,f cells as indicated by the kinetics of insulin release recorded from islets temporarily

Transcription

1 J. Physiol. (1982), 328, pp With 5 text-figures Printed in Great Britain EVIDENCE FOR A SLOWLY EXCHANGEABLE POOL OF CALCIUM IN THE PANCREATIC ft CELL PLASMA MEMBRANE BY ERIK GYLFE AND BO HELLMAN From the Department of Medical Cell Biology, Biomedicum, Univer8ity of Uppsala, S Uppsala, Sweden (Received 13 November 1981) SUMMARY 1. Exposure to media deprived ofca2+ resulted in prompt and transient stimulation of 45Ca efflux from,/ cell-rich pancreatic islets microdissected from ob/ob-mice and to some extent also from the isolated neurohypophysis. 2. Particular high efflux rates were reached when the Ca2+-deficient medium contained EGTA, but there was no effect of the chelator on the total amount of radioactivity mobilized from the islets. 3. The removal of extracellular Ca2+ was less effective in promoting the 45Ca efflux in the absence of Na+ and no stimulatory response was seen in the presence of 1 mm-la The 45Ca washout was stimulated whether or not the media used for the loading or subsequent perifusion of the islets were supplemented with 20 mm-d-glucose. However, there was no response to a second exposure to a Ca2+-deficient medium even subsequent to redistribution of intracellular calcium induced by temporary lowering of the temperature. 5. It is suggested that the islet 45Ca released by the removal of extracellular Ca2+ originates from a distinct plasma membrane pool which is exchanged slowly compared to most of the calcium at the fi cell periphery. INTRODUCTION It is generally accepted that Ca2+ plays a decisive role in coupling the stimulus recognition to the discharge of the insulin secretary granules. Evidence has been provided that Ca2+ serves not only as a prerequisite for the proper function of the secretary machinery but that the accumulation of this cation in the cytoplasm actually initiates the discharge of insulin (Malaisse, Herchuelz, Devis, Somers, Boschero, Hutton, Kawazu, Sener, Atwater, Duncan, Ribalet & Rojas, 1978a; Hellman, Andersson, Berggren Flatt, Gylfe & Kohnert, 1979). The extracellular Ca2+ has both stimulatory and inhibitory effects on the secretary activity of the pancreatic,f cells as indicated by the kinetics of insulin release recorded from islets temporarily exposed to high concentrations ofca2+ (Hellman et al. 1979). It has been demonstrated in recent studies that the /8 cell plasma membrane accommodates binding sites for cations with direct inhibitory effects on insulin secretion (Flatt, Berggren, Gylfe & Hellman, 1980; Flatt, Boquist & Hellman, 1980).

2 286 E. GYLFE AND B. HELLMAN Substantial amounts of Ca2+ can be displaced from the surface of the fa cells by exposure to La3+. The mobility of this Ca2+ is sufficient to meet the requirements for a short-term modulation of the secretary process (Hellman, Sehlin & Tiljedal, 1976). When exploring the functionally significant membrane calcium it is also important to pay attention to how the various plasma membrane pools are affected by D-glucose, the major natural stimulator of insulin release. This sugar has been found to diminish both the membrane binding of the non-penetrating lanthanide thulium (Flatt, Gylfe & Hellman, 1981) and to suppress the fluorescence of chlortetracycline used as a probe for the La3+-displaceable calcium in the plasma membrane (TAljedal, 1978, 1979). It has been reported that the removal of extracellular Ca2+ from rat islets results in a transient mobilization of 45Ca supposed to be associated with the plasma membrane and that this effect is abolished by D-glucose (Malaisse, Hutton, Sener, Levy, Herchuelz, Devis & Somers, 1978 b). The present paper describes effects of Ca2+ removal from the fi cell-rich pancreatic islets microdissected from ob/ob-mice. The results are compatible with the existence of a limited pool of slowly exchangeable plasma membrane Ca2+. However, this pool was found to be sensitive to omission of extracellular Ca2+ also in the presence of D-glucose. METHODS Adult ob/ob-mice were taken from a non-inbred colony (Hellman, 1965) and starved overnight. The animals were killed by decapitation and pancreatic islets were isolated by microdissection. For comparative purposes, some experiments included also studies of the neurohypophysis. The basal medium used for the isolation and the subsequent measurements of the 4"Ca efflux was a HEPES buffer, ph 7 4, with C1- as the sole anion (Hellman, 1975). The Ca2+ concentration was determined by working in the known addition mode (KA/5) of an Orion 901 microprocessor ionalyzer (Orion Research Inc., Cambridge, MA, U.S.A.) equipped with F21 10 Ca2+ and K4040 calomel electrodes (Radiometer A/S, Copenhagen, Denmark). The kinetics of4"ca efflux were studied using previously described procedures (Gylfe & Hellman, 1978). The specimens were incubated with 1-28 mm-4'ca (390 Ci/mol.) for 90 min. After two serial 5 min washes, batches of 8-10 islets or a single neurohypophysis were transferred to a 10 1I. chamber and perifused at a constant rate of about 40 #Il./min with non-radioactive medium supplemented with 1 mg/mi. albumin. With the removal of Cal+ the final concentration of this ion was reduced to 15FM (in the absence of EGTA) or < 001FM (in the presence of EGTA). Details regarding the concentration of Ca2+ and other additives to the perifusion media are given in the legends to the Figures. In each experiment, two or three chambers were loaded with islets from the same animal and run in parallel. The perifusate was collected over successive periods of 2 or 5 min and samples analysed for radioactivity by liquid scintillation counting. After perifusion, the specimens were removed from the chambers, freeze-dried overnight and weighed on a quartz fibre balance. To facilitate comparisons with previous studies of4"ca efflux from the islets of the ob/ob-mice, the data were expressed per kilogram dry weight, assuming the same specific radioactivity as in the loading medium. It was checked whether the stimulation of45ca efflux obtained by combining removal of Ca2+ with addition of EGTA could be accounted for by the mobilization of4"ca adhering to the inner walls of the perifusion apparatus. Removal of islets from the chambers resulted in a prompt disappearance of the radioactivity irrespective of the medium concentrations of Ca2+ and EGTA. A rapid washout of4"ca from the perifusion system was demonstrated also by direct introduction of large amounts of radioactivity into chambers lacking islets (Fig. 1). As indicated from the semi-logarithmic plot there was only a minimal increase above the background radioactivity when Ca2+ was removed andegta added to the perifusion medium.

3 45Ca EFFLUX FROM PANCREATIC fi CELLS 287 RESULTS Removal of Ca2+ after 60 min of perifusion resulted in a prompt stimulation of the islet 4"Ca efflux with a considerable variation in the response. Particularly high efflux rates were reached when the Ca2+-deficient medium was supplemented with 0 5 mm- EGTA (Fig. 2A). Since the addition ofegta also shortened the period of stimulation, the presence of the chelator did not increase the total amounts of radioactivity Ca2+ removal +EGTA C E 1000 I II~~~~L"I I Fig. 1. Washout of radioactivity after introducing 1 jul of the 4"Ca loading medium (- 900,000 counts/min) in the perifusion chamber. Replacement of a perifusion medium containing 2-56 mm-cal+ with one deficient in Ca'+ and supplemented with 0-5 mm-egta did not result in apparent increase of radioactivity above the background values reached after min of perifusion. Mean values +±.E. for three experiments. mobilized. The amounts of 45Ca (mmol/kg dry weight) released during the period of CaO+ removal shown in Fig. 2A was in the presence of EGTA as compared with in the absence of the chelator. A 40 min delay in the removal of Ca2+ resulted in a reduction of the amounts of 45Ca released. There was no efflux response when the islets were exposed to the EGTA-containing medium during a second period (Fig. 2B). Neither was the response re-established when the second exposure to EGTA was preceded by a period of temperature reduction to +2 IC (Fig. 3A). The "Ca efflux in response to Ca2+ removal disappeared in the presence oft mm-la3+ both when the lanthanide was present throughout the perifusion (not shown) or added as replacement for Ca2+ (Fig. 3B). The combination of CaO+ removal and addition of EGTA resulted in a prominent stimulation of the 45Ca efflux whether or not the

4 288 E. GYLFE AND B. HELLMAN media used for the loading or subsequent perifusion of the islets were supplemented with 20 mm-d-glucose (Fig. 4). The removal of extracellular Ca2+ was less effective in stimulating the 45Ca efflux when the initial concentration of Ca2+ was lowered to 0-26 mm (Fig. 5A) or when the perifusion medium was deficient in Na+ (Fig. 5B). The exposure to media deprived A Ca2+ removal + EGTA B Ca2+ removal + EGTA 120 Ca2' removal Ca2+ removal + EGTA Z A2 0) E x Fig. 2. Effects of Ca2+ removal on the efflux of 4"Ca from pancreatic islets. The islets were labelled with 45Ca in the absence of glucose and perifused with a glucose-free medium containing 2-56 mm-ca2+. Ca2+ was removed from the medium during the periods indicated by the horizontal bars. A, the removal of Ca2+ was combined (0) or not (A) with the addition of 05 mm-egta. B, shows the result of combining removal of Ca2+ with the addition of 0-5 mm-egta when this procedure had been preceded (0) or not (0) by a similar period of treatment. Mean values+s.e. for four experiments. of Ca2+ promoted the efflux of 45Ca also from the neurohypophysis although the effect was less pronounced than that obtained with the islets (Fig. 5C). DISCUSSION Extracellular Ca2+ can promote the washout of 45Ca from pancreatic islets by Ca2+/Ca2+ exchange (Hellman et al. 1979; Herchuelz, Couturier & Malaisse, 1980; Abrahamsson, Gylfe & Hellman, 1981). Nevertheless, the removal of Ca2+ was found to stimulate the efflux of 45Ca from the,d cell-rich islets of the ob/ob-mice. It was

5 45Ca EFFLUX FROM PANCREATIC fl CELLS conclusively shown in the control experiments that the stimulation of the 45Ca efflux obtained with the sudden deprivation of extracellular Ca2+ was not an experimental artifact. Evidently the observed effects have more general implications as indicated from the previous reports of an increased 45Ca washout from rat islets (Malaisse et al. 1978b) and the fact that stimulated efflux was also observed from the neurohypo- 120 A8 Ca2+ removal + EGTA Ca2+ removal Ca2+ removal + La X 100 E 2~ 80 0) E -:3 60 x S Fig. 3. Effects of Ca2+ removal from a glucose-free perifusion medium on the efflux of 46Ca from pancreatic islets. In A the 45Ca loading was followed by 20 min of exposure of the islets to a Ca2+-deficient medium containing 0 5 mm-egta. During the perifusion the temperature was either maintained at 37 0C (0) or temporarily reduced (between 10 and 45 min) to + 2 0C (@). The horizontal bar indicates replacement of 2-56 mm-ca2+ with 05 mm-egta. In B, the perifusion medium with 2-56 mm-ca2+ was replaced with a Ca2+-deficient medium supplemented (El) or not (U) with 1 mm-la3+ during the period indicated by the horizontal bar. Mean values +S.E. for four experiments. physis. A detailed comparison between islets and the neurohypophysis is difficult in view of their size differences. The later and more prolonged response of the neurohypophysis may consequently be accounted for by slower exchange of Ca2+ in the extracellular space. In contradistinction to the observations reported for rat islets (Malaisse et al. 1978b), sudden exposure to media deprived of Ca2+ resulted in a transient mobilization of islet 45Ca also when 20 mm-d-glucose was present in the perifusion medium. Since this sugar is known to suppress the efflux of 45Ca during perifusion with Ca2+-deficient medium (Malaisse, Brisson & Baird, 1973; Gylfe, Buitrago, Berggren, Hammarstrom & Hellman, 1978; Kikuchi, Wollheim, Cuendet, 10 PHY 328

6 290 E. GYLFE AND B. HELLMAN Renold & Sharp, 1978; Hellman et al. 1979), it should be emphasized that the stimulatory effect of Ca2+ removal was at least as prominent in the presence of D-glucose. When the Ca2+-deficient medium was supplemented with EGTA the stimulated 45Ca efflux was intensified although of shorter duration. The fact that EGTA altered A Ca2' removal + EGTA B Ca2' removal + EGTA E 80 0 E 360 X 0) VU 1W Fig. 4. Effects of Ca2+ removal on the efflux of 4"Ca from pancreatic islets. During the periods indicated by the horizontal bars the ordinary perifusion medium with 2-56 mm-ca"+ was replaced with medium deficient in Ca2+ and supplemented with 0 5 mm-egta. In A, labelling of the islets was performed either in the absence (0) or presence (0) of 20 mm-glucose. B shows the results of perifusion in the absence (@) and presence (a) of 20 mm-glucose after labelling in a glucose-free medium. Mean values ± s.e. for four to five experiments. the pattern of 45Ca mobilization from the islets rather than the total amounts released during Ca2+-removal suggests that the radioactive calcium originates from a pool of limited size. Evidently there was some turnover ofthis pool also at high concentrations of extracellular Ca2+ as indicated by the lower stimulatory peak observed with extension of the first period of perifusion with medium containing 2-56 mm-ca2+. The dependence on the extracellular Ca2+ was demonstrated also by lowering the initial concentration ofca2+ in the perifusion medium. Less 45Ca was consequently mobilized during the exposure to the EGTA-containing medium when the initial concentration of extracellular Ca2+ was 0-26 instead of 2-56 mm.

7 45Ca EFFLUX FROM PANCREATIC f CELLS The pool of 45Ca mobilized by Ca2+ removal appeared to be independent of the radioactive calcium sequestered in the intracellular organelles. There was no stimulation of the 45Ca efflux during a second exposure to a Ca2+-deficient medium even after the redistribution of intracellular calcium obtained by temporary lowering of the temperature to + 2 'C. Furthermore, the 45Ca efflux was stimulated to a similar A 8 C Ca2" removal + EGTA Ca2' removal + EGTA Ca2+ removal + EGTA ~80 -oc o 60 E % 40 Ca Fig. 5. Effects of Ca2+-removal on the efflux of 46Ca from pancreatic islets and neurohypophysis. The specimens were labelled with 45Ca in the absence of glucose and perifused with glucose-free medium. During the periods indicated by the horizontal bars the compositions of the media were altered by combining the removal ofca2+ with the addition of 0-5 mm-egta. A refers to experiments with islets using an original medium concentration of Ca2+ of either 0-26 mm (0) or 2-56 mm (M). B shows similarly designed islet experiments with an original Ca2+ concentration of 2-56 mm in perifusion media deficient (A) or not (0) in Na+. When preparing the Na-deficient variety of the medium this ion was reduced to 12 mm by equimolar replacement with choline+. C refers to experiments with islets (M) and neurohypophysis (LI) using an original medium concentration of Ca2+ of 1-28 mm. Mean values + S.E. for four to five experiments. extent when the islets had been loaded in the presence and absence of20 mm-d-glucose. Previous studies have indicated that the exposure to D-glucose results in intracellular accumulation of 45Ca in the pancreatic fi cells by favouring uptake by mitochondria and secretary granules (Kohnert, Hahn, Gylfe, Borg & Hellman, 1979; Hellman et al. 1979). The observations that Na+ deficiency diminished the efflux of 45Ca in response to Ca2+ removal does not necessarily imply that the mobilization involves 10-2

8 292 E. GYLFE AND B. HELLMAN radioisotopic calcium of intracellular origin. extrusion from the pancreatic, cells by Na+/Ca2+ counter transport (Hellman, Andersson, Berggren & Rorsman, 1980), Na+ has been found to compete for the superficial Ca2+ sites in many cells (Rubin, 1974) including the pancreatic /3 cells (Tiljedal, 1974). In constituting a pool without significant exchange with intracellular stores it is likely that the islet 45Ca mobilized by Ca2+ removal originates from the plasma membrane. The validity of this postulate is reinforced by the promptness of the stimulatory effect and its limited duration. The 45Ca efflux obtained in response to Ca2+ removal was inhibited by La3+. This calcium pool also differed from the superficial/, cell Ca2+ displaced by La3+ (Hellman et al. 1976; Hellman, 1978) in being slowly exchanged at physiological concentrations of extracellular Ca2+. The 45Ca released with the exposure to Ca2+-deficient medium may consequently originate from a distinct plasma membrane pool dependent on the readily exchangeable Ca2+ known to be present at the /3 cell periphery (Hellman et al. 1979). Although different classes of Ca2+ binding sites have been demonstrated in islet cell plasma membranes (Naber, McDonald, Jarett, McDaniel, Ludvigsen & Lacy, 1980), there is so far no evidence for a positive co-operativity in the binding of Ca2+ to the /3 cell surface in analogy to what has been demonstrated with plasma membranes from lymphocytes (Mikkelsen & Freerksen, 1980). Apart from participating in the Ca2+ This work was supported by the Swedish Medical Research Council (12x-562 and 12x-6240) and the Swedish Diabetes Association. The authors are indebted to Inger Ronnberg and Kristina Mustajiirvi for excellent technical assistance. REFERENCES ABRAHAMSSON, H., GYLFE, E. & HELLMAN, B. (1981). Influence of external calcium ions on labelled calcium efflux from pancreatic fl-cells and insulin granules in mice. J. Physiol. 311, FLATT, P. R., BERGGREN, P.-O., GYLFE, E. & HELLMAN, B. (1980). Calcium and pancreatic fl-cell function. IX. Demonstration of lanthanide-induced inhibition of insulin secretion independent of modifications in transmembrane Ca2+ fluxes. Endocrinology 107, FLATT, P. R., BoQUIST, L. & HELLMAN, B. (1980). Calcium and pancreatic fl-cell function. The mechanism of insulin secretion studied with the aid of lanthanum. Biochem. J. 90, FLATT, P. R., GYLFE, E. & HELLMAN, B. (1981). Use of thulium as a probe for cation binding sites in the pancreatic fl-cell membrane. Endocrinology 108, GYLFE, E., BuITRAGo, A., BERGOREN, P.-O., HAMMARSTR6M, K. & HELLMAN, B. (1978). Glucose inhibition of 4"Ca efflux from pancreatic islets. Am. J. Phy8iol. 235, E191-E196. GYLFE, E. & HELLMAN, B. (1978). Calcium and pancreatic fl-cell function. 2. Mobilization of glucose-sensitive 45Ca from perifused islets rich in fl-cells. Biochim. biophy8. Acta 538, HELLMAN, B. (1965). Studies in obese-hyperglycemic mice. Ann. N. Y. Acad. Sci. 131, HELLMAN, B. (1975). The significance of calcium for glucose stimulation of insulin release. Endocrinology 97, HELLMAN, B. (1978). Calcium and pancreatic f-cell function. 3. Validity of the La3+-wash technique for discriminating between superficial and intracellular 4"Ca. Biochim. biophy8. Acta 540, HELLMAN, B., ANDERSSON, T., BEROGREN, P.-O., FLATT, P., GYLFE, E. & KOHNERT, K.-D. (1979). The role of calcium in insulin secretion. In Hormone8 and Cell Regulation, vol. 3, ed. DUMONT, J. & NUNEZ, J., pp Amsterdam: Elsevier/North Holland Biomedical Press. HELLMAN, B., ANDERSSON, T., BERGGREN, P.-O. & RORSMAN, P. (1980). Calcium and pancreatic fl-cell function. XI. Modification of 45Ca fluxes by Na+ removal. Biochem. Med. 24, HELLMAN, B., SEHLIN, J. & TXLJEDAL, I.-B. (1976). Calcium and secretion: distinction between two pools of glucose-sensitive calcium in pancreatic islets. Science, N. Y. 194,

9 45Ca EFFLUX FROM PANCREATIC fi CELLS HERCHUELZ, A., COUTURIER, E. & MALAISSE, W. J. (1980). Regulation of calcium fluxes in pancreatic islets: glucose-induced calcium-calcium exchange. Am. J. Phy8iol. 238, E96-E103. KIKUCHI, M., WOLLHEIM, C. B., CUENDENT, G. S., RENOLD, A. E. & SHARP, G. W. G. (1978). Studies on the dual effects of glucose on 45Ca efflux from isolated rat islets. Endocrinology 102, KOHNERT, K.-D., HAHN, H.-J., GYLFE, E., BORG, H. & HELLMAN, B. (1979). Calcium and pancreatic fl-cell function. 6. Glucose and intracellular 46Ca distribution. Mol. & cell. Endocr. 16, MALAISSE, W. J., BRISSON, G. R. & BAIRD, L. E. (1973). Stimulus-secretion coupling of glucoseinduced insulin release. X. Effect of glucose on 46Ca efflux from perifused islets. Am. J. Phy8iol. 224, 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. & ROJAS, E. (1978a). Regulation of calcium fluxes and their regulatory roles in pancreatic islets. Ann. N. Y. Acad. Sci. 307, MALAISSE, W. J., HUTTON, J. C., SENER, A., LEVY, L., HERCHUELZ, A., DEVIS, G. & SOMERS, G. (1978b). Calcium antagonists and islet function. VII. Effect of calcium deprivation. J. Membrane Biol. 38, MIKKELSEN, R. B. & FREERKSEN, D. L. (1980). Calcium binding to plasma membranes from normal and SV40 transformed hamster lymfocytes. J. Recept. Re8. 1, NABER, S. P., McDoNALD, J. M., JARETT, L., MCDANIEL, M. L., LUDVIGSEN, C. W. & LACY, P. E. (1980). Preliminary characterization of calcium binding in islet cell plasma membranes. Diabetologia 19, RUBIN, R. P. (1974). Calcium and the Secretory Proce88. New York: Plenum Press. TALJEDAL, I.-B. (1974). Interaction of Na+ and Mg2+ with Ca2+ in pancreatic islets as visualized by chlortetracycline fluorescence. Biochim. biophy8. Acta 372, TALJEDAL, I.-B. (1978). Chlortetracycline as a fluorescent Ca2+ probe in pancreatic islet cells. Methodological aspects and effects of alloxan, sugars, methylxanthines and Mg2+. J. cell Biol 76, TALJEDAL, I.-B. (1979). Polarization of chlortetracycline fluorescence in pancreatic islet cells and its response to calcium ions and D-glucose. Biochem. J. 178,