Modulation of insulin secretion and 45Ca2+ efflux by dopamine in glucose-stimulated pancreatic islets

Pergamon

0306-3623(94)E0030-P

Gen. Pharmac.Vol.25, No. 5, pp. 909-916, 1994 Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0306-3623/94$7.00+ 0.00

Modulation of Insulin Secretion and 45Ca2+ Efflux by Dopamine in Glucose-stimulated Pancreatic Islets C E L I A R. N O G U E I R A , U B I R A T A N F. M A C H A D O , R U I C U R I a n d A N G E L O R. C A R P I N E L L I * Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sdo Paulo, 05508-900 S~o Paulo, SP, Brazil [TeL (011) 818 7363 Fax (011) 813 0845] (Received 22 December 1993)

Abstract--l. The effect of dopamine on calcium eftlux and insulin secretion is examined in the present study. For this purpose, islets isolated from adult Wistar rats were perfused or incubated at 37°C for 60 min. 2. The results obtained from perfused islets indicate that 100/~M dopamine, in the presence of 5.6 mM glucose, increases insulin secretion and causes a modest elevation of 45Ca2+ efflux. However, glucose stimuli (from 5.6 to 16.7mM) provoked an unexpected reduction of insulin release, with no alteration in calcium efilux, when 100 #M dopamine was present in the perfusion medium. 3. Similar findings were obtained in incubated islets when the prolonged effect of dopamine was investigated. 4. The observations described above led us to conclude that bioactive amines might play an important role in the modulation of the glucose-induced insulin secretion. Key Words: Insulin secretion, pancreatic islets, dopamine, beta-cell

INTRODUCTION It is well known that glucose is the most important physiological insulin secretagoge and its action is directly related with glucose metabolism in pancreatic islets. Glucose metabolism initiates several signals in islet B cells which trigger secretory process. One of which is the increase in ATP/ADP ratio that inhibits the ATP-dependent potassium channels (Ashcroft et aL, 1984), provoking cell depolarization and the opening of the voltage-sensitive calcium channels (VSCC). In this manner the extra cellular calcium enters the cell through the VSCC following the calcium electrochemical gradient (Arkhammar et al., 1989; Malaisse, 1973; Wollheim et al., 1981). Moreover, glucose metabolism induces the inositol 1,4,5-triphosphate synthesis which produces endoplasmic reticulum calcium extrusion (Wolf et al., 1988). The last event and the opening of VSCC increases the cytosolic ionic calcium which triggers the granular extrusion. *To whom all correspondence should be addressed.

On the other hand, the increased glucose-6-phosphate concentration caused by glucose phosphorylation raises the endoplasmic reticulum calcium uptake (Wolfet al., 1988). This effect partially counteracts the inositol 1,4,5-triphosphate action. The metabolic effect of glucose on insulin secretion, described above, is modulated by the autonomic nervous system. In addition to noradrenaline and acetyicholine, dopamine (Feldman et al., 1971) has been assumed to play a role as one of the neurotransmitters which control pancreatic cell function (Lorenzi et al., 1979). Dopamine was detected in A and B pancreatic islet cells of a large number of mammals (Cegrell, 1968), but was not detected in rat, mouse or rabbit pancreas (Cegrell, 1968; Ericson et al., 1977; Gylfet al., 1973). However, dopamine synthesis occurs when a biochemical precursor is administered to these animals (Cegrell, 1968; Ericson et aL, 1977; Yu et al., 1984). Furthermore, pre-treatment of these animals with monoamine oxidase inhibitors increases the amount of dopamine in pancreatic B cells (Cegrell, 1970). The administration of dopamine to humans

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increases the plasma concentration of glucagon and insulin and this effect is not inhibited by alpha or beta adrenergic blockers (Lorenzi et aL, 1979). These results indicate that dopamine is not metabolized to noradrenaline or adrenaline before acting on pancreatic islet secretion. It was found by Ahren and Lundquist (1985) that, in the presence of basal glucose concentration (2.8 mM), a short lasting dopamine stimulus promotes 4SCa2÷ efflux from the endoplasmic reticulum and through the islet B cell membrane. These authors also postulated that a prolonged B cell exposure to dopamine decreases cytosolic ionic calcium content and consequently insulin secretion. Furthermore, however, the effect of dopamine on insulin release in the presence of high glucose concentrations have not been investigated yet. To elucidate some of these points, the mechanisms by which dopamine affects the calcium efflux and insulin secretion to 5.6 and 16.7 mM glucose stimuli are examined in this study. MATERIALS AND METHODS Animals Male and female, 2-month old Wistar rats were maintained in an environment at 23 + 2°C and 14 light: 10 dark photoperiod.

gyline and dietyl carbamate; 200 mM each) were added to assay medium; this concentration has been shown to prevent the metabolism of dopamine by more than 90%. Insulin concentration in the incubation medium was determined by radioimmunoassay (Morgan and Lazarow, 1963; Pupo and Marreiro, 1970).

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Chemicals Collagenase Type V and albumin bovine-fraction V were purchased from Sigma Chemical, St Louis, Mo, U.S.A. and 45Ca2+ from Dupont NEN products, Boston, Mass. ~25I-insulinwas furnished by courtesy of the laboratory of Dr A. A. Pupo, Faculdade de Medicina da Universidade de Sho Paulo, Brazil. Rat insulin standards and anti-rat insulin antibody were a courtesy of Dr Leclercq, Universit~ Libre de Bruxeiles, Belgium.

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Pancreatic islets were isolated from pancreas by the collagenase method as previously described (VaUar and Meldolesi, 1989). After isolation, the islets were picked up and kept in Krebs-Henseleit solution. The buffer contained albumin (5%) and glucose 5.6 mM was gassed with 95% O 2 / 5 0 / 0 C O 2 for 10min and adjusted to pH 7.4.

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Incubation experiments Batches of five islets were incubated for 60 min at 37°C in Krebs-Henseleit (Krebs et al., 1932) solution containing different glucose concentrations (0, 2.2, 4.4, 5.6, 8.3, ll.1 and 16.7mM) in the absence and presence of 100/~M dopamine. To avoid dopamine conversion to adrenaline, enzymatic inhibitors (par-

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Fig. 1. Effect of glucose load on insulin secretion and 4SCa2÷ outflow rate (FOR) in perifused islets. The islets were pre-incubated in Krebs-Henseleit buffer having 5.6 mM glucose. Afterwards, the islets were perfused in the presence of 5.6 mM glucose during 40 min. At this time, as indicated by vertical interrupted line, the perfusion medium was changed: (1) control conditions were maintained at 5.6 mM glucose and (2) 16.7mM glucose was added. The values are expressed as mean + SEM of 4 perfusions.

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and the aliquots counted using a liquid scintillation counter. Aliquots were also stored at - 2 0 ° C for the insulin assay. At the end of the perfusion, the radioactivity of the islets was also determined. 45Ca2+ efl~ux is expressed as the fractional outflow

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T i m e (rain) Fig. 2. Effect of dopamine (100/1 M) on insulin secretion and 45Ca+2 ouflow rate (FOR) in perfused islets. The islets were pre-incubated in Krebs-Henseleit buffer having 5.6 mM glucose. Afterwards, the islets were perfused in the presence of 5.6 mM glucose during 40 min. At this time, as indicated by vertical interrupted line, the perfusion medium was changed: (1) control conditions were maintained at 5.6 mM glucose and (2) dopamine (100 #M) was added. The values are expressed as mean __+SEM of 4 perfusions.

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Perfusion experiments 100 islets were pre-incubated for 6 0 m i n in 0.2 ml Krebs-Henseleit solution containing glucose (5.6mM) and 45Ca2+ (0.2mCi/ml). After preincubation, the islets were transferred to a perfusion chamber and retained on a filter (cellulose a c e t a t e - S C W P I00, 8.0 # m pore size). The perfusion medium was pumped through the filter at a constant rate ( l m l / m i n ) and collected with a fraction collector (Ultrorac II, LBK, Broma 2070). During the first 40min, the islets were perfused with one medium which was replaced by another one for the following 30 min. The differences between media are detailed in the figures. The perfusate was collected at 1 min intervals from 30 to 60 min,

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T i m e (rain) Fig. 3. Effect of glucose load and dopamine (100#M) on insulin secretion and 45Ca2+ outflow rate (FOR) in perfused islets. The islets were pre-incubated in Krebs-Henseleit buffer having 5.6 mM glucose. Afterwards, the islets were perfused in the presence of 5.6mM glucose and 100/~M dopamine during 40min. At this time, as indicated by vertical interrupted line, the perfusion medium was changed: (1) control conditions were maintained at 5.6 mM glucose and 100 #M dopamine and (2) 16.7 mM glucose plus dopamine (100 #U) was perfused. The values are expressed as mean + SEM of 4 perfusions.

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Fig. 4. Effect of 20 mM potassium on insulin secretion and 45Ca2+ outflow rate (FOR) in perfused islets. The islets were pre-incubated in Krebs-Henseleit buffer having 5.6 mM glucose. Afterwards, the islets were perfused in the presence of 5.6 mM glucose during 40 min. At this time, as indicated by vertical interrupted line, the perfusion medium was changed: (1) control conditions was maintained at 5.6 mM glucose and (2) 20 mM Potassium was added with (B side) and without (A side) dopamine (100 #M). The values are expressed as means +__SEM of 4 perfusions.

rate (FOR), which represents the ratio of the isotope efflux to the islet isotope content for a given time interval. Statistical analysis

Statistical analysis was made by A N O V A to compare the mean values of incremental integrated areas for each interval of time. The significance level was set for P < 0.05.

RESULTS Figure 1 shows that 16.7 m M glucose increased insulin secretion and 45Ca2+ F O R from islets preincubated for 6 0 m i n in the presence of 5.6 m M glucose and 2 0 0 # C i 45Ca2+. When dopamine (100/~M) was added, instead of 16.7mM glucose (Fig. 2), an increase in insulin secretion and 4SCa2" F O R was also observed. When dopamine was present

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throughout the perfusion period (Fig. 3), insulin secretion, in the presence of 5.6 mM glucose, was much higher than the control values (compare Figs 1 and 3). The glucose load markedly decreased insulin secretion near to the values obtained for basal glucose concentration and in the absence of dopamine. Figure 4(A) shows the stimulatory effect of 20 mM potassium on insulin secretion [Fig. 4(A), upper panel] and 45Ca2+ FOR [Fig. 4(A) lower panel]. When 100/~ M dopamine was added at the same time as 20mM potassium [Fig. 4(B)] no alterations on insulin secretion [Fig. 4(B), upper panel] and a modest decrease in 45Ca2+ F O R were detected [Figure 4(B), lower panel]. The addition of 16.7 mM glucose and 100/zM dopamine markedly increased insulin secretion and tended to decrease 45Ca2+ FOR (Fig. 5). Figure 6 shows insulin release by islets incubated for 60 min at 37°C in the presence of increasing glucose concentrations. When 100 p M dopamine was added, insulin secretion was inhibited except in the presence of 5.6 mM glucose when there was a significant increase. The presence ofinhibitors of dopamine catabolism did not alter insulin response. DISCUSSION

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Time (min) Fig. 5. Effect of glucose load and dopamine on insulin secretion and 4SCa:+ outflow rate (FOR) in perfused islets. The islets were preincubated in Krebs-Henseleit buffer having 5.6 mM glucose. Afterwards, the islets were perfused in the presence of 5.6 mM glucose during 40 rain. At this time, as indicated by vertical interrupted line, the perfusion medium was changed: (I) control conditions were mainmined at 5.6 mM glucose and (2) 16.7 mM glucose plus dopamine (100 #U) was added. The values are expressed as mean + SEM of 4 perfusions.

The experiments were planned to be as close as possible to in vivo conditions: islets were pre-incubated for 45Ca2+ labeling in the presence of 5.6 instead of 16.7 mM glucose as usually used by several research groups (Malaisse et al., 1978; Malaisse, 1973; Wollhelm et al., 1981) and the perfusion experiments were performed by increasing glucose concentrations in the perfusion medium from 5.6 to 16.7 mM glucose. In agreement with our previous observations (Santos et al., 1988), glucose load (16.7 mM) provoked an increase in 45Ca2+ FOR (which reflects the cellular calcium enter) without the usual initial decrease (Fig. 1) observed when glucose load was raised from 0 to 16.7 mM (Malaisse et al., 1978; Malaisse, 1973). Figure 2 shows an increase in insulin secretion and a modest elevation of 45Ca2+ F O R when 100 # M dopamine was added to the perfusion medium containing 5.6 mM glucose. Although dopamine acutely inhibits islet calcium uptake (Lindstrom et al., 1982) the present results could be explained by the fact that, it would provoke, in the presence of low glucose concentration, organelle calcium release enhancing cytosolic ionic calcium (Ahren and Lindquist, 1985) with a consequent increase in 45Ca2+ outflow. The prolonged exposure of islets (30 min) to dopamine in the presence of 5.6 mM glucose clearly increased basal insulin (Fig. 3, as compared with Fig. 1). Possibly, chronical dopamine stimulation causes removal and depletion of calcium from organelles

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Glucose concentration (mM) Fig. 6. Insulin secretion in isolated islets incubated in the presence of different glucose concentrations. The incubations were performed in Krebs-Henseleit medium with no addition (Control), in the presence of dopamine (100#M) and in the presence of dopamine metabolism inhibitors; pargyline and dietyl carbamate; 200 mM each (dopamine + inhibitors), and control conditions plus dopamine metabolism inhibitors (Control + inhibitor). The values are expressed as mean -t- SEM of 10 incubations.

(Ahren and Lindquist, 1985), such as endoplasmic reticulum, increasing cytosolic ionic calcium which in turn will lead to insulin release. This may explain the high levels of insulin release in the presence of dopamine and low glucose levels as compared to the control conditions but needs more experiments to be confirmed. However, glucose load of 16.7 mM under these conditions (Fig. 3) provoked a surprising decrease in insulin secretion with no alteration in 45Ca2+ FOR. The paradoxical effect of glucose load on insulin output (Carpinelli et al., 1992) is not fully understood. In a speculative manner it could be postulated that a reduction in the availability of cytosolic calcium might play an important role for this process takes place. The lowering of cytosolic calcium would be caused by a sequence of 3 events: (a) the gradual loss of calcium removed from the organeUes dopamine, during insulin release by 5.6 mM glucose stimulus, would decrease total calcium content in the B-ceils, (b) formation of glucose-6-phosphate by glucose phosphorylation would increase calcium uptake by endoplasmic reticulum (Wolf et al., 1988) and (c) low calcium uptake by the cells due to dopamine inhibiting effect on voltagesensitive calcium channels. The latter possibility was

confirmed by using potassium (20 mM) to depolarize B-cells opening VSCC, increasing 45Ca2+ FOR [Fig. 4(A)]. Indeed, this potassium effect was abolished [Fig. 4(B)] when 100 # M dopamine was added into the medium which could contribute for the decrease of intra-ceilular calcium concentration. The reduced content of calcium in the organelles might avoid the replenishment of cytosolic calcium by mediators such as inositol-triphosphate, produced during glucose metabolism in B cells. The establishment of a low cytosolic calcium state would enable B-cells to secrete insulin. The measurements of cytosolic calcium concentration has to be performed to follow this speculative proposition. The results presented in Fig. 5 led us to consider two possible mechanisms for the acute and prolonged control of insulin release by dopamine. A prolonged stimulus of dopamine would result in low cytosolic calcium state being responsible for the paradoxical glucose effect shown in Fig. 3; as proposed above in a speculative manner. Under acute stimulation, however, this amine might increase cytosolic calcium due to its removal from the organelles (Ahren and Lindquist, 1985; Lindquist et al., 1980), potentiating

Dopamine and insulin secretion insulin release by 5 . 6 m M (Fig. 3) and 16.7mM (Fig. 5) glucose stimuli. Therefore, it is conceivable that dopamine determines consecutive high and low cytosolic calcium states in the islets. The duration of these two conditions may vary as a function of the glucose concentration. In order to better study the prolonged effect of dopamine on insulin secretion, islets were incubated in the presence of 100/~M dopamine, inhibitors of dopamine metabolism and increasing glucose concentrations during 1 hr. In agreement with the results of the perfusion experiments (Fig. 3), insulin secretion by incubated islets at 5.6 m M glucose was increased by dopamine and decreased by enhancing glucose concentrations. It is not clear why this amine did not increase insulin secretion in 0, 2.2 and 4.4 m M glucose, however, under these conditions, several metabolic parameters are modified and insulin release could be impaired (Malaisse et al., 1979). Although systematic study on dopamine receptors in pancreatic islets has not been carried, Takeuchi et al. (1990) proposed the presence of DA1 and DA2 receptors by analyzing the effect of different catecholamine agents on pancreatic islets. The stimulation and inhibiting effects of dopamine on insulin secretion in the present studies are compatible with this proposition. Based on studies in other tissues (Vallar and Meldolesi, 1989), DA1, by increasing intracellular cyclic AMP, would enhance insulin release, whereas DA2, by decreasing c A M P levels and opening potassium channels, would provoke a reduction. These points taken together led us to conclude that bioactive amines have an important role in the modulation of the process of glucose-induced insulin secretion. The occurrence of paradoxical changes in insulin output, as often observed in non-insulin dependent diabetes and experimental conditions in which hyperglycemia is present, must be considered as one possible consequence of a dopamine modulating effect. Nevertheless, the mechanisms involved have to be investigated. Acknowledgements--The authors are grateful to A. F.

Meira for the revision of the manuscript style and to M. S. Rocha, J. C. B. Gonqalves, R. S. Nascimento and R. R. Valentin for technical assistance. REFERENCES

Ahren B. and Lundquist, I. (1985) Effects of 1-dopa induced dopamine accumulation on 45Ca2+ effiux and insulin secretion in isolated rat islets Pharmacology 30, 71-82. Arkhammar P., Nilson T. and Berggren P. O. (1989) Glucose-induced changes in cytoplasmic free Ca 2+ concentration and the significance for the regulation of GP

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insulin release. Measurements with Fura-2 in suspensions and single aggregate of mouse pancreatic B-cells. Cell Calcium 10, 17-27. Aschroft F. M., Harrinson D. E. and Ashroft S. J. H. (1984) Glucose induces closure of single potassium channels in isolated rat pancreatic beta cells. Nature 312, 446-448. Carpinelli A. R., Nogueira C. R., Machado U. F., Curl R. and Malaisse W. J. (1992) Paradoxical inhibition of insulin release by D-glucose in islets exposed to dopamine. Horm. Metabol. Res. 24, 452-433. Cegrell L. (1968) The occurrence of biogenic monoamines in the mammalian endocrine pancreas. Acta PhysioL Scand. 314, 1-60. Cegrell L. (1970) Monoaminergic mechanisms in the pancreatic alfa- cells. The Structure and Metabolism of the Pancreatic lslets. (Edited by Falkner S., Hellman B. and Talijedal I. B.) Wenner Gren Center International Symposium Series 'Vol. 16. London, Pergamon, p. 131. Ericson L. E., Hakason R. and Lundquist I. (1977) Accumulation of dopamine in mouse pancreatic B-cells following injection of 1-dopa. Localization to secretory granules and inhibition of insulin secretion. Diabetologia 13, 117-27. Feldman J. M., Boyd A. E. III and Lebovitz H. E. (1971) Structural determinants of catecholamine action on in vitro insulin release. J. Pharmacol. Exp. Ther. 176, 611-21. Gylfe E., Hellman B. and Shelin J. (1973) Aminoacid conversion into 5-hydroxytriptamine in pancreatic beta cells. Endocrinology 93, 932-1023. Krebs H. A. and Henseleit K. (1932) Untersuchnge uber die harnstoffbildung in tierkorper Hoppe-Seyler's. Z Phy. Chem. 210, 33-66. Lindstrom P. (1982) Modification of mouse islets function by 5-hydroxytryptamine, dopamine and the precursors. Acta BioL Med. Germ. 41, 1185-90. Lorenzi M., Karan J. H., Tsalikian E., Bohannon N. V., Gerich J. E. and Forsham P. H. (1979) Dopamine during alfa or beta adrenergic blockade in man. J. Clin. Invest. 63, 310-7. Lundquist I., Ahren B., Hakanson R. and Sundler F. (1980) The role of intracellular amines in islet regulation. In Waldhausl, W. K. Diabetes (Edited by Waldhausl W. K.), pp. 57-63. Excerpta Medica, Amsterdam. Malaisse W. J. (1973) Insulin secretion-multifactorial regulation for a single process of release. Diabetologia 9, 167-73. Malaisse W. J., Herchuelz A., Devis G., Somers G., Boschero A. C., Hutton J. C., Kawazu S., Sener A., Atwater I. J., Duncan G., Ribalet B. and Rojas E. (1978) Regulation of calcium fluxes and their regulatory roles in pancreatic islets. A. N.Y. Acad. Sci. 307, 562-582. Malaisse W. J., Sener A., Herchuelz A. and Hutton J. C. (1979) Insulin release: the fuel hypothesis. Prog. Endocrinol. Metab. 28, 373-86. Morgan C. R. and Lazarow A. (1963) Immunoassay of insulin: two antibody systems: plasma insulin levels of normal, subdiabetic and diabetic rats. Diabetes 12, 115-21. Pupo A. A. and Marreiro D. (1970) Dosagem de insulina pelo radioimunoensaio com duplo antiocorpo. Rev. Assoc. Med. Bras. 16, 153-55. Santos A. Jr Villela F. G., Machado U. F., Curi R. and Carpinelli A. R. (1988) Insulin secretion in the isolated islets of single-, regular-fasted and fed rats. Physio. Behav. 45, 923-927. Takeuchi S., Ocamura T., Shonai K. and Imamura S. (1990) Distribution of catecholaminergic receptors in the rat's pancreas islets. Nippon Ika Daigaku Zasshi 57, 119-26.

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Vallar L. and Meldolesi J. (1989) Mechanisms of transduction at the dopamine D 2 receptor. Trends Pharmacol. Sci. 10, 74-77. Wolf B. A., Colca J. A., Turk J., Florholmen J. and McDaniel M. L. (1988) Regulation of Ca :+ homeostasis in islets endoplasmic reticulum and its role in insulin secretion. Am. J. Physiol. E121-E132.

Wollheim C. B. and Sharp G. W. G. (1981) Regulation of insulin release by Calcium. Physiol. Rev. 61, 91-73. Yu E. W.-T,, Stern L. and Tenenhouse A. (1984) Decarboxylation of l-dopa and 5-hydroxytryptophan in dispersed rat pancreas cells. Pharmacology 29, 185-92.