Thus, these features could explain the discrepancies between electrophysiological results obtained in normal and tumoral insulin secreting cells. Alternatively, it could also be proposed that differences in cytosolic pH might, at least in part, affect the B cell responses to "K + channel openers". This speculative proposal is based on the recent demonstration that the gating of ATP-dependent K + channels can be modulated by intracellular pH (46,47). W h a t h e v e r the exact mechanism underlying the sometimes divergent responses to "K + channel openers", this situation implies that data emerging from studies conducted exclusively on tumoral islet cells should be analyzed with great care especially when discussing implications for the therapeutic application of drugs. The effects of pinacidil, cromakalim, nicorandil, minoxidil sulfate and RP 49356 on ionic and secretory events in insulin secreting cells were usually observed at higher concentrations than those required to affect cardiovascular function "in vitro". This is in agreement with the view that the affinity of the different compounds for KAT P channels may vary between different tissues (11, 31, 33) and that hypoglycaemic sulfonylureas exhibit some selectivity for the B-cell KAT P channel (35,36). Lastly, experiments conducted on normal pancreatic B-cells clearly revealed that some drugs depicted as "K + channel openers" reduced the K + channel activity. These findings may be taken as further evidence that the pharmacological profile of drugs acting on K + channels may vary from tissue to tissue. Moreover, the above observations indicate that the existence of several subtypes of KAT P channels should not be overlooked. Acknowledgements The authors are indebted to P. Surardt for secretarial help. This work was supported in part by grants from the National Fund for Scientific Research.
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MINIREVIEW K + C H A N N E L OPENERS A N D INSULIN RELEASE P. Lebrun, M.-H. Antoine and A. Herchuelz. Laboratory of Pharmacology - Brussels Free University - School of Medicine, 808 route de Lennik, B-1070 Brussels, Belgium. (Received
in final
form July 6, 1992)
Summary Recent in vivo and in vitro experiments suggested that the smooth muscle relaxation mediated by diverse pharmacologic agents resulted from K + channel opening. Pinacidil, cromakalim, nicorandil, RP 4 9 3 5 6 , minoxidil sulfate and diazoxide belong to this n e w group of smooth muscle relaxants : the "K + channel openers". Because modifications in the K + permeability are k n o w n to represent a critical event in the insulin-releasing process, numerous studies have been performed in order to examine the putative effects of K + channel openers on B-cell function. The aim of the present review is to summarize these experimental data which are sometimes divergent. Under physiological conditions, the maintenance of homeostatic blood glucose concentrations is mainly dependent on an appropriate secretion of insulin from the pancreatic B-cell. The primary physiological stimulant and metabolic substrate of the B-cell is the molecule of glucose. Today, it is generally believed that the metabolism of the nutrient in the pancreatic B-cell leads to an e x t e n s i v e remodeling of ionic fluxes across the plasma membrane (1). Radioisotopic and electrophysiological experiments revealed that a rise in the extracellular glucose concentration initially provokes a decrease in membrane permeability to K + (2,3). This reduction in B-cell membrane K + permeability appears to reflect the closure of ATP-sensitive K + channels resulting from the increase in the ATP/ADP ratio mediated by glucose metabolism (4). Notably, the activity of the ATP-regulated K + channels equipping the B-cell plasma membrane is also reduced by hypoglycemic sulfonylureas (4-6). Because the resting potential of the pancreatic B-cell is mainly determined by a high permeability to K + ions, the decrease in K + channels activity leads to membrane depolarization.
Correspondence address : P. Lebrun, Laboratory of Pharmacology, Brussels Free University, School of Medicine, Route de Lennik, 808, B-1070 Brussels Belgium, Fax : 3 2 / 2 / 5 5 5 . 6 3 . 7 0 .
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The reduction of the electrical potential difference across the plasma membrane provokes, in turn, the opening of voltage-sensitive Ca 2 + channels with subsequent Ca 2+ inflow. The enhanced Ca 2+ entry would then raise the cytosolic concentration of ionized Ca 2 + and, hence, triggers exocytosis by affecting enzyme activities, electrostatic membrane charges and/or the effector system responsible for the translocation of secretory granules (7). Theoretical studies on the electrical activity of pancreatic B-cells also predicted this sequence of events leading to insulin release (8,9). In the last few years, great interest has been focused on new pharmacological agents exhibiting a n t i h y p e r t e n s i v e a c t i v i t y "in v i v o " and vasorelaxant properties "in vitro". Several studies have suggested that such compounds could activate membrane K + channels thereby controlling the membrane potential and the excitability of smooth muscle cells (10,11). Cromakalim (BRL 34915), pinacidil, nicorandil, minoxidil sulfate, RP 49356 and diazoxide belong to this new group of smooth muscle relaxants : the K + channel openers (10,11). These drugs present several potential clinical applications including the treatment of hypertension, angina pectoris, asthma, irritable bladder syndrome, cerebral ischemia and peripheral vascular diseases (10,11 ). Because modifications in K + permeability represent a crucial step in the sequence of events leading to insulin release (2,3,7), numerous radioisotopic, fluorimetric, electrophysiological and pharmacological studies have been performed in order to examine the putative effects of K + channel openers on pancreatic B-cell function. In these investigations, whole islets as well as isolated cells from different insulin secreting cell lines have been used. The experimental data emerging from these studies are sometimes divergent and the purpose of the present commentary is to review them. Moreover, an attempt is made to show that the excitable pancreatic B-cell is an excellent model system which may provide new clues to the understanding of drug action. "in v i t r o " effects o f diazoxide
Diazoxide ( 3 - m e t h y l - 7 - c h l o r o - l , 2 , 4 - b e n z o t h i a d i a z i n e known to have vasodilator and hyperglycemic properties.
1,1-dioxide)is
The hyperglycemic activity of diazoxide is primarily attributed to its ability to inhibit insulin secretion from pancreatic B-cells. From measurements of 86Rb outflow, used as a tracer for K +, it was concluded about ten years ago that the sulfonamide increased the K + permeability of the B-cell membrane (12,13). The resultant hyperpolarisation was proposed to close the voltageresponsive Ca 2 + channels, to reduce Ca 2 + influx and ultimately to inhibit the secretory process (1 2,13). Patch clamp studies conducted on single B-cells confirmed the capacity of diazoxide to increase K + currents and suggested that the drug activated the ATP-sensitive K + channels equiping the B-cell plasma membrane (14-22). The
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stimulatory effect of diazoxide on the KAT P channels appeared to be specific. Indeed, the drug was shown neither to affect the delayed rectifier-type K + channel (19) nor the calcium and voltage-activated K + channel (16,19). Although the diazoxide-induced inhibition of insulin release is currently ascribed to the activation of a class of K + channels characterized by their sensitivity to ATP, the interaction of the sulfonamide derivative with its target site is not fully understood. The drug appears to have an internal site of action and analysis of single channel data showed that diazoxide provoked an increase in both the individual open state probability and the number of functional KAT P channels (20). A more detailed analysis of the kinetic behavior of the KAT P channels revealed that diazoxide increased channel burst duration and reduced the interburst closed intervals (19). Patch clamp experiments also indicated that the presence of ATP at the intracellular membrane surface was required in order for diazoxide to activate the KAT P channels (14-17, 19,20). On the other hand, experiments conducted on RINm5F cells suggested that Mg 2+ ion was not a necessary cofactor (15) whilst studies performed on CRIG1 cells concluded that the presence of Mg 2+ was essential to detect a stimulatory effect of diazoxide on KAT P channels (20). The latter finding and the failure of diazoxide to increase KAT P channel activity in the presence of a non-hydrolysable ATP analogue, whether Mg 2 + was present or not, led to the proposal that a phosphorylation process was involved in the diazoxide-induced KAT P channel activation (20-22). In cells from the rat pancreatic cell line CRIG1, diazoxide (600 pM) was also shown to progressively inhibit KAT P currents when the solution bathing the cytosolic membrane surface was devoid of both ATP and Mg 2 + (20). "in vitro" effects of pinacidil Pinacidil ( N " - c y a n o - N - 4 - p y r i d y I - N ' - 1 , 2 , 2 - t r i m e t h y l p r o p y l g u a n i d i n e monohydrate) is a cyanoguanidine derivative belonging to the new group of smooth muscle relaxants : the K + channel openers (10,11). The first observation of an effect of pinacidil on B-cell function came from experiments conducted on whole rat pancreatic islets (23). It was shown that, whether in the presence or absence of extracellular Ca 2+, pinacidil increased 86Rb outflow from islets perifused in the presence of glucose, 2-ketoisocaproate or tolbutamide. Moreover, the drug inhibited 45Ca outflow (reflecting an inhibition of 4°Ca entry into the islet cells) and the glucoseinduced insulin release. These findings suggested that, in endocrine islet cells like in smooth muscle cells, the primary effect of ~inacidil was to increase the K + permeability. Because pinacidil increased 86Rb outflow only under experimental conditions where the activity of KAT P channels was inhibited (4), it was proposed that the drug activated such K * channels (23). A more detailed study indicated that, in whole islets, pinacidil and diazoxide similarly affected the ionic (45Ca outflow, 86Rb outflow, 86Rb inflow, [Ca2+]i ) and secretory responses to glucose or tolbutamide (24). It should also be stressed that pinacidil such as diazoxide failed to affect the increase in 45Ca efflux, insulin
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release and cytosolic free Ca 2 + concentration resulting from KCI depolarization (24). Taken together, these observations substantiated the hypothesis that the primary effect of pinacidil was to increase the B-cell K + permeability through the activation of KAT P channels. The hy•erpolarisation resulting from K + channel activation is likely to impede Ca z+ inflow through voltagesensitive Ca 2+ channels and consequently inhibit the secretory process (23,24). The recording of bioelectrical activity in intact mouse islets indicated that pinacidil, like diazoxide, abolished the regular membrane potential fluctuations elicited by intermediate concentrations of glucose (18). On the other hand, whole cell ATP-sensitive K + currents were enhanced by pinacidil in a way similar to diazoxide (18,25). The cyanoguanidine derivative was also shown to activate single KAT P currents from excised outside-out membrane patches (25).
Thus, the electrophysiological data confirm that the pinacidil-induced inhibition of insulin release is due to the opening of B-cell ATP-sensitive K + channels with subsequent membrane hyperpolarisation and reduction of Ca 2+ influx. Incidentally, in RINm5F cells, the application of pinacidil to the cytosolic side of the membrane in the absence of ATP unexpectedly provoked a reduction in the KATP channel activity (25). This finding was taken as evidence that pinacidil-induced channel activation required protein phosphorylation (25).
Pinacidil/Diazoxide I_Cro___makali______m_ ~//~ / ~
KATP i h anne' ~on ~ Membrane Polarizati
I RP 49356 I
,.-
Ca
channel
[Nicorandil] Minoxidilsulfate intracellular Ca 2+ redistribution
Ca 2+ influx CystolicCa 2+ concentration Insulinrelease Fig.1 : "In vitro" effects of putative "K + channel openers" on ionic and secretory events in pancreatic B-cells.
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"in vitro" effects of cromakalim Cromakalim [BRL 34915; 6-cyano-3,4-dihydro-2,2-dimethyl- trans-4-(2oxo-l-pyrollidyl)-2H-benzo(b)pyran-3-ol] appears to be the most potent smooth muscle relaxant belonging to the "K + channel openers". However, the effects of cromakalim recorded on normal pancreatic B cells and on tumoral cell lines are conflicting. In rat and/or mouse pancreatic islets, the drug unexpectedly decreased rather than increased 86Rb outflow (18,26). The cromakalim-induced inhibition of 86Rb outflow was a concentration-dependent phenomenon and several findings suggested that this inhibitory effect could reflect a reduction in the activity of the B-cell ATP-sensitive K + channels (26). First, cromakalim decreased 86Rb outflow in islets exposed to a glucose-free medium; an experimental condition where 86Rb extrusion is mainly mediated by ATPdependent K + channels. Second, the inhibitory effect of cromakalim was attenuated by glibenclamide, a hypoglycemic sulfonylurea known to selectively block the ATP-sensitive K + channels in islet cells (4-6). Third, patch clamp studies on mouse pancreatic B-cells revealed that cromakalim reduced the current through KAT P channels (18). The experiments conducted on whole islets further indicated that cromakalim also inhibited a Ca2+-sensitive modality of 86Rb extrusion (26). Indeed, when the B-cell KAT P channels were almost fully inhibited by the presence of high glucose concentrations in the medium (4), the drug still decreased 86Rb outflow. Moreover, the cromakalim-induced-reduction in 86Rb outflow was markedly reduced in islets exposed to a Ca 2 +-free medium. If the target site of cromakalim was restricted to the K + channels, the drug-induced decrease in 86Rb outflow, which mimics the effect of most insulin secretagogs, should cause facilitation of insulin release. Cromakalim, however, elicited a concentration-dependent inhibition of the glucose-induced insulin release (18,26). Radioisotopic data further showed that cromakalim provoked a dose-dependent reduction in 45Ca outflow (reflecting the isotopic exchange between influent 40Ca and effluent 45Ca) from islets perifused in the presence of glucose and extracellular Ca 2+ (26). In islets exposed to a glucose free or a Ca 2+ free medium, the drug was ineffective. Moreover, cromakalim inhibited the increased 45Ca outflow in response to K + depolarisation, an effect reminiscent of that evoked by Ca 2 + channel blockers (26, 27). These observations were interpreted as being due to an inhibitory effect of cromakalim on the B-cell Ca 2+ channels. A decrease in Ca 2+ entry as mediated by cromakalim may explain the effect of the drug on the insulin releasing process as well as its action on the Ca2+-sensitive modality of 86Rb extrusion. In summary, the experiments conducted on normal rat and/or mouse B cells revealed that cromakalim had mixed effects. On the one hand, the drug reduces the activity of the KAT P channels and indirectly inhibit the Ca 2+activated K + channels. On the other hand, cromakalim inhibits Ca 2 + channels
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and subsequently reduces insulin secretion. The results of electrophysiological experiments carried out on insulin secreting cells isolated from tumoral cell lines contrast with the above mentioned findings. At 100 pM, a concentration reported to affect 86Rb outflow, 45Ca outflow and insulin secretion from rat pancreatic islets, cromakalim was found to have no effects on the whole cell or single channel ATP-K + currents recorded in CRI-G1 cells (20). In whole cell patch clamp experiments performed on the clonal insulin-secreting cell line RINm5F, cromakalim (100-200 #M) was found to hyperpolarise the cell membrane and to inhibit the spiking activity generated by low glucose concentrations (21). The concomitant measurement of single cell intracellular calcium concentration demonstrated that the drug reduced the glucose-induced rise in [Ca2+]i (21). Cromakalim, in the same range of concentrations as those used in normal rat and/or mouse B cells, also enhanced the whole cell KAT P current (21). In excised outside-out membrane patches of RINm5F, the drug activated single KAT P channels; an effect counteracted by tolbutamide (21). Lastly, using the open-cell variation of the patch clamp technique, it was shown that application of cromakalim (200800 #M) to the inside of the RINm5F membrane in the complete absence of internal ATP either did not affect or reduced the activity of K ATP channels (28). "in v i t r o " effects o f R P 4 9 3 5 6
RP49356 (N-methyl-2-(3-pyridil)-tetrahydrothiopyran-2-carbothiamide- 1oxide) has been reported to provoke relaxation of K + depolarized rat aortic rings (29), to relax airway smooth muscle (30) and to activate KAT P channels in isolated cardiac cells (31). The drug was recently shown to counteract the depolarizing effect of glucose in RINm5F cells and to enhance the KAT P currents recorded in the same clonal insulinoma cell line (25). When open cell experiments were conducted in the absence of ATP on the inside of the plasma membrane of RINm5F cells, RP49356 usually provoked an inhibition of the KAT P channel activity characterized by a decrease in the single channel current amplitude (25). Radioisotopic experiments conducted on whole rat pancreatic islets challenge the view that RP49356 could increase the membrane permeability to K +. Indeed, the drug did not stimulate but rather reduced 86Rb outflow from prelabeled and perifused pancreatic islets (32). The drug-induced reduction in 86Rb outflow was recorded in islets exposed to a high glucose concentration, when most ATP-sensitive K + channels are closed and Ca2+-activated K + channels are stimulated (4). Furthermore, glibenclamide, a specific blocker of ATP-modulated K + channels (4-6), did not reverse the inhibitory effect of RP49356 whereas the absence of extracellular Ca 2 + did. These findings were taken as indication that RP49356 inhibited a Ca2+-sensitive modality of 86Rb extrusion. Because the drug also inhibited insulin output and 45Ca outflow
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from islets perifused in the presence but not the absence of extracellular Ca 2+, it was proposed that RP49356 could reduce the activity of voltagedependent Ca 2+ channels (32). A decrease in Ca 2+ entry as mediated by RP49356 would, in turn, reduce the insulin output and the Ca2+-dependent 86Rb outflow. "in v i t r o " effects o f nicorandil
Few studies have been devoted to the effect of nicorandil (N-(nitroxy-2 ethyl)pyridinecarboxamide-3) on B-cell function. In RINm5F cells, nicorandil behaved like pinacidil and RP49356 (25). The drug provoked a membrane hyperpolarisation which was inhibited by tolbutamide. Nicorandil was also shown to enhance the current flowing through the KAT P channels (25). The stimulatory effect of nicorandil on KAT P channel activity appeared to require the presence of ATP at the cytosolic aspect of the membrane. Indeed, when added to the inside of the membrane in the absence of ATP, the drug exhibited the capacity to inhibit the K + channels (25). Patch clamp experiments in B-cells from mouse pancreatic islets failed to confirm the above results (18). Whole cell recordings indicated that nicorandil was without effect on the ATP-sensitive K + current (18). The same study revealed that high concentrations of nicorandil slightly reduced the glucoseinduced insulin release and marginally accelerated 86Rb outflow from whole mouse islets perifused in the presence of glucose (18). The same concentrations of nicorandil also inhibited 45Ca outflow from glucose-stimulated rat pancreatic islets (Lebrun et al., unpublished observations). Thus, these results suggested that the inhibition of insulin output mediated by nicorandil could be dependent, in part only, on an increase in membrane K + permeability with subsequent reduction in 40Ca entry. However, the experiments conducted on normal B-cells do not confirm that the modifications in membrane K + permeability reflect alterations in KAT P channel activity (18). "in v i t r o " effects o f m i n o x i d i l sulfate
Minoxidil sulfate, the active metabolite of minoxidil (2,4-diamino-6piperidinyl-pyrimidine 3-oxide), is known to provoke arterial dilatation by activating membrane K + channels (33). The drug was unexpectedly found to increase the release of insulin from glucose-stimulated rat and/or mouse pancreatic islets (18,34). These observations contrast with the effects of other K + channel openers on B-cell function. Minoxidil sulfate was also shown to provoke a concentration-dependent reduction in 86Rb outflow (18,34). The magnitude of this inhibitory effect was reduced in a concentration-dependent manner by glucose, tolbutamide and glibenclamide (34). Such insulin secretagogs are known to reduce the activity of the KAT P channels (4-6). Further patch clamp studies conducted on single mouse B-cells also revealed an inhibitory effect of the drug on the ATP-K + cur-
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rents (18). These combined data were taken as an indication that minoxidil sulfate potentiated insulin secretion by primarily decreasing the activity of the ATP-sensitive K + channels (18, 34). However, measurements of 45Ca outflow and cytosolic free Ca 2+ concentrations clearly indicated that, in addition to its effect on the K + permeability, minoxidil sulfate promoted an intracellular calcium redistribution (34). It is thus conceivable that this calcium translocating effect of minoxidil sulfate may account, at least in part, for the stimulatory effect of the drug on the insulin-releasing process. "in v i v o " e f f e c t s o f K + c h a n n e l ooeners
Diazoxide, at doses producing a marked reduction in blood pressure, is known to cause a decrease in plasma insulin levels with subsequent hyperglycemia (35-37). Because of such properties, the drug has been used in clinical practice to treat both hypertensive emergencies and various forms of hypoglycemia (38). Regarding the other K + channel openers, the concentrations required to affect insulin release "in vitro" were far superior to that necessary to relax a large variety of smooth muscles. Hence, it is unlikely that such compounds may cause or favor hyperglycemia, at least in patients with normal glucose tolerance and receiving recommended cardiovascular therapeutic doses of the drugs. The effects of cromakalim, nicorandil and RP52891 (the active enantiomer of the racemate RP 49356) on glucose and insulin plasma concentrations have been investigated in rats. In vivo, cromakalim was found to have minimal effects on plasma glucose and insulin levels at concentrations producing large falls in blood pressure (35). Moreover, at concentrations exerting hypotensive effects, cromakalim, nicorandil and RP 52891 were found not to affect the plasma insulin concentration in hyperglycemic rats (37). The effects of pinacidil administered during two weeks at doses recommended in clinical antihypertensive therapy (25-50 mg once a day) was investigated in six healthy humans (39). The vasodilator pinacidil, which in healthy subjects failed to affect heart rate and blood pressure, did not modify glucosestimulated insulin secretion or impair the oral glucose tolerance test. This short term treatment with pinacidil also failed to change the fasting blood levels of glucose or insulin. Although most drugs reviewed here (except diazoxide), appear unlikely to perturb glucose homeostasis, no studies are available in diabetic patients. Discussion Pinacidil, diazoxide, cromakalim, nicorandil and RP 4 9 3 5 6 were reported to inhibit whereas minoxidil sulfate was found to potentiate the release of insulin from glucose-stimulated B-cells. Among the different drugs, only diazoxide and pinacidil appeared to behave, in the B-cell, as pure K + channel openers. Indeed, these two
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compounds only have been shown, under physiological conditions, to specifically activate the KAT P channels equiping the B-cell plasma membrane. The resulting hyperpolarization may in turn restrict the opening of voltage-sensitive Ca 2+ channels, reduce Ca 2+ inflow, decrease the cytosolic free Ca 2+ concentration and ultimately inhibit the secretory process. Cromakalim and minoxidil sulfate were found to reduce rather than to increase the activity of the ATP-sensitive K + channels in normal B cells. Nicorandil was reported to have no effect on the ATP-sensitive K + current in mouse B-cells whereas RP 4 9 3 5 6 was shown to inhibit a sulfonylureainsensitive modality of 86Rb outflow from pancreatic rat islets. Incidentally, radioisotopic and fluorimetric studies conducted in whole islets further revealed that cromakalim as well as RP 49356 might exhibit antagonistic actions on B-cell Ca 2+ channels whilst minoxidil sulfate could promote an intracellular translocation of Ca 2 +. By contrast, experiments conducted on insulin secreting cells isolated from tumoral cell lines indicated that cromakalim had either no effect or activated single KAT P channels. Furthermore, RP 4 9 3 5 6 and nicorandil were shown to enhance the KAT P current recorded in the clonal insulinoma cell line RINm5F. At present, the origin of the different reactions of normal and tumoral cells to "K + channel openers" appears unclear. It has long been known that t h e islets of Langerhans represent highly organized functional units consisting of a central core of B-cells surrounded by at least three different cell types. This structural organization allows islets hormones to exert paracrine and/or autocrine effects (40). Moreover, there is now ample evidence that the cells within an islet act in concert and display close contacts with homologous and heterologous cells (3,40). The gap junctions interconnecting B and non-B islet cells represent an intracellular pathway for metabolic and ionic signals. Such a functional organization of the islets appears to be a prerequisite for an appropriate glucose-induced insulin release (41). Thus, the use of isolated islets has the potential advantage to respect the intraislet arrangement of pancreatic B-cells and represents a useful approach to the study of B cell physiology and pharmacology. The perifusion of intact islets has been found to be an appropriate method to characterize the dynamics of insulin secretion and parallel changes in ionic fluxes. When radioisotopic and conventional bioelectric data (intracellular impalements in whole islets) collected under close-to-identical experimental conditions are combined, a fine understanding of changes in ionic fluxes evoked by physiological and/or pharmacological agents can be reached. This experimental approach can still be improved by the use of the patch clamp method which allows the study of channel properties for individual ion channels. Although the technique fails to respect the integrity of the pancreatic islets, whole cell recordings performed on multicellular clusters of cells initially dispersed from normal islets of Langerhans can help to characterize the type of ionic channel involved in the response to physiological and/or pharmacological stimuli.
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Such a combination of techniques has been used to characterize the effects of putative "K + channel openers" on normal pancreatic B-cells. The results of the different investigations led to identical conclusions regarding the different drugs. In contrast, electrophysiological data obtained on clonal B-cells are often different from those recorded in B-cells isolated from pancreatic islets. Cell lines established from islet cell tumour have been developed to provide a easy supply of insulin secreting cells. However, tumoral islet cells present defects of signal transduction and differ from normal islet cells in several distinct respects (42). In RINm5F cells, the most studied line of tumoral islet cells, anomalies in hexose transport, in the generation and channeling of hexose phosphates, in phosphofructokinase activation, in mitochondrial, oxidative events, in the anomeric specificity of glucose metabolism, in insulin biosynthesis, storage and release have been described (42). Radioisotopic data also revealed that the cationic response of RINm5F cells to nutrient secretagogs was impaired (43,44). Moreover, cell lines may change their properties during culture (45). Thus, these features could explain the discrepancies between electrophysiological results obtained in normal and tumoral insulin secreting cells. Alternatively, it could also be proposed that differences in cytosolic pH might, at least in part, affect the B cell responses to "K + channel openers". This speculative proposal is based on the recent demonstration that the gating of ATP-dependent K + channels can be modulated by intracellular pH (46,47). W h a t h e v e r the exact mechanism underlying the sometimes divergent responses to "K + channel openers", this situation implies that data emerging from studies conducted exclusively on tumoral islet cells should be analyzed with great care especially when discussing implications for the therapeutic application of drugs. The effects of pinacidil, cromakalim, nicorandil, minoxidil sulfate and RP 49356 on ionic and secretory events in insulin secreting cells were usually observed at higher concentrations than those required to affect cardiovascular function "in vitro". This is in agreement with the view that the affinity of the different compounds for KAT P channels may vary between different tissues (11, 31, 33) and that hypoglycaemic sulfonylureas exhibit some selectivity for the B-cell KAT P channel (35,36). Lastly, experiments conducted on normal pancreatic B-cells clearly revealed that some drugs depicted as "K + channel openers" reduced the K + channel activity. These findings may be taken as further evidence that the pharmacological profile of drugs acting on K + channels may vary from tissue to tissue. Moreover, the above observations indicate that the existence of several subtypes of KAT P channels should not be overlooked. Acknowledgements The authors are indebted to P. Surardt for secretarial help. This work was supported in part by grants from the National Fund for Scientific Research.
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