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Carbachol has opposite effects to glucose in raising the sodium content of pancreatic islets
European Journal of Pharmacology, 204 ( 1991) 211-2 ! 5
211
© 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50
EJP 52114
Carbachol has opposite effects to glucose in raising the sodium content of pancreatic islets S a m i r S a h a a n d Bo H e l l m a n Department of Medical Cell Biology, Unicersityof Uppsala. Biomedicum. Box 571. S-751 23 Uppsala. Sweden
Received 23 May 1991, revised MS received 19 July 1991, accepted 13 August 1991
Integrating flame photometry was used for measuring sodium in single pancreatic islets from ob/ob mice. Exposure to 100 .aM carbachol resulted in a 25-40% increase in sodium without any effect on potassium during incubation with 0-5 mM glucose in media deficient or not in Ca 2+. This action of carbachol was abolished by 10 ,aM atropine or by raising the glucose concentration to 20 mM. A minor increase of the steady state content of sodium occurred in the presence of 200 ,aM ATP or 10 nM tetradecanoylphorbol 13-acetate (TPA). Carbachol differed from TPA in markedly stimulating sodium accumulation after ouabain inhibition of the Na/K pump. The results indicate that muscarinic receptor activation has opposite effects to glucose in inducing a rise of the islet content of sodium. It is suggested that the cholinergic control of the endocrine pancreas involves entry, of Na + in addition to the Na + entry mediated by protein kinase C activation of Na+/H ÷ countertransport. Carbachol; Muscarinic receptors; Na +; Pancreatic islets
1. Introduction It is generally believed that the insulin-releasing E-cells are sensitized to the glucose stimulus by acetylcholine released from vagal efferents within the pancreatic islets during the cephalic phase of digestion (Miller, 1981; Ahr6n et al., 1986; Rasmussen et al., 1990). Previous studies in our laboratory have indicated that muscarinic receptor activation results in biphasic mobilization of intracellular calcium from the /3-cellrich pancreatic islets of o b / o b mice (Hellman and Gylfe, 1986; Hellman et al., 1987). The two phases differed with respect to their dependence on glucose, the sugar being a prerequisite for the initial phase but suppressing the other. Whereas the first phase was attributed to the inositol-l,4,5-trisphosphate (IP 3) generated during polyphosphoinositide breakdown, the second one was proposed to result from increased entry to Na +. Although radioisotope studies have provided support for a rise of islet sodium after muscarinic receptor activation (Gagerman et al., 1980; Henquin et al., 1988), this increase remains to be demonstrated by direct measurements. The high sensitivity offered by integrat-
Correspondence to: B. Heliman, Department of Medical Cell Biology, Biomedicum, Box 571, S-751 23 Uppsala, Sweden. Tel. 46.18.174 424. fax 46.18.556 401,
ing micro flame photometry allowed us to show that the muscarinic receptor agonist, carbachol, does induce a substantial rise of islet sodium and that this effect is co-nteracted by glucose. Evidence is provided that receptor activation is coupled to an entry of Na ÷ additional to that mediated by stimulated N a + / H ~countertransport.
2. Materials and methods Deionized water and reagents of analytical grade were used. ATP, carbachol, tetradecanoylphorbol-13acetate (TPA), dimethyl sulphoxide (DMSO), ouabain, E G T A and bovine serum albumin (fraction V) were from Sigma Chemical Co. (St. Louis, MO). Collagenase was obtained from Boehringer Mannheim GmbH (Mannheim, Germany). Adult o b / o b mice were from a non-inbred colony (Hellman, 1965) and were sta~'ed overnight. The animals were killed by decapitation under ether anesthesia and pancreatic islets were isolated with collagenase. Previous studies have indicated that these islets contain more than 90%/t-cells, which respond normally to glucose and other stimulators of insulin release (Hellman, 1970; Hahn et al., 1974). The basal medium used for the experiments was buffered with 25 mM HEPES and physiologically balanced cations (raM: Na + 137, K + 5.9, Mg 2÷ 1,2 and Ca'-" 1.28)with CI- as the sole
2~2
anion (HeUman. 1975). A Ca-'*-deficient variety of this medium was obtained by omission of CaCI_, and supplementing the medium with 1 mM EGTA. All media were equilibrated with ambient air and contained 1 m g / m l albumin. The islets were preincubated for 45 min at 37 ° C with 3 mM glucose then exposed to media modified as indicated in the legends to the tables and figures. At the end of the experiments the islets were washed for 2 min with an ice-cold solution of sucrose (Wessldn et al., 1986). Subsequent freeze-drying and weighing of the islets made it possible to express the sodium and pota~ium contents in terms of dry weight. Single islets (dry weight 0.8-2.5 /.tg) were analysed for sodium and potassium by means of integrating flame photometr3." according to previously described procedures (Wessldn et al., 1986; Bergsten et al., 1986). Each observation was based on five to eight measurements. Statistical significances for differences between paired observations were evaluated by means of Student's t-test.
3. Results The islet content of sodium after 60 min of incubation with or without glucose is shown in table 1. The addition of 5 or 20 mM of the sugar resulted in 30-40% lowering of sodium. The reductions were equivalent to 40 + 6 and 27 + 4 m m o l / k g dry weight respectively (P < 0.001). Whereas carbachol (100 /.tM) induced a rise of sodium in media with or without 5 mM glucose, this cholinergic agent had no effect when the concentration of the sugar was raised to 20 mM. ATP (200 # M ) was effective in raising sodium only in the presence of 5 mM glucose. Table 2 presents the results of similar analyses in a medium deficient in
TABLE 2 Effects of carbachol and ATP on islet content of sodium in a medium deprived of Ca-"÷. The islets were incubated for 60 min with or without 100 # M carbachol or 200 g M ATP at different concentrations of glucose in a Ca-'+-deficient medium supplemented with 0.5 mM EGTA. The sodium contents are given as m m o l / k g dry weight and represent the mean values+S.E, for eight experiments. Statistical significances for carbachol or ATP included in the media are indicated by a p < 0.025. Glucose (mM)
Additions
Sodium content Observed
Effect of additions
0
-
136+
7
0
0
Carbachol ATP
193+24 135+ 12
+57+-21 a -2+ 8
-
5 5 5
Carbachoi ATP
103+ 8 125+ 10 108+ 7
+22+- 11 +5+- 9
20 21) 20
Carbachol ATP
95 + 5 114+ 10 95-+ 8
+ 19+ 9 0_ 6
Ca 2+, an experimental situation associated with a general increase of sodium. In this case also there was a lowering of sodium in the presence of glucose (P < 0.001). In the CaE+-deficient medium carbachol induced a rise of sodium only in the absence of glucose, and ATP had no effect irrespective of whether the sugar was present or not. Figure 1 shows simultaneous measurements of islet sodium and potassium at different glucose concentra-
SODIUM
I-,"1-
o
POTASSIUM
200
B
IJJ >n"
a
o~ 150
TABLE ! Effects of carbachol and ATP on islet content of sodium. The islets were incubated for 60 rain with or without 100/.tM carbachol or 200 # M A'I-P at different concentrations of glucose. The sodium contents are given as m m o l / k g dry weight and represent the mean values+ S.E. for seven experiments. Statistical significances for carbachol or ATP included in the media are indicated by ~ P < 0.01; h p < 0.005. Glucose CmM)
Additions
0 0 0 5 5 5 20 20 20
Carbachol ATP Carbachol ATP Carbachoi ATP
lOO
50
Sodium content Observed
Effect of additions
101-4-_ 10 129 + 7 96+_ 8 61_+ 5 93 + 7 76+ 5 73_+ 7 83+ 11 69+ 7
+ 29 + 8 a -64-6 + 32 + 6 h + 15_+4 a + 10±6 -4-+2
0
3
20
0
3
20
GLUCOSE CONCENTRATION (mM) Fig. 1. Effects of carbachol on islet contents of sodium and potas-
sium at different glucose concentrations. The islets were incubated for 15 min with (hatched bars) or without (open bars) 100 ,ttM carbachol in media containing 0, 3 or 20 mM glucose. Mean values + S.E. for nine experiments. The presence of carbachol resulted in significant increases of sodium in the media lacking (P < 0.025) or containing (P < 0.001) 3 mM glucose.
213 TABLE 4 Effects of carbachol and TPA on the increase of islet sodium in response to ouabain. The islets were exposed for 5 min to 1 mM ouabain in the presence or absence of 100 ttM carbachol or 10 nM TPA in media containing 3 or 20 mM glucose. The increases of sodium from an initial value of 58 + 4 mmol/kg dry weight are given as mean values + S.E. for seven experiments, Statistical significances for carbachol in the medium are indicated by " P < 0.02; b p < 0.001.
30
Glucose
20
Additions
(raM) 10
0.1
1
10
100
Sodiumincrease Observed
Effect of additions
3 3 3
Carbachol TPA
21 +3 50 + 3 23 + 3
+ 29 + 2 b + 2+ 3
20 20
Carbachol
40+3 54+2
+ 14+4 a
1000
CARBACHOL (pM)
Fig. 2. The increase of islet sodium obtained with different concentrations of carbachol. The islets were incubated for 45 min with 0.1-1000/zM of carbachol in the presence of 3 mM glucose. The bars indicate the mean values+S.E, for the rise of sodium from a control value of 74 + 3 mmol/kg dry weight in five experiments.
tions after 15-min exposure to carbachol. W h e r e a s the potassium c o n t e n t r e m a i n e d unaffected, additional evidence was o b t a i n e d for a c a r b a c h o l - i n d u c e d rise of sodium c o u n t e r a c t e d by 20 m M glucose. Studies of the dose d e p e n d e n c e revealed that the maximal s~dium increase was a t t a i n e d with 100/.LM carbachol (fig. 2). T h e effect was already evident after 2 min (not shown) and was s u p p r e s s e d by atropine. A f t e r 45 min of exposure to 100/.~M carbachol the rise of sodium observed at 3 m M glucose in the absence and presence of 10 # M a t r o p i n e was 24 + 6 and 7 + 5 m m o l / k g dry weight respectively (n = 5). T h e sodium c o n t e n t could also be raised with the phorbol ester, T P A . T a b l e 3 illustrates this effect after 15 min of exposure to 10 n M T P A in a m e d i u m
TABLE 3 Effects of carbachol and q'P~ on islet content of sodium. The islets were incubated for 15 min with or without 100 #M carbachol or 10 nM TPA in media containing 3 or 20 mM glucose. All media cop.rained 0.1% DMSO, used for preparing a stock solution of TPA. The sodium contents are given as mmol/kg dry weight and represent the mean values±S.E, for the number of experiments shown in parentheses. Statistical significances for carbachol and/or TPA ipcluded in the media are indicated by " P < 0.01; b p < 0.901. Glucose (mM)
Additions
Sodium content Observed
Effect of additions
Carbachol TPA Carbachol + TPA
63+3 (17) 82+9 (8) 70±4 (17) 82 + 7 (8)
+19±5" +7±i h +19±2 h
20
-
57+5
20
TPA
61±5 (7)
3 3 3 3
(7)
+5+2
c o n t a i n i n g 3 m M glucose. T P A was less effective than carbachol to raise sodium and no additive effects were seen when the two c o m p o u n d s were combined. T h e r e was no further rise of sodium when the T P A concentration was raised to 100 nM or the exposure time e x t e n d e d to 60 min (not shown). Table 4 shows the net increase in sodium during the 5-min period following inhibition of the N a / K p u m p with ouabain. W h e r e a s the presence of 100 t t M carbachol more than d o u b l e d the sodium u p t a k e in the presence of 3 m M glucose the addition of T P A had no effect. Raising glucose to 20 mM resulted per se in an increased net uptake of sodium, a process a c c e n t u a t e d by the simultaneous presence of carbachol. T h e carbachol effects were less at 3 mM than at 20 mM glucose (P < 0.05).
4. D i s c u s s i o n
P a r a s y m p h a t h e t i c nerves form peri-insular plexuses allowing direct innervation of the islet cells (Miller, 1981). T h e functional significance of this i~nervation is e m p h a s i z e d by the observation of a 10-fold higher c o n c e n t r a t i o n of choline acetyltransfetase in islets than in the exocrine p a n c r e a s (Godfrey and Matschinsky, 1975). Acetylcholine has been reported to have stimulatory actions not only on insulin secretion but, in some species, also on the release of glucagon, somatostatin and pancreatic p o l y p e p t i d e (AhrEn et al., 1986). It is well established that cholinergic agents act by binding to muscarinic receptors (Grill and 0 s t e n s o n , 1983), and there are reasons to believe that M 3 is the pred o m i n a t i n g receptor subtype in the islets (Verspohl et al., 1990). Various observations suggest that muscarinic receptors are important for islet handling of Na ÷. Both the delayed mobilization of intracellular islet calcium initiated by carbachol ( H e l l m a n and Gylfe, 1986; Hellman
214
et al,, 1987), and the acetylcholine-induced depolarization of the/3-cells (Henquin et ai., 1988) require extracellular Na + Moreover, acetylcholine has been reported to stimulate the uptake of 22Na in isolated mouse islets (Gagerman et al., 1980; Henquin et al., 1988). It is evident from the present study that these radioisotope data reflect a muscarinic-induced increase of the sodium content and not simply a stimulated exchange with non-radioactive sodium. Accordingly, cholinergic agents differ from glucose with regard to their effect on islet handling of sodium. As suggested from a radioisotope approach (Kavazu et al., 1978), and confirmed by previous (Wessl6n et ai., 1986; Ali and Hellman, 1991) and present measurements of sodium, the overall effect of glucose is to increase the turnover of sodium, implying that an enhanced entry of Na* is overcompensated by stimulated extrusion, Although the observed values for islet sodium compare favourably with those obtained with the radioactive approach (Sehlin and T~iljedal, 1974; Kavazu et ai., 1978: Gagerman et al., 1980), they are considerably higher than estimates of Na + activity based on membrane potential recordings (Atwater et al., 1978; Meissner et ai., 1978) or from direct measurements with the fluorescent indicator SPFI (Ali et al., 198%). Accordingly, it is likely that only a minor part of the /3-cell sodium occurs in an ionized form. External ATP mediates a similar mobilization of calcium from the pancreatic/3-cells, as noted on stimulation of polyphosphoinositide breakdown with carbachol (Gylfe and Hellman, 1987). By demonstrating an ATP-induced rise of sodium in the presence of 5 mM glucose, the present study provides further evidence that purine P2 receptors mimic muscarinic ones with regard to effects on the internal ionic milieu. A similar relationship seems to exist in Ehrlich ascites tumour cells, which have been reported to respond to both ATP and carbachol with a rise of intracellular sodium based on activation of amiloride-sensitive N a + / H ÷ countertransport (Wiener et al., 1986). In addition to providing definite evidence that muscarinic receptor activation is an effective means of raising islet sodium, this increase was found to be counteracted by 20 mM glucose. It is not surprising that such an opposing action had escaped detection in previous measurements of the radioactive 22Na uptake (Gagerman et al., 1980; Henquin et al., 1988), since these studies were restricted to glucose concentrations up to 11 mM. The observation that glucose opposes the carbachol-induced rise of sodium may explain why the sugar inhibits the Na+-dependent efflux of Ca 2+ induced by muscarinic receptor activation (Hellman and Gylfe, 1986; Hellman et al., 1987). Glucose also suppresses the Ca 2÷ mobilization obtained when intraceilular sodium is raised by removal of extracellular K ÷ or addition of ouabain or veratridine (Hellman et al.,
1982). The ability of glucose to prevent an increase in sodium is not restricted to situations of raised intracellular contents of the element. As repeatedly observed in our laboratory (Wessl6n et al., 1986; Ali and Hellman, 1991) and also as shown from the present data, a rise of glucose to 5 mM results iit a reduction of the islet content of sodium. This effect, as well as the interference by glucose with the carbachol-induced rise of sodium, was found to persist in a medium depleted of Ca 2÷. The glucose-induced lowering of the sodium content has been attributed to an increased outward transport mediated at least in part by mechanisms other than stimulation of the N a / K pump (Ali et al., 1989b; Ali and Hellman, 1991). It seems likely from the present observations that glucose suppression of the sodium response to muscarinic receptor activation also involves interference with the influx of the ion. it is well established that activation of nicotinic receptors results in an enhanced Na ÷ permeability in various types of cells (Hucho, 1986). The fact that muscarinic receptors also may sometimes initiate a ~ubstantial Na ÷ influx was clearly demonstrated from the present islet measurements. A similar increase occurs in the exocrine pancreas (Petersen, 1976; Putney et al., 1980), suggesting that Na ÷ is involved in the vagal control of both parts of the gland. Muscarinic receptors are potent stimulators of the islet hydrolysis of phosphoinositides, providing diacylglycerol for stimulation of protein kinase C (Peter-Riesch et al., 1988; Arkhammar et al., 1989). Activation of this enzyme often promotes N a ÷ / H ÷ countertransport (Rothstein, 1989). However, it is difficult to believe that such an activation is a major explanation for the rise of islet sodium evoked by muscarinic receptor activation. After TPA stimulation of protein kinase C the sodium content was only slightly elevated and the phorbol ester did not promote an additional rise of the element following inhibition of the N a / K pump. Whereas cholinergic agents do not affect basal insulin release, they markedly potentiate the secretion obtained when the glucose concentration is raised (Gagerman et al., 1980; Ahr6n et al., 1986). In discussing the functional significance of the muscarinic effects on /3-ceU handling of Na ÷ it is necessary to consider both the results of depolarization mediated by the enhanced entry of the ion and a rise of its intracellular activity. As emphasized by Henquin et al. (1988) a contribution to the glucose-induced depolarization can be expected to aid in the opening of voltage-dependent Ca 2÷ channels, which in turn stimulates insulin release. An increase of cytoplasmic Na ÷ as such may also initiate secretory activity by mobilizing Ca 2÷ from intracellular stores and interfere with its outward transport (Hellman, 1986). With regard to the observation that glucose opposes the increase of islet sodium it seems likely that it is the depolarization rather than
215
the rise of intracellular Na ÷ which is essential for muscarinic potentiation of the stimulatory action of the sugar on insulin release.
Acknowledgements This study was supported by grants from the Swedish Medical Research Council (12x-562), the Swedish Diabetes Association, the Nordic Insulin Foundation, the Swedish Hoechst and Novo-Nordisk Companies and the Family Ernfors Foundation. Samir Saha is a scientific officer from the Bangladesh Institute for Research and Rehabilitation on Diabetes, Endocrine and Metabolic Disorders (BIRDEM), working as a visiting research fellow sponsored by the International Program in the Chemical Sciences.
References Ahr6n, B., G.J. Taborsky, Jr. and D. Porte, Jr., 1986, Neuropeptidergic versus cholinergic and adrenergic regulation of islet hormone secretion, Diabetologia 29, 827. All, L. and B. Hellman, 1991, Glucose stimulation of ouabain-resistant efflux of Na + from rat pancreatic islets, J. Physiol. 435, 295. Ali, L., E. Grapengiesser, E. Gylfe, B. Hellman and P.-E. Lund, 1989a, Free and bound sodium in pancreatic g-cells exposed to glucose and tolbutamide, Biochem. Biophys, Res. Commun, 164, 212. Ali, L., N. Wessldn and B. Hellman, 1989b, Effects of depolarizing agents on the sodium content of rat pancreatic islets, Biochim. Biophys. Acta 1010, 283. Arkhammar, P., T. Nilsson, M Welsh, N. Welsh and P.O. Berggren, 1989, Effects of protein kinase C activation on the regulation of the stimulus-secretion coupling in pancreatic g-cells, Biochem. J. 264, 207. Atwater, I., B. Ribalet and E. Rojas, 1978, Cyclic changes in potential and resistance of the g-cell membrane induced by glucose in islets of Langerhans from mouse, J. Physiol. 278, 117. Bergsten, P., F. Rorsman and B, Hellman, 1986, Modifications of the pancreatic g-cell function after lowering their potasJium content, Acta Physiol. Scand. 128, 619. Gagerman, E., J. Sehlin and I.-B. T~iljedal, 1980, Effects of acetylcholine on ion fluxes and chlortetracycline fluorescence in pancreatic islets, J. Physiol. 300, 505. Godfrey, D.A. and F.M. Matschinsky, 1975, Enzymes of the cholinergic system in islets of Langerhans, J. Histochem. Cytochem. 23, 645. Grapengiesser, E., E. Gylfe and B. Hellman, 1989, Regulation of pH in individual pancreatic g-cells as evaluated by fluorescence ratio microscopy, Biochim. Biophys. Acta 1014, 219. Grill, V. and C.G. tgstenson, 1983, Muscarinic receptors in pancreatic islets of the rat. Demonstration and dependence on long-term glucose environment, Biochim. Biophys. Acta 756, 159. Gylfe, E. and B. Hellman, 1987, External ATP mimic~-carbachol in initiating calcium mobilization from pancreatic g-cells conditioned by previous exposure to glucose, Br. J. Pharmacol. 92, 281. Hahn, H.-J,, B. Hellman, A. Lernmark, J. Sehlin and I.-B. Tiiljedal, 1974, The pancreatic /3-cell recognition of insulin secretagogues. Influence of nel)raminidase treatment on the release of insulin and the islet content of insulin, sialic acid and cyclic adenosine 3':5'-monopho:;phate, J. Biol. Chem 249, 5275.
Hellman, B., 1965, Studies in obese-hyperglycemic mice, Ann. N.Y. Acad. Sci. 131,541. Hellman, B., 1970, Methodological approaches to studies on the pancreatic islets, Diabetologia 6, 110. Hellman, B., 1975, The significance of calcium for glucose stimulation of insulin release, Endocrinology 97, 392. Heliman, B., 1986, Calcium transport in pancreatic g-cells: Implications for glucose regulation of insulin release, Diabet. Metab. Rev. 2, 215. Hellman, B. and E. Gylfe, 1986, Mobilization of different intracellular calcium pools after activation of muscarinic receptors in pancreatic/]-cells, Pharmacology 32, 257. Hellman, B., T. Honkanen and E. Gylfe, 1982, Glucose inhibits insulin release induced by Na + mobilization of intracellular calcium, FEBS Lett. 148, 289. Hellman, B., E. Gylfe and P. Bergsten, 1987, Mobilization of different pools of glucose-incorporated calcium in pancreatic g-cells after muscarinic receptor activation, in: Biophysics of the Pancreatic g-Cell, eds. I. Atwater, E. Rojas and B. Soria (Plenum Press, New York) p. 325. Henquin, J.C., M.C. Garcia, M. Bozem, M.P. Hermans and M. Nenquin, 1988, Muscarinic control of pancreatic g-cell function involves sodium-dependent depolarization and calcium influx, Endocrinology 122, 2134. Hucho, F., 1986, The nicotinic acetylcholine receptor and its ion channel, Eur. J. Biochem. 158, 211. Kavazu, S., A.C. Boschero, C. Delcroix and W.J. Malaisse, 1978, The stimulus-secretion coupling of glucose-induced insulin release. XXVII. Effect of glucose on Na + fluxes in isolated islets, Pfliigers Arch. 375, 197. Meissner, H.P., J.C. Henquin and M. Preissler, 1978, Potassium dependence of the membrane potential of pancreatic g-cells, FEBS Lett. 94, 87. Miller, R.E., 1981, Pancreatic neuroendocrinology: Peripheral neural mechanisms in the regulation of the islets of Langerhans, Endocrinol. Rev. 2, 471. Peter-Riesch, B., M. Fathi, W. Schlegel and C.B. Wollheim, 1988, Glucose and carbachol generate 1,2-diacylglycerols by different mechanisms in pancreatic islets, J. Clin. Invest. 81, 1154. Petersen, O.H., 1976, Electrophysiology of mammalian gland cells, Physiol. Rev. 56, 535. Putr~cv, J.W., C.A. Landis and C.M. Van de Walle, 1980, Effect of carbachol on radiosodium uptake by dispersed pancreatic acinar cells, Pfliigers Arch. 385, 131. Rasmussen, H., K.C. Zawalich, S. Ganesan, R. Calle and W.S. Zawalich, 1990, Physiology and pathophysiology of insulin secretion, Diabet. Care 13, 655. Rothstein, A., 1989, The N a + / H + exchange system in cell pH and volume control, Rev. Physiol. Biochem. Pharmacol. 112, 235. Sehlin, J. and I.-B. T~iljedal, !974, Sodium uptake by microdissected pancreatic islets. Effects of ouabain and chloromercuribenzenep-sulphonic acid, FEBS Lett. 39, 209. Verspohl, E.J., R. Tacke, E. Mutschler and G. Lambrecht, 1990, Muscarinic receptor subtypes in rat pancreatic islets: binding and functional studies, Eur. J. Pharmacol. 178, 303. Wessldn, N., P. Bergsten and B. Hellman, 1986, Glucose-induced reduction of the sodium content in g-cell-rich pancreatic islets, Biosci. Rep. 6, 967. Wiener, E., G. Dubyak and A. Scarpa, 1986, Na ÷ H + exchange in Ehrlich ascites tumor cells. Regulation by extracellular ATP and 12-O-tetradecanoylphozbol 13-acetate, J. Biol. Chem. 261, 4529.
211
© 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50
EJP 52114
Carbachol has opposite effects to glucose in raising the sodium content of pancreatic islets S a m i r S a h a a n d Bo H e l l m a n Department of Medical Cell Biology, Unicersityof Uppsala. Biomedicum. Box 571. S-751 23 Uppsala. Sweden
Received 23 May 1991, revised MS received 19 July 1991, accepted 13 August 1991
Integrating flame photometry was used for measuring sodium in single pancreatic islets from ob/ob mice. Exposure to 100 .aM carbachol resulted in a 25-40% increase in sodium without any effect on potassium during incubation with 0-5 mM glucose in media deficient or not in Ca 2+. This action of carbachol was abolished by 10 ,aM atropine or by raising the glucose concentration to 20 mM. A minor increase of the steady state content of sodium occurred in the presence of 200 ,aM ATP or 10 nM tetradecanoylphorbol 13-acetate (TPA). Carbachol differed from TPA in markedly stimulating sodium accumulation after ouabain inhibition of the Na/K pump. The results indicate that muscarinic receptor activation has opposite effects to glucose in inducing a rise of the islet content of sodium. It is suggested that the cholinergic control of the endocrine pancreas involves entry, of Na + in addition to the Na + entry mediated by protein kinase C activation of Na+/H ÷ countertransport. Carbachol; Muscarinic receptors; Na +; Pancreatic islets
1. Introduction It is generally believed that the insulin-releasing E-cells are sensitized to the glucose stimulus by acetylcholine released from vagal efferents within the pancreatic islets during the cephalic phase of digestion (Miller, 1981; Ahr6n et al., 1986; Rasmussen et al., 1990). Previous studies in our laboratory have indicated that muscarinic receptor activation results in biphasic mobilization of intracellular calcium from the /3-cellrich pancreatic islets of o b / o b mice (Hellman and Gylfe, 1986; Hellman et al., 1987). The two phases differed with respect to their dependence on glucose, the sugar being a prerequisite for the initial phase but suppressing the other. Whereas the first phase was attributed to the inositol-l,4,5-trisphosphate (IP 3) generated during polyphosphoinositide breakdown, the second one was proposed to result from increased entry to Na +. Although radioisotope studies have provided support for a rise of islet sodium after muscarinic receptor activation (Gagerman et al., 1980; Henquin et al., 1988), this increase remains to be demonstrated by direct measurements. The high sensitivity offered by integrat-
Correspondence to: B. Heliman, Department of Medical Cell Biology, Biomedicum, Box 571, S-751 23 Uppsala, Sweden. Tel. 46.18.174 424. fax 46.18.556 401,
ing micro flame photometry allowed us to show that the muscarinic receptor agonist, carbachol, does induce a substantial rise of islet sodium and that this effect is co-nteracted by glucose. Evidence is provided that receptor activation is coupled to an entry of Na ÷ additional to that mediated by stimulated N a + / H ~countertransport.
2. Materials and methods Deionized water and reagents of analytical grade were used. ATP, carbachol, tetradecanoylphorbol-13acetate (TPA), dimethyl sulphoxide (DMSO), ouabain, E G T A and bovine serum albumin (fraction V) were from Sigma Chemical Co. (St. Louis, MO). Collagenase was obtained from Boehringer Mannheim GmbH (Mannheim, Germany). Adult o b / o b mice were from a non-inbred colony (Hellman, 1965) and were sta~'ed overnight. The animals were killed by decapitation under ether anesthesia and pancreatic islets were isolated with collagenase. Previous studies have indicated that these islets contain more than 90%/t-cells, which respond normally to glucose and other stimulators of insulin release (Hellman, 1970; Hahn et al., 1974). The basal medium used for the experiments was buffered with 25 mM HEPES and physiologically balanced cations (raM: Na + 137, K + 5.9, Mg 2÷ 1,2 and Ca'-" 1.28)with CI- as the sole
2~2
anion (HeUman. 1975). A Ca-'*-deficient variety of this medium was obtained by omission of CaCI_, and supplementing the medium with 1 mM EGTA. All media were equilibrated with ambient air and contained 1 m g / m l albumin. The islets were preincubated for 45 min at 37 ° C with 3 mM glucose then exposed to media modified as indicated in the legends to the tables and figures. At the end of the experiments the islets were washed for 2 min with an ice-cold solution of sucrose (Wessldn et al., 1986). Subsequent freeze-drying and weighing of the islets made it possible to express the sodium and pota~ium contents in terms of dry weight. Single islets (dry weight 0.8-2.5 /.tg) were analysed for sodium and potassium by means of integrating flame photometr3." according to previously described procedures (Wessldn et al., 1986; Bergsten et al., 1986). Each observation was based on five to eight measurements. Statistical significances for differences between paired observations were evaluated by means of Student's t-test.
3. Results The islet content of sodium after 60 min of incubation with or without glucose is shown in table 1. The addition of 5 or 20 mM of the sugar resulted in 30-40% lowering of sodium. The reductions were equivalent to 40 + 6 and 27 + 4 m m o l / k g dry weight respectively (P < 0.001). Whereas carbachol (100 /.tM) induced a rise of sodium in media with or without 5 mM glucose, this cholinergic agent had no effect when the concentration of the sugar was raised to 20 mM. ATP (200 # M ) was effective in raising sodium only in the presence of 5 mM glucose. Table 2 presents the results of similar analyses in a medium deficient in
TABLE 2 Effects of carbachol and ATP on islet content of sodium in a medium deprived of Ca-"÷. The islets were incubated for 60 min with or without 100 # M carbachol or 200 g M ATP at different concentrations of glucose in a Ca-'+-deficient medium supplemented with 0.5 mM EGTA. The sodium contents are given as m m o l / k g dry weight and represent the mean values+S.E, for eight experiments. Statistical significances for carbachol or ATP included in the media are indicated by a p < 0.025. Glucose (mM)
Additions
Sodium content Observed
Effect of additions
0
-
136+
7
0
0
Carbachol ATP
193+24 135+ 12
+57+-21 a -2+ 8
-
5 5 5
Carbachoi ATP
103+ 8 125+ 10 108+ 7
+22+- 11 +5+- 9
20 21) 20
Carbachol ATP
95 + 5 114+ 10 95-+ 8
+ 19+ 9 0_ 6
Ca 2+, an experimental situation associated with a general increase of sodium. In this case also there was a lowering of sodium in the presence of glucose (P < 0.001). In the CaE+-deficient medium carbachol induced a rise of sodium only in the absence of glucose, and ATP had no effect irrespective of whether the sugar was present or not. Figure 1 shows simultaneous measurements of islet sodium and potassium at different glucose concentra-
SODIUM
I-,"1-
o
POTASSIUM
200
B
IJJ >n"
a
o~ 150
TABLE ! Effects of carbachol and ATP on islet content of sodium. The islets were incubated for 60 rain with or without 100/.tM carbachol or 200 # M A'I-P at different concentrations of glucose. The sodium contents are given as m m o l / k g dry weight and represent the mean values+ S.E. for seven experiments. Statistical significances for carbachol or ATP included in the media are indicated by ~ P < 0.01; h p < 0.005. Glucose CmM)
Additions
0 0 0 5 5 5 20 20 20
Carbachol ATP Carbachol ATP Carbachoi ATP
lOO
50
Sodium content Observed
Effect of additions
101-4-_ 10 129 + 7 96+_ 8 61_+ 5 93 + 7 76+ 5 73_+ 7 83+ 11 69+ 7
+ 29 + 8 a -64-6 + 32 + 6 h + 15_+4 a + 10±6 -4-+2
0
3
20
0
3
20
GLUCOSE CONCENTRATION (mM) Fig. 1. Effects of carbachol on islet contents of sodium and potas-
sium at different glucose concentrations. The islets were incubated for 15 min with (hatched bars) or without (open bars) 100 ,ttM carbachol in media containing 0, 3 or 20 mM glucose. Mean values + S.E. for nine experiments. The presence of carbachol resulted in significant increases of sodium in the media lacking (P < 0.025) or containing (P < 0.001) 3 mM glucose.
213 TABLE 4 Effects of carbachol and TPA on the increase of islet sodium in response to ouabain. The islets were exposed for 5 min to 1 mM ouabain in the presence or absence of 100 ttM carbachol or 10 nM TPA in media containing 3 or 20 mM glucose. The increases of sodium from an initial value of 58 + 4 mmol/kg dry weight are given as mean values + S.E. for seven experiments, Statistical significances for carbachol in the medium are indicated by " P < 0.02; b p < 0.001.
30
Glucose
20
Additions
(raM) 10
0.1
1
10
100
Sodiumincrease Observed
Effect of additions
3 3 3
Carbachol TPA
21 +3 50 + 3 23 + 3
+ 29 + 2 b + 2+ 3
20 20
Carbachol
40+3 54+2
+ 14+4 a
1000
CARBACHOL (pM)
Fig. 2. The increase of islet sodium obtained with different concentrations of carbachol. The islets were incubated for 45 min with 0.1-1000/zM of carbachol in the presence of 3 mM glucose. The bars indicate the mean values+S.E, for the rise of sodium from a control value of 74 + 3 mmol/kg dry weight in five experiments.
tions after 15-min exposure to carbachol. W h e r e a s the potassium c o n t e n t r e m a i n e d unaffected, additional evidence was o b t a i n e d for a c a r b a c h o l - i n d u c e d rise of sodium c o u n t e r a c t e d by 20 m M glucose. Studies of the dose d e p e n d e n c e revealed that the maximal s~dium increase was a t t a i n e d with 100/.LM carbachol (fig. 2). T h e effect was already evident after 2 min (not shown) and was s u p p r e s s e d by atropine. A f t e r 45 min of exposure to 100/.~M carbachol the rise of sodium observed at 3 m M glucose in the absence and presence of 10 # M a t r o p i n e was 24 + 6 and 7 + 5 m m o l / k g dry weight respectively (n = 5). T h e sodium c o n t e n t could also be raised with the phorbol ester, T P A . T a b l e 3 illustrates this effect after 15 min of exposure to 10 n M T P A in a m e d i u m
TABLE 3 Effects of carbachol and q'P~ on islet content of sodium. The islets were incubated for 15 min with or without 100 #M carbachol or 10 nM TPA in media containing 3 or 20 mM glucose. All media cop.rained 0.1% DMSO, used for preparing a stock solution of TPA. The sodium contents are given as mmol/kg dry weight and represent the mean values±S.E, for the number of experiments shown in parentheses. Statistical significances for carbachol and/or TPA ipcluded in the media are indicated by " P < 0.01; b p < 0.901. Glucose (mM)
Additions
Sodium content Observed
Effect of additions
Carbachol TPA Carbachol + TPA
63+3 (17) 82+9 (8) 70±4 (17) 82 + 7 (8)
+19±5" +7±i h +19±2 h
20
-
57+5
20
TPA
61±5 (7)
3 3 3 3
(7)
+5+2
c o n t a i n i n g 3 m M glucose. T P A was less effective than carbachol to raise sodium and no additive effects were seen when the two c o m p o u n d s were combined. T h e r e was no further rise of sodium when the T P A concentration was raised to 100 nM or the exposure time e x t e n d e d to 60 min (not shown). Table 4 shows the net increase in sodium during the 5-min period following inhibition of the N a / K p u m p with ouabain. W h e r e a s the presence of 100 t t M carbachol more than d o u b l e d the sodium u p t a k e in the presence of 3 m M glucose the addition of T P A had no effect. Raising glucose to 20 mM resulted per se in an increased net uptake of sodium, a process a c c e n t u a t e d by the simultaneous presence of carbachol. T h e carbachol effects were less at 3 mM than at 20 mM glucose (P < 0.05).
4. D i s c u s s i o n
P a r a s y m p h a t h e t i c nerves form peri-insular plexuses allowing direct innervation of the islet cells (Miller, 1981). T h e functional significance of this i~nervation is e m p h a s i z e d by the observation of a 10-fold higher c o n c e n t r a t i o n of choline acetyltransfetase in islets than in the exocrine p a n c r e a s (Godfrey and Matschinsky, 1975). Acetylcholine has been reported to have stimulatory actions not only on insulin secretion but, in some species, also on the release of glucagon, somatostatin and pancreatic p o l y p e p t i d e (AhrEn et al., 1986). It is well established that cholinergic agents act by binding to muscarinic receptors (Grill and 0 s t e n s o n , 1983), and there are reasons to believe that M 3 is the pred o m i n a t i n g receptor subtype in the islets (Verspohl et al., 1990). Various observations suggest that muscarinic receptors are important for islet handling of Na ÷. Both the delayed mobilization of intracellular islet calcium initiated by carbachol ( H e l l m a n and Gylfe, 1986; Hellman
214
et al,, 1987), and the acetylcholine-induced depolarization of the/3-cells (Henquin et ai., 1988) require extracellular Na + Moreover, acetylcholine has been reported to stimulate the uptake of 22Na in isolated mouse islets (Gagerman et al., 1980; Henquin et al., 1988). It is evident from the present study that these radioisotope data reflect a muscarinic-induced increase of the sodium content and not simply a stimulated exchange with non-radioactive sodium. Accordingly, cholinergic agents differ from glucose with regard to their effect on islet handling of sodium. As suggested from a radioisotope approach (Kavazu et al., 1978), and confirmed by previous (Wessl6n et ai., 1986; Ali and Hellman, 1991) and present measurements of sodium, the overall effect of glucose is to increase the turnover of sodium, implying that an enhanced entry of Na* is overcompensated by stimulated extrusion, Although the observed values for islet sodium compare favourably with those obtained with the radioactive approach (Sehlin and T~iljedal, 1974; Kavazu et ai., 1978: Gagerman et al., 1980), they are considerably higher than estimates of Na + activity based on membrane potential recordings (Atwater et al., 1978; Meissner et ai., 1978) or from direct measurements with the fluorescent indicator SPFI (Ali et al., 198%). Accordingly, it is likely that only a minor part of the /3-cell sodium occurs in an ionized form. External ATP mediates a similar mobilization of calcium from the pancreatic/3-cells, as noted on stimulation of polyphosphoinositide breakdown with carbachol (Gylfe and Hellman, 1987). By demonstrating an ATP-induced rise of sodium in the presence of 5 mM glucose, the present study provides further evidence that purine P2 receptors mimic muscarinic ones with regard to effects on the internal ionic milieu. A similar relationship seems to exist in Ehrlich ascites tumour cells, which have been reported to respond to both ATP and carbachol with a rise of intracellular sodium based on activation of amiloride-sensitive N a + / H ÷ countertransport (Wiener et al., 1986). In addition to providing definite evidence that muscarinic receptor activation is an effective means of raising islet sodium, this increase was found to be counteracted by 20 mM glucose. It is not surprising that such an opposing action had escaped detection in previous measurements of the radioactive 22Na uptake (Gagerman et al., 1980; Henquin et al., 1988), since these studies were restricted to glucose concentrations up to 11 mM. The observation that glucose opposes the carbachol-induced rise of sodium may explain why the sugar inhibits the Na+-dependent efflux of Ca 2+ induced by muscarinic receptor activation (Hellman and Gylfe, 1986; Hellman et al., 1987). Glucose also suppresses the Ca 2÷ mobilization obtained when intraceilular sodium is raised by removal of extracellular K ÷ or addition of ouabain or veratridine (Hellman et al.,
1982). The ability of glucose to prevent an increase in sodium is not restricted to situations of raised intracellular contents of the element. As repeatedly observed in our laboratory (Wessl6n et al., 1986; Ali and Hellman, 1991) and also as shown from the present data, a rise of glucose to 5 mM results iit a reduction of the islet content of sodium. This effect, as well as the interference by glucose with the carbachol-induced rise of sodium, was found to persist in a medium depleted of Ca 2÷. The glucose-induced lowering of the sodium content has been attributed to an increased outward transport mediated at least in part by mechanisms other than stimulation of the N a / K pump (Ali et al., 1989b; Ali and Hellman, 1991). It seems likely from the present observations that glucose suppression of the sodium response to muscarinic receptor activation also involves interference with the influx of the ion. it is well established that activation of nicotinic receptors results in an enhanced Na ÷ permeability in various types of cells (Hucho, 1986). The fact that muscarinic receptors also may sometimes initiate a ~ubstantial Na ÷ influx was clearly demonstrated from the present islet measurements. A similar increase occurs in the exocrine pancreas (Petersen, 1976; Putney et al., 1980), suggesting that Na ÷ is involved in the vagal control of both parts of the gland. Muscarinic receptors are potent stimulators of the islet hydrolysis of phosphoinositides, providing diacylglycerol for stimulation of protein kinase C (Peter-Riesch et al., 1988; Arkhammar et al., 1989). Activation of this enzyme often promotes N a ÷ / H ÷ countertransport (Rothstein, 1989). However, it is difficult to believe that such an activation is a major explanation for the rise of islet sodium evoked by muscarinic receptor activation. After TPA stimulation of protein kinase C the sodium content was only slightly elevated and the phorbol ester did not promote an additional rise of the element following inhibition of the N a / K pump. Whereas cholinergic agents do not affect basal insulin release, they markedly potentiate the secretion obtained when the glucose concentration is raised (Gagerman et al., 1980; Ahr6n et al., 1986). In discussing the functional significance of the muscarinic effects on /3-ceU handling of Na ÷ it is necessary to consider both the results of depolarization mediated by the enhanced entry of the ion and a rise of its intracellular activity. As emphasized by Henquin et al. (1988) a contribution to the glucose-induced depolarization can be expected to aid in the opening of voltage-dependent Ca 2÷ channels, which in turn stimulates insulin release. An increase of cytoplasmic Na ÷ as such may also initiate secretory activity by mobilizing Ca 2÷ from intracellular stores and interfere with its outward transport (Hellman, 1986). With regard to the observation that glucose opposes the increase of islet sodium it seems likely that it is the depolarization rather than
215
the rise of intracellular Na ÷ which is essential for muscarinic potentiation of the stimulatory action of the sugar on insulin release.
Acknowledgements This study was supported by grants from the Swedish Medical Research Council (12x-562), the Swedish Diabetes Association, the Nordic Insulin Foundation, the Swedish Hoechst and Novo-Nordisk Companies and the Family Ernfors Foundation. Samir Saha is a scientific officer from the Bangladesh Institute for Research and Rehabilitation on Diabetes, Endocrine and Metabolic Disorders (BIRDEM), working as a visiting research fellow sponsored by the International Program in the Chemical Sciences.
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