J. Physiol. (1978), 283, pp. 409-424 With 9 text-figuree Printed in Great Britain

409

REGULATION OF CALCIUM FLUXES IN PANCREATIC ISLETS: DISSOCIATION BETWEEN CALCIUM AND INSULIN RELEASE

BY A. HERCHXYELZ AND W. J. MALAISSE From the Laboratories of Pharmacology and Experimental Medicine, Brussels University School of Medicine, Brussels, Belgium

(Received 6 March 1978) SUMMARY

1. The release of 45calcium from prelabelled pancreatic islets is rapidly and almost totally inhibited by lanthanum. 2. Glucose provokes an initial fall followed by a secondary rise in 45calcium efflux. The latter rise occurs concomitantly with insulin release. Its magnitude is reduced whenever the secretary response to glucose is inhibited, e.g. in the absence of extracellular calcium, presence of Verapamil, or at high magnesium concentration. 3. However, under suitable conditions, the glucose-induced secondary rise in 45calcium efflux is not totally suppressed whilst insulin release is totally abolished. 4. Inversely, when calcium is replaced by barium in the perifusate, glucose increases insulin output without causing any obvious secondary rise in 45calcium efflux. 5. It is concluded that this secondary rise, which originates from a lanthanumnondisplaceable calcium pool, does not correspond solely to an exocytotic release of 45calcium. It could represent, in part at least, a displacement of 45calcium from cellular sites and reflect a glucose-induced increase in the rate of calcium entry in islet cells. INTRODUCTION

It is generally accepted that calcium plays an essential role in the coupling of stimulus recognition and insulin release by the pancreatic fl-cell (Malaisse, 1973). The exact localization of the calcium pool which controls insulin release, and the detailed mechanism by which glucose affects calcium movements in the fl-cell remain a matter of debate. The measurement of 45calcium net uptake by isolated islets (Malaisse-Lagae & Malaisse, 1971; Hellman, Sehlin & Thljedal, 1976a), the assay by atomic absorption of 40calcium in the islets (Malaisse et al. 1978a), and the ultrastructural localization and quantification of calcium in the fl-cell by the pyronantimonate technique (Herman, Sato & Hales, 1973; Schafer & Kloppel, 1974; Ravazzola, Malaisse-Lagae, Amherdt, Perrelet, Malaisse & Orci, 1976) all indicate that glucose provokes an intracellular accumulation of calcium. This may be due to either an increased rate of calcium entry in the fl-cell, and/or a decreased outward transport of calcium. Dean & Matthews (1970), on the basis of their electrical data, concluded that glucose indeed increases calcium influx in the fl-cell. The effect of glucose on 45calcium efflux from prelabelled and perifused islets was interpreted as an indication that glucose

A. HERCHUELZ AND W. J. MALAISSE decreases calcium efflux from the islets cells (Malaisse, Brisson & Baird, 1973). However, in further work, no effect of glucose on 45calcium efflux could be detected, when judged from the fall in the total 45calcium islet content during static incubation (Hellman et al. 1976a; Naber, MacDaniel & Lacy, 1977). Moreover, it was argued that the lanthanum-non-displaceable intracellular calcium pool measured in some of these studies (Hellman et al. 1976a) does not display a sufficient mobility to provide the initial signal for insulin release, and that a more labile calcium pool, possibly located in the fl-cell plasma membrane appears as a better candidate for such a role (Hellman et al. 1976a, b). In order to get additional information on these issues, we decided to further explore the regulation of calcium fluxes in pancreatic islets. In the present study, the effect of glucose on "calcium efflux from perifused islets was therefore re-evaluated. 410

METHODS

Incubation, washing and perifu8ion media The media used for incubation, washing or perifusion of the islets consisted of a KrebsRinger bicarbonate buffered solution having the following composition: NaCl 115 mm, KCl 5 mm, CaCl 1 MM, MgCl2 I mm, NaHCO3 24 m . Albumin (fraction V; Sigma Chemical Company, St Louis, Mo.) dialysed before use was added to a final concentration of 5 mg/ml. The media were equilibrated against a mixture of 02 (95 %) and CO2 (5 %) and maintained at pH 7-4. In some perifusion media, to which no calcium chloride was incorporated, the calcium concentration averaged 37 ±44um as measured by atomic absorption. When lanthanum was included in the media, the latter contained no albumin, and bicarbonate was replaced by HEPES (N-2hydroxyethylpiperazine-N'-2-ethane sulphonic acid) 25 mm, the pH being adjusted to 7-4 with NaOH 1 mm and the media equilibrated with 02. The perifusate also contained when required, glucose, Verapamil (Knoll A. G., Ludwigshafen, Germany) and barium. All reagents were of analytical grade. Experimental procedure Isolated islets were obtained from fed female albino rats by collagenase digestion of the pancreas. The islets were collected individually. Each batch of islets was washed 5 times in order to remove by decantation small pieces of acinar tissue occasionally collected together with the islets. In each experiment, two groups of one hundred islets were incubated for 60 min at 37 "C in the presence of glucose (16.7 mm) and calciumm (1.12 mm: 0-2 mc/ml.). After incubation the islets were submitted to five new washes in order to remove extracellular radioactivity. The washing medium contained no calciumm and its temperature was close to 10 'C. Each group of islets was then placed on a cellulose filter (1 SMWPO 1300; Millipore S. A., 67120 Molsheim, France) which was inserted in a perifusion chamber (Swinnex 0001300; Millipore S.A., 78530 Buc, France) connected to two reservoirs through a Y-shaped valve. The reservoirs contained media kept at 38 'C and continuously mixed with 02 (95 %) and CO2 (5 %). The perifusate was delivered at a constant rate (1-0 ml./min) by means of a peristaltic pump (12,000 varioperpex; LKB, S 16125 BROMMA, Sweden). The temperature in the perifusion chamber was kept constant, averaging 37 'C. The inside pressure corresponded to the atmospheric pressure with no significant modification above this value during the perifusion. The perifusate was derived from the first reservoir until the end of the 43rd min and from the second reservoir thereafter, so that the new medium was collected from the 45th min onwards. The switch at the end of the 43rd min was always performed even when both reservoirs contained identical media (control experiments). From the 31st to the 70th min, the effluent was collected over successive periods of 1 min each in plastic tubes kept at 0 "C with ice. An aliquot of 0-4 ml. was then transferred to a counting vial and mixed with 10 ml. scintillation fluid (Instagel; Packard instrument Co., Downers Grove, Illinois). The remainder of the effluent was stored at -20 "C for insulin assay. In each individual experiment, calcium efflux (c.p.m. per minute) was expressed in percentage of the mean value found within the same experiment between the 40th and 44th min of perifusion.

.

CALCIUM FLUXES IN PANCREATIC ISLETS

411

Inrwin release Insulin was radioimmunologically assayed in duplicate with rat insulin as a standard and using a dextran-coated charcoal method for the separation of free and antibody-bound insulin (Herbert, Lau, Gotthieb & Bleicher, 1965). Because secretory rates varied to some extent among individual perifusions, the results are occasionally expressed as the difference in insulin output (A insulin) relative to the mean value recorded, within the same experiment, at the end of the equilibration period (min 30 to min 44). The latter reference values are reported in the legend of the Figures. Radiosootopes

'5Calcium was obtained from the Radiochemical Centre (Amersham) and [12'I]insulin from I.R.E. (Fleurus, Belgium). G300

Go- La2

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50

G300 - La2

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Time (min)

Fig. 1. Effect of lanthanum (2 mM) on 'calcium efflux. The composition of the perifusate administered respectively from 0 to 44 and from 45 to 70 min is shown in the upper part of the Figure. Mean values (± S.E. of mean) for calciumm efflux are expressed in percentages (see Methods) and refer to three individual experiments.

RESULTS

Effect of lanthanum on 45calcium efflux

Lanthanum 2 mm (Fig. 1) dramatically reduced 45calcium efflux whether in the absence (left panel) or presence of glucose 16-7 mm (right panel). Within 5-10 min after the addition of lanthanum, the efflux averaged no more than approximately

412 A. HERCHUELZ AND W. J. MALAISSE 10 % of the control value recorded between the 40th and 44th min. We were unable to detect any initial increase in effluent radioactivity upon addition of lanthanum to the perifusate. ;:- Go Go G'..-----O_-

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Fig. 2. Effect of glucose (16.7 mM) on 4-calcium efflux (upper panel) and insulin release 0). Control experiments ( O- - -0) were performed in the absence (lower panel; * of glucose. Mean values (± S.E. of mean) for 45calcium efflux are expressed as in Fig. 1 and refer to eighteen individual experiments. Mean values (+ s.E. of mean) for insulin output is expressed in ments.

4su/islet. min and refer to the same eighteen individual experi-

Effect of glucose on 45calcium efflux Fig. 2 illustrates the effect of glucose 16-7 mm on 45calcium efflux and insulin release. The upper panel shows that addition of glucose, after 44 min perifusion in the

413 CALCIUM FLUXES IN PANCREATIC ISLETS absence of any metabolic substrate, provoked a biphasic modification of 45calcium efflux as previously reported (Malaisse et al. 1973; Bukowiecki & Freinkel, 1976). An initial fall observed during the first 3 min of exposure to glucose, was followed by a dramatic increase in 45calcium efflux, this secondary rise being concomitant to G300

G300

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50

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70

Fig. 3. Left part: effect of glucose suppression on "calcium efflux (upper panel) and *). Right part: effect of Verapamil (10 FM) on insulin release (lower panel; * *). Control "calcium efflux (upper panel) and insulin release (lower panel; * -0) were performed in the presence of glucose (16-7 mM). Mean values perifusions (0 ( ± S.E. of mean) for "calcium efflux are expressed as in Fig. 1 and refer to four individual experiments. Mean changes in insulin output (± s.E. of mean) relative to the mean insulin output recorded at the end of the equilibration period (min 30 to min 44) are expressed in Izu./islet, and refer to the same four individual experiments. The mean 0-05 ,uu./islet.min. insulin output at the end of the control period averaged 1-17 - -

414 A. HERCHUELZ AND W. J. MALAISSE insulin release (Fig. 2, lower panel). The secondary rise in 45calcium efflux itself displayed a biphasic pattern, which is reminiscent of that seen for insulin release with a peak at the 50th min and a trend towards a rebound at about the 55th-57th min. When glucose was withdrawn from the perifusate an immediate inhibition of 45calcium efflux was noticed (Fig. 3, upper and left panel). Five minutes after no Ca-G0

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40

50

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Time (min)

Fig. 4. Effect of D-gluCoSe (16.7 mM; * 0*) and L-glucose (O- --) at the same concentration on 45calcium efflux (upper panel) and insulin release (lower panel), when no calcium chloride was incorporated in the perifusion medium. Mean values (+ s.E. of mean) for calcium efflux and mean changes in insulin output ( + s.E. of mean) are expressed as in Fig. 3, and refer to the same four individual experiments. The mean insulin output at the end of the control period averaged 0 12 ± 0 01 #tsu./islet. min).

415 CACIIUM FLUXES IN PANCREATIC ISLETS withdrawal of glucose, the efflux averaged 52-5 + 3*7 % as distinct from 82-7 + 22 0/ i.e. the value found at the same time in control experiments performed throughout in the presence of glucose. This fall in calcium efflux was paralleled by a reduction of

insulin release (Fig. 3, lower and left panel) with was of the same order of magnitude (51-8 + 1.80/ as distinct from 98 6 + 0 5 0/ at the 50th min).

Effect of glucose in the absence of extracellular calcium In the absence of extracellular calcium (Fig. 4, upper panel), the addition of glucose only caused the initial fall in 45calcium efflux, the secondary rise being almost completely abolished. Four minutes after the addition of glucose 16-7 mM, the efflux averaged 46-7 + 80 0/ as opposed to 88-0 + 3-2 % in the control experiment. During the first 3 min after the addition of glucose, the reduction in 45calcium efflux was of the same orderof magnitude as that observed at normal calcium concentration. Although extremely blunted and delayed the secondary rise in 45calcium efflux may not be totally abolished, as suggested by a slow increase in efflux after the 54th min. No stimulation of insulin release was observed (Fig. 4, lower panel). On the contrary the basal insulin release which was of the same order of magnitude in the absence of extracellular calcium than at normal calcium concentration, was transiently reduced by glucose. This effect of D-glucose was mimicked by L-glucose which however had no effect on 45calcium efflux.

Effect of Verapamil Verapamil is known to abolish glucose-induced insulin release presumably by inhibiting calcium entry into the fl-cell (Malaisse, Herchuelz, Levy & Sener, 1977). Fig. 5 (lower panel) shows that Verapamil at a concentration of 1O/AM markedly inhibited and delayed glucose-induced insulin release. In terms of integrated output between the 45th and 70th min, the inhibition of insulin release averaged 72 %. In the presence of Verapamil the effect of glucose on 45calcium efflux remained biphasic (Fig. 5, upper and right panel). The initial fall was as steep as in the control experiment, but apparently lasted 1 min more. The secondary rise was delayed and blunted. The delay was the same for both insulin release and the secondary rise in 45calcium efflux, but Verapamil seemed to reduce more markedly the release of insulin than it affected the secondary rise in effluent radioactivity. Indeed, as judged by planimetry and after correction for the efflux seen in response to glucose but absence of extracellular calcium (Fig. 4), it can be calculated that Verapamil decreased the magnitude of the secondary rise by only 21-6 %. When Verapamil 1 0/M was added to the islets already stimulated by glucose, both insulin release and 45calcium efflux were inhibited (Fig. 3, right panel). The inhibition of insulin release again appeared more marked than the inhibition of 45calcium efflux. When used at a 20 /M concentration, Verapamil completely suppressed the secretary response to glucose (Fig. 6, right panel). The initial fall in 45calcium efflux was not affected. Although inhibited by 38-6 %, the secondary rise in 45calcium efflux was still present.

A. HERCHUELZ AND W. J. MALAISSE

416

Effect of magnesium 10 mm Magnesium is a well known inhibitor of insulin release, presumably because it competes with calcium for a common carrier system (Miner & Hales, 1967). In the presence of magnesium 10 mm, the capacity of glucose to stimulate insulin release was strongly inhibited and delayed as compared to the control experiment (Fig. 7, Go

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Fig. 5. Right part: effect of glucose (16-7 mM) upon calcium efflux (upper and right panel) and insulin release (lower and right panel) in the presence of Veiapamil (10 tM). The control experiments performed in the absence of Verapamil are shown in the panel to the left. Mean values ( ± s.E. of mean) for calcium efflux and insulin output are expressed as in Fig. 2, and refer to four individual experiments.

417 CALCIUM FLUXES IN PANCREATIC ISLETS lower panel). The inhibition by magnesium of glucose-induced insulin release averaged 85 %. The efflux of 45calcium remained biphasic (Fig. 7, upper panel). The Go -V20

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Fig. 6. Effect of glucose (16.7 mM) upon calcium efflux and insulin release in the in Fig. 5. presence of Verapamil (20 FM). Same presentation as

initial fall was present and lasted apparently 2 min more than in the control experiment. As previously outlined in the case of Verapamil, it can be calculated that magnesium reduced the secondary rise in 45calcium efflux by 55-1 %. Both insulin release and the secondary rise were again delayed for the same period of time.

14

PHY

283

A. HERCHUELZ AND W. J. MALAISSE

418

Effed of barium In the absence of glucose and extracellular calcium, barium is known to provoke a prompt but rapidly decreasing stimulation of insulin release (Malaisse et al. 1973; Somers, Devis, Van Obberghen & Malaisse, 1976). When the islets had been exposed Go

GOMg1O G300 -Mg 10

G 300

200 , 180 160 k.-I

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Fig. 7. Effect of glucose (16.7 mM) upon calciumm efflux and insulin release in the presence of MgCl2 (10 mM). Same presentation as in Fig. 5.

for 44 min to such a glucose-free and calcium-free medium containing barium 2 mm, the introduction of glucose provoked a marked and almost immediate increase in insulin output, whereas the efflux of 45calcium remained apparently unaffected

(Fig. 8).

CALCIUM FLUXES IN PANCREATIC ISLETS no Ca-Ba-G0 -O--O no0 G3

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Fig. 8. Effect of glucose (16-7 mM) upon 45calcium offlux (upper panel) and insulin release (lower panel) in islets exposed to a medium in which calcium was replaced by barium (2 mM; * *). Control experiments were performed in the absence of glucose (--- -0). Mean values (± s.E. of mean) for 45calcium efflux and changes in insulin output are expressed as in Fig. 3, and refer to four individual experiments. The mean insulin output at the end of the control period averaged 0-46 ± 0-04 ,u./islet min, a value significantly higher than that found at normal calcium concentration in the absence of barium (04 1 ± 0 04 #u./islet.min). .

420

A. HERCHUELZ AND W. J. MALAISSE DISCUSSION

Source of effluent radioactivity In our experiments, the islets, after exposure to calcium, are submitted to five successive washes and then perifused for 30 min before that any measurement of 45calcium efflux is performed. Therefore, it is fairly probable that the effluent radioactivity originates from intracellular site(s). Indeed, when isolated pancreatic islets are preloaded during 60 min incubation with 45calcium and thereafter submitted to successive washes, the radioactivity eventually released by the islets during the last wash is proportional to the final calcium content of the islets (Malaisse-Lagae & Malaisse, 1971). Moreover, when tritiated sucrose is used as an extracellular marker, up to 98 % of the radioactivity is released within 60 see (Nabel et al. 1977). It is nevertheless conceivable that 45calcium originating from intracellular sites reequilibrates with superficially or even extracellular calcium pools in such a way that the pattern of 45calcium efflux reflects the changes in affinity for calcium of both intracellular and extracellular structures. Because of its high affinity for calcium binding sites and its poor penetration in the intracellular space, lanthanum has been successfully used by different authors to inhibit transmembrane calcium fluxes and to displace calcium from extracellular binding sites (Weiss, 1974; Hellman et al. 1976a). In our system lanthanum failed to increase effluent radioactivity and, instead almost completely abolished 45calcium efflux from the islets. This finding supports the view that, under the present experimental conditions, the effluent radioactivity is exclusively derived from intracellular site(s).

Effect of glucose on 45calcium efflux Confirming previous work (Malaisse, 1973; Bukowiecki & Freinkel, 1976), our results illustrate that glucose provokes dramatic and rapid modifications of 45calcium efflux, namely an initial fall, which precedes the onset of insulin release and, 3-4 min later, a secondary rise which is concomitant to insulin release. These modifications might reflect intracellular redistribution or altered permeability to calcium of the plasma membrane. In any case the glucose-induced changes in 45calcium efflux, and especially the initial fall, occur with a sufficient rapidity to meet the kinetic requirement of a pool (or pools) participating in the initiation of stimulus-secretion coupling. At least our findings do not support the view that the 'lanthanum-non-displaceable' or intracellular calcium pool is not sufficiently labile to play such a role (Hellman et al. 1976a, b). Nature of the glucose-induced secondary rise in 45calcium efflux It was proposed that the glucose-induced secondary rise in 45calcium efflux corresponds, in part at least, to 45calcium released at the time and site of exocytosis; for instance, it could correspond to the release of calcium sequestered in secretary granules (Malaisse et al. 1973). Because the secondary rise would then merely be the consequence of insulin release, it has received much less attention than the initial fall in 45calcium efflux thought to reflect an inhibition of calcium outward transport across the plasma membrane (Malaisse et al. 1973). Recently, Kikuchi and others observed a dissociation between the rise in 45calcium efflux and the

421 CALCIUM FLUXES IN PANCREATIC ISLETS stimulation of insulin release, when the secretary response to glucose was blocked by use of a low temperature or in the presence of a low concentration of either somatostatin or epinephrine (Kikuchi, Cuendet, Renold & Sharp, 1976; Kikuchi, Wollheim, Renold & Sharp, 1977). This observation led us to re-evaluate the nature of the glucose-induced secondary rise in 45calcium efflux. At normal extracellular calcium concentration the secondary rise, which indeed occurs concomitantly to insulin release, displays a biphasic pattern reminiscent of that seen for insulin release. It is likely that, whatever its nature, the phenomenon which is responsible for the secondary rise persists throughout the period of exposure to glucose. Indeed, when glucose is removed from the perifusate, a fall in effluent radioactivity is observed, which again closely parallels the falling pattern of insulin

release. Despite these close temporal analogies between the pattern of '45calcium efflux and insulin release, the secondary rise in calcium efflux may not be merely the consequence of insulin release. The two phenomena can indeed be dissociated. For instance, Verapamil 10/UM, which strongly inhibits insulin release, poorly affects the secondary rise in calcium efflux. Furthermore, at a higher concentration of Verapamil (20 mM) glucose was still able to evoke a secondary rise in calcium efflux, despite complete inhibition of insulin release. Under the latter experimental condition, the secondary rise was both retarded and blunted. In the experiments conducted by Kikuchi et al. (1976, 1977), the secondary rise remained unaffected when glucose-induced insulin release was suppressed by either somatostatin or epinephrine. We are forced, therefore, to consider an alternative explanation for the glucose-induced increase in calcium efflux. Is the secondary rise in 45calcium efflux due to entry of calcium in islet cells? As can be seen from the results obtained in the presence of Verapamil or in the absence of extracellular calcium, the secondary rise seems to be affected by conditions known to interfere with calcium entry into the fl-cell. It was previously shown that, when the perifusate is devoided of calcium and enriched with EGTA 1 mm, the secondary rise is completely suppressed (Malaisse et al. 1973). At a slightly higher extracellular calcium concentration, namely in the absence of EGTA, we observed a small and extremely retarded secondary rise. In the presence of increasing Verapamil concentrations, the secondary rise was gradually inhibited. These converging data are compatible with the hypothesis that the secondary rise somehow reflects the rate of calcium influx into the fl-cell. To further explore this hypothesis, experiments were carried out in the presence of a high magnesium concentration, thought to inhibit calcium influx into the fl-cell by competing with calcium for a common carrier system (Milner & Hales, 1967; Malaisse, Devis, Herchuelz, Sener, & Somers, 1976). The results show that magnesium at a 10 mm concentration, indeed inhibits the glucose-induced secondary rise in 45calcium efflux. Our working hypothesis, according to which the glucose-induced increase in 45calcium efflux may be the consequence of an increased rate of 40calcium entry in the islet cells, is also supported by the highly significant correlation (Fig. 9) observed between (i) the magnitude of the secondary rise in calcium efflux, as documented by the present study, and (ii) the values for the glucose-stimulated net uptake of

422 A. HERCHUELZ AND W. J. MALAISSE 45calcium by the islets, as previously measured (Malaisse et al. 1976, 1977, 1978b). In considering this correlation, it should be stressed that we have purposely restricted our comparison to experimental situations in which the agent under study (i.e. extracellular calcium, extracellular magnesium, Verapamil) does not exert any obvious primary effect upon calciumm efflux (Malaisse et al. 1976, 1977, 1978b). 100

*

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100

Fig. 9. Correlation between the magnitude of the glucose-induced secondary rise in "calcium efflux and the values for glucose-stimulated "calcium net uptake (Malaisse et al. 1976, 1977, 1978 b). The magnitude of the secondary rise was judged from the area located below the "calcium efflux curve (45th -70th min) and above the level reached at the 54th min of perifusion in the absence of extracellular calcium. The latter value was chosen because it corresponded to the nadir in "calcium efflux observed in this condition (see Fig. 4, upper panel). The mean values for both parameters were recorded in the presence of Verapamil 10 ,M (E), Verapamil 20 /Sm (@), magnesium 10 mM (V) and media to which no calcium was added (V), and are expressed in percentage of the mean reference value found under normal experimental conditions (*).

Stimulation of insulin release without a rise in 45calcium efflux We have so far stressed experimental conditions in which glucose provokes a secondary rise in 45calcium efflux despite suppression of insulin release. In the absence of calcium but presence of barium, the opposite situation was encountered: glucose increased insulin output without causing any obvious change in 45calcium efflux

(Fig. 8).

It is known that barium can substitute for calcium in the process of insulin release evoked by glucose and other insulinotropic agents (Malaisse, Brisson & MalaisseLagae, 1970). Barium is even able to stimulate insulin release in the absence of any other secretagogue, provided that little or no calcium is present in the extracellular milieu (Hales & Milner, 1968; Malaisse et al. 1973; Wollheim, Blondel, Trueheart, Renold & Sharp, 1975). Radioisotopic studies suggest that the effect of barium upon insulin release depends on its accumulation within the islet cells (Somers et at. 1976).

423 CALCIUM FLUXES IN PANCREATIC ISLETS A recent ultrastructural study confirms that barium accumulates in the fl-cell, mostly in mitochondria and to a lesser extent in the plasma membrane and secretary granules (Howell & Tyhurst, 1976). Our results are compatible with the view that barium enters the islet cells and, in doing so, may displace calcium from intracellular sites (Malaisse et al. 1973). Indeed, when calcium was replaced by barium, the curve for 45calcium efflux displayed a steeper pattern, suggestive of an increased fractional turnover rate. Under this condition, the introduction of glucose and the resultant stimulation of insulin release was not accompanied by any rise in 45calcium efflux, as if barium had already depleted the pool of 45calcium normally mobilized in

response to glucose. In conclusion, the present study indicates that the efflux of45calcium from isolated pancreatic islets, although entirely derived from intracellular sites, may reflect several distinct regulatory processes such as the intracellular redistribution of this cation, its true outward transport accros the plasma membrane and its displacement by influent divalent cations. This work was supported in part by grants 3.4527.75 and 3.4537.76 from the Belgian Foundation for Scientific Medical Research, and a contract of the Belgian Ministery of Scientific Policy within the framework of the association Euratom-TJniversities of Brussels and Pisa. The authors are indebted to C. Remion and S. Procureur for skilful technical assistance.

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