Role of calcium in exocrine pancreatic secretion I. Calcium movements in the rabbit pancreas

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Biochimica et Biophysica Acta, 404 ( 1 9 7 5 ) 2 5 7 - - 2 6 7 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 2 7 7 3 4

ROLE OF CALCIUM IN EXOCRINE PANCREATIC SECRETION I. CALCIUM MOVEMENTS IN THE RABBIT PANCREAS* V.V.A.M. SCHREURS, H.G.P. SWARTS, J.J.H.H.M.D E P O N T and S.L. B O N T I N G

Department of Biochemistry, University of Nijmegen, Nijmegen (The Netherlands) (Received March 11th, 1975)

Summary 1. Calcium movements in the isolated rabbit pancreas and in rabbit pancreas fragments have been studied with the aid of 4 s Ca2+. 2. Addition of 4 s Ca2÷ to the incubation medium of the isolated rabbit pancreas results in an immediate appearance of isotope in the secreted fluid reaching a constant specific activity in 30 min. The absolute activity in the secreted fluid is 30--40% of that in the incubation medium. 3. Addition of 10 -s M carbachol after 2 h preincubation with 4 s Ca2+ results in enzyme secretion accompanied by calcium release. There is also an increase in 4 s Ca2+ secretion, but this is maximal 10 min after the protein and total calcium peaks. 4. Partial removal of 4 s Ca2+ from the bathing medium, before stimulation, reduces the increase in 4 s Ca2+ secretion nearly proportionally. 5. [3 H] Mannitol, added to the bathing medium, appears in the secreted fluid and behaves upon carbachol stimulation similarly to 4 s Ca2.. 6. Upon repeated stimulation with 10 -5 M acetylcholine, a 45 Ca2÷ peak appears, even in virtual absence o f enzyme secretion. In this case the peak coincides with a small total calcium peak. 7. Efflux studies of rabbit pancreas fragments, preloaded with 4 SCa2+ ' show a carbachol-stimulated 4 s Ca2+ efflux in addition to a release of amylase. 8. These studies indicate that there are three calcium movements in rabbit pancreas which can all be influenced by cholinergic agents: (a) an extracellular route for calcium and other small molecules and ions; (b) a calcium release across the apical membrane along with the enzymes, originating from a pool which does not freely exchange with 4 s Ca2+ in the bath; (c) a calcium flux across the serosal membrane, which involves calcium exchanging freely with

* Presented in preliminary form at the 7th meeting of the European Pancreatic Club, Dundee, 3--6 July, 1974.

258 4 s Ca2+ from the bath. The third flux is thought to result from an increase in cytoplasmic calcium, which may be involved in the stimulus-secretion coupling of pancreatic enzyme secretion.

Introduction Calcium metabolism seems to be involved in pancreatic functioning at different levels. Omission of calcium from the bathing medium or perfusion fluid reduces the basal electrolyte secretion in the perfused cat pancreas [1], and abolishes the secretory effects of secretagogues in pigeon pancreas slices [ 2 ] , rat pancreas fragments [3] and the perfused cat pancreas [1]. The basal calcium concentration of the pancreatic fluid is always dependent on and generally lower than the calcium concentration of the medium [1,4--6]. Increases in enzyme secretion, caused b y various stimuli of the exocrine pancreas, are always accompanied b y increases in the secretion of calcium in the secreted fluid in cat [ 1 ] , dog [4,5] and rabbit [6]. These observations suggest that the calcium content of the fluid is closely related to the amount of digestive enzymes present in it. This cannot be the full picture, since in none of these studies a constant calcium:protein ratio is observed. This indicates that the calcium in the secreted fluid cannot merely represent a single pool of protein-associated calcium, b u t must originate from one or more other calcium pools as well. The calcium should at least be secreted in t w o fractions, one associated with the digestive enzymes and the other with the electrolytes of the fluid [1,5]. From experiments with the perfused cat pancreas [1] it is further concluded that Ca 2+ may play an important role in the stimulus-secretion coupling of the pancreatic acinar cells, as t h e y do in the secretion from mast cells, chromaffin cells and neurohypophysis [ 7 ] . Since the pattern of calcium movements in relation to stimulation may yield information a b o u t the role of calcium in the secretory process, we have tried to identify these movements in the pancreas. In these experiments we have used b o t h the intact, isolated rabbit pancreas and fragments of this pancreas. In the former system the stimulatory and secretory compartments are retained separately and thus 4s Ca2. can be added to the first compartment alone. The fragments, preloaded with 4SCa:+, permit the study of 4s Ca2* efflux on the stimulatory side, although in combination with that on the secretory side (Fig. 6). The conclusions, which can be drawn a b o u t the calcium movements in the rabbit pancreas, are discussed in relation to a role of calcium in the stimulus-secretion coupling of the pancreatic enzyme secretion. Materials and Methods Chemicals. Carbachol, the carbamyl analogue of acetylcholine, was purchased from Brocades-acf Holland. 4 s CaC12 (1 ~Ci/0.037 mg calcium per ml) and 3H-labelled inulin (1 pCi]0.0058 mg inulin) were supplied by Amersham and [3H] mannitol (1 /~Ci/0.0688 mg mannitol per ml) b y New England Nu-

259

clear. Insta-gel and Aquasol were obtained from Packard and New England Nuclear, respectively. Preparation of the isolated rabbit pancreas. Male and female New Zealand white rabbits, weighing 2--3 kg, are fasted 24 h before the start of each experiment. The animals are killed by a blow on the neck, immediately followed by carotic exsanguination. The rabbit pancreas is further prepared and m o u n t e d essentially according to R o t h m a n [8]. Preparation o f pancreas fragments. Fragments of about 250 mg wet weight are cut from the pancreatic tissue stretched between the spleen and the rectum. Incubation medium. Both preparations of the rabbit pancreas are incubated in a balanced salt solution, containing (in mmol/1): Na ÷, 143.5; K ÷, 4.9; Ca 2+, 2.5; Mg2÷, 1.2; HCO~, 25.0; H2PO:,, 1.2; CI-, 130.7; and glucose, 5.5. Before incubation the pH of the solution is adjusted to 7.2 by addition of HC1. During incubation the medium is continuously gassed with O:/CO2 (95 : 5, v/v) and maintained at 37 ° C. Incubation and fraction collection for isolated pancreas. The isolated rabbit pancreas is incubated for 1 h after mounting in a bath, containing 300 ml incubation medium, to reach a steady-state condition. After this period, the incubation m e d i u m is replaced by a medium containing 4 s Ca2+ and occasionally [3 HI mannitol plus 2 mM mannitol. In single label experiments 15 #l of the undiluted 4s CaC12 solution is added and in double label experiments 25 ~1 combined with 25 #1 of the undiluted [3 H] mannitol solution. The pancreatic fluid is collected in 5-min fractions in pre-weighed plastic counting vials. From each fraction 10-pl samples are taken for protein and total calcium determination. The remaining volume, determined by weighing, is mixed with 10 ml Aquasol and subjected to radioactive counting. During incubation the secretion stimuli are applied as indicated in the table and figures. Incubation and sample collection for pancreas fragments. The pancreas fragments are preloaded for 2 h in 10 ml incubation medium, containing 100 ~1 of the undiluted 4 s CaCI~ solution. The fragments are then washed for 15 min in 300 ml fresh m e d i u m to remove adhering radioactivity. They are then transferred after fixed periods from one plastic counting vial, containing 5 ml fresh medium, to another. In the initial phase of the experiment we have used 15-min periods and in the later phase 5-rain periods. From each efflux medium 200-pl samples are taken for the amylase assay. The radioactivity left in each vial is measured after mixing the m e d i u m with 10 ml Insta-gel. The stimulus is present in the efflux vials used during the period indicated by the bar in the figure. The radioactivity left in the tissue is determined by means of the internal standard m e t h o d and the efflux rate of each fraction is calculated. Assay methods. Protein is determined according to Lowry et al. [9] on a micro scale, bovine serum albumin (Behringwerke) serving as a standard. Total calcium concentrations are determined on a micro scale with a calcium rapid stat kit (Pierce Chemical Co., U.S.A.}, by which the blue color o f the calcium complex of m e t h y l t h y m o l blue is measured. Amylase activity is determined according to Bernfeld [10]. One unit of amylase activity is defined as 1 mg maltose liberated in 3 min at 30 ° C. The radioactivity present in the collected fractions is measured in a liquid

260 scintillation analyzer (Philips). In dual label experiments, the radioactivity was calculated b y means o f the external standard channels ratio method. Results

Isolated pancreas in the resting state The pancreatic fluid is collected from the cannulated duct of the isolated rabbit pancreas in 5-min fractions. These are analyzed for total weight and the concentrations of * 5 Ca~÷, total calcium and protein. The average results for six experiments are presented in Table I. A typical experiment is shown in Fig. 1. After addition of the tracer ion to the bathing medium, 4 s Ca2÷ is immediately secreted into the pancreatic fluid. After a b o u t 30 min the specific activity becomes constant and is then equal to that of the bathing medium, while the total calcium concentration amounts to only 30--40% of the bath concentration. The t w o levels remain constant for at least 4 h in the absence of stimulation. Only when the volume flow decreases below 250 pl/h do the t w o concentrations increase. Effects o f carbachol When after a preincubation period of 2 h the enzyme secretion is stimulated b y adding 10 -s M carbachol to the 4 s Ca~+ containing medium, the protein concentration in the secreted fluid increases within 5 min and reaches a maximum in a b o u t 10 min (Fig. 1). The same is true for the total calcium concentration. However, the 4 s Ca2÷ concentration begins to rise only after about 8 min and reaches a maximum after 20 min. Another difference is that the 45 Ca2÷ concentration in the secreted fluid increases only 2-fold, which is less than the increase in the total calcium. 2 h after stimulation the protein concentration has nearly returned to its basal level, while the * s Ca2÷ and total calcium levels b e c o m e equal again, b u t remain higher than they were before stimulation.

TABLE I E F F E C T S O F 10 -5 M C A R B A C H O L O N T H E C O M P O S I T I O N O F T H E S E C R E T E D F L U I D C O L L E C T ED FROM THE ISOLATED RABBIT PANCREAS M e a n v a l u e s w i t h s t a n d a r d e r r o r s o f the m e a n f o r e i g h t c o n s i s t e n t e x p e r i m e n t s . T h e e a r b a e h o l is a d d e d t o the 4 5 C a 2 + - c o n t a t n i n g m e d i u m at the end o f the s e c o n d h o u r ( p e r i o d 4). Half-hour period

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Effects of repeated stimulation In order to check whether the increase in 4 s Ca2+ is due to an increased calcium exchange upon stimulation, the effect of repeated stimulation has been studied. Acetylcholine has been used in this experiment instead of carbachol, which is a poor substrate for acetylcholinesterase [ 1 1 ] . In the case of stimulation with 10 -s M acetylcholine the initial effects are comparable to that of carbachol, as is seen by comparing Fig. 2 with Fig. 1. Addition of a second dose of acetylcholine 80 min later gives only a very minor increase in protein secretion. However, the 4 s Ca2+ secretion increases to about the same level as upon 150

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262

the first stimulation. It is remarkable that the increase in total calcium now coincides with that of 4 s Ca~+. Both peaks are delayed with respect to the protein peak. R e m o v a l o f 4 s Ca~+ before stimulation

Partial removal (down to 25% of the original level) of 4 s Ca~+ f r o m the bathing medium after 90 min equilibration in the presence of the isotope causes the specific activity of the secreted fluid to drop proportionally in 30 min. Stimulation at that time also gives a reduced stimulation of the ~ s Ca~+ secretion, b u t its peak value is again about double the level just before stimulation. The curves for total calcium and protein show the normal characteristics (Fig. 3). Complete removal of ~ s Ca~÷ has also been attempted. It was not possible to obtain a completely isotope-free medium, due to leakage o f radioactivity from the tissue, but the level decreased to less than 5%. In four such experiments the ~ s Ca~÷ level in the secreted fluid decreased to 4.2% (S.E.: 0.6) of the original bath concentration. The 4 s Ca~+ peak after stimulation reached a maxim u m of 8.7% (S.E.: 0.5), which is again double the level before stimulation (not shown). Studies with extracellular markers

The possible occurrence of an extracellular 45 Ca2+ movement has been studied by comparing the 4 s Ca~+ movements with those of the radioactive extracellular marker [3 H] mannitol [ 1 2 ] . In these experiments (Fig. 4) the 4s Ca2÷ and the [ ~ H ] m a n n i t o l are added simultaneously at time zero to the bathing medium, which contains also 2 mM non-labeled mannitol. The similarity between the concentration curves for 4s Ca~÷ and [~H]mannitol in the 150

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pancreatic fluid is striking. The equilibrium between the stimulatory and secre: t o r y compartments is reached within half an hour for both substances, while t h e y both show a maximal value 20 min after stimulation with 10 -s M carbachol. The two curves differ only in the levels of 4 s Ca~+ and [3 H] mannitol, expressed as percentage of their concentrations in the bathing medium. The constant mannitol concentration is about twice the 4 S Ca2+ concentration before stimulation. On stimulation, the [3 H] mannitol concentration increases less than t h a t of 4 s Ca2+ and then returns to its original Value, while the 4 s Ca2+ concentration remains increased relative to its original concentration. When in similar studies the larger extracellular marker 3 H-labeled inulin was used, hardly any 3H radioactivity could be detected in the secreted fluid. 4 s Ca2+ efflux from pancreas fragments The pancreas fragments are incubated for 2 h in the presence of 4 s Ca2+ and then transferred to a series of plastic counting vials with 4 s Ca2÷.free medium to determine the 4 s Ca2+ efflux rate. The experiment of Fig. 5 shows a m o n o t o n e efflux of 4 s Ca2+ in the absence of carbachol. When 10 -s M carbachol is added to the efflux medium, there is an immediate, large increase in 4 s Ca2+ efflux. The normal efflux rate is restored about 30 min after stimulation. The increased 4 s Ca2+ efflux is accompanied by a large increase of the amylase release. The amylase efflux remains relatively high during the rest of the experiment.

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Discussion Since the stimulus-secretion coupling in pancreatic enzyme secretion is thought to be mediated b y an increase of the cytoplasmic calcium concentration, we have tried to obtain more detailed information a b o u t the calcium movements in this organ. We have used the isolated rabbit pancreas, which because of its sheetlike structure is quite suitable for in vitro incubation studies. Upon cannulation of the main pancreatic duct separate stimulatory and secretory compartments are obtained, and additions to the stimulatory compartment can be made w i t h o u t necessitating organ perfusion. The preparation is stable for at least 5 h and through the application of micro-analytical methods it is possible to analyze successive 5-min fractions. In the basal situation, where the enzyme secretion is very low, the calcium concentration is normally about 30% of that in the medium. Only when the volume flow decreases below 250 gl/h does the calcium concentration in the secreted fluid tend to that of the medium. This is in agreement with the findings of Argent et al. [1] in cat pancreas and Goebell et al. [5] in dog. Upon addition o f 4 s Ca:+ to the bathing medium, the isotope immediately appears in the secretory fluid and its specific activity reaches that of the medium in about 30 min (Fig. 1). The 4 s Ca2+ content in the secreted fluid decreases proportionally with the level in the medium, when the specific activity of the bathing medium is lowered after equilibration (Fig. 3). This suggests the existence o f an extracellular route for calcium, possibly b y the so-called tight junctions between adjacent cells. This hypothesis seems to be confirmed b y the fact that upon addition of the extracellular marker mannitol to the bathing medium this

265 substance also appears immediately in the secreted fluid and reaches its equilibrium level in the same time as 4 s Ca2÷. A similar observation has been made, when sodium in the incubation medium is partially replaced b y potassium. In that case, the sodium and potassium concentrations in the secreted fluid equilibrate again in 30 min with their concentrations in the medium (Case and Scratcherd [ 1 4 ] ; Schreurs et al., unpublished observations), suggesting an extracellular route for these monovalent cations. The low calcium concentration in the secreted fluid may be due to t w o antagonistic effects: a low diffusion constant for calcium in the extracellular route, and a high flow rate for water in the isolated rabbit pancreas. This suggestion finds support in our observation that the calcium concentration tends to increase when the flow rate decreases. The latter observation argues against a re-uptake process for calcium in the ductular cells as an explanation for the low calcium concentration in the secreted fluid. When the pancreas is stimulated with 10 -s M carbachol (Fig. 1) or 10 -s M acetylcholine (Fig. 2) the protein secretion is accompanied by a simultaneous secretion of calcium. This has been reported previously in several pancreatic preparations [1,4--6]. There is a positive correlation between the increase in calcium and the magnitude of the enzyme secretion, but the ratio is not constant. Although in our experiments the calcium and protein peaks coincide in time, the enzyme concentration returns to its original level faster than the calcium concentration does (Figs 1 and 2, and Table I). An unexpected observation in this study is the fact that the increase in 4 s Ca2÷ secretion is always less than that of total calcium and that the 4 s Ca2÷ peak is delayed with respect to the total calcium peak. This indicates that the calcium secreted along with the enzymes does not exchange with the 4 s Ca2÷ from the medium during the 2-h incubation period. The origin of this calcium flux is not clear. It probably represents calcium sequestered b y zymogen granules as occurs in the parotid gland [ 1 5 ] , b o u n d to the enzymes or to the membranes, which contain a high a m o u n t of calcium [ 1 6 ] . In any case, it is an intracellular pool which does not freely exchange with 4 s Ca2÷ in the incubation medium. These results support the suggestion of Argent et al. [1] and of Goebell et al. [5] that the calcium content of the pancreatic fluid is produced b y distinct calcium movements. What is the meaning of the delayed 4 s Ca2÷ peak found u p o n stimulation? The experiment of Fig. 2 with repeated stimulation has been undertaken in order to check whether the increase in 4 s Ca2÷ is due to an increased exchange of 4 s Ca2+ with protein-associated calcium u p o n stimulation. The figure enables us to conclude that this is not the case, since the delayed 4 s Ca2÷ movement after the second stimulation is paralleled b y a similar increase of non-labeled calcium, b u t in the virtual absence of enzyme secretion (Fig. 2). This observation pleads for a distinct calcium movement u p o n stimulation, which is normally masked b y the large a m o u n t of protein-associated calcium. Its presence can, however, be derived from the fact that the total calcium decreases slower than the protein. Most likely this calcium movement is due to an increase in the permeability of the extracellular route. This is supported b y the fact that the secretion of [3 H] mannitol is simultaneously enhanced u p o n stimulation with 10 -s M carbachol (Fig. 4). It is not clear whether such an increase in the extra-

266

iSOLATED ORGAN 3-COMP SYSTEM

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COMPARTMENTS

C A L C I U M FLUXES

I I ST I MU L A T OR Y COMPARTMENT (BLOOD) 2. SECRETORY TISSUE 3. SECRETORY COMPARTMENT (DUCT)

I EXTRACELLULAR CALCIUM FLUX [I SECRETORY CALCrUM FLUX I][ STIMULATORY CALCIUM FLUX

Fig. 6. S c h e m a t i c r e p r e s e n t a t i o n o f t h e c a l c i u m f l u x e s in the t w o d i f f e r e n t rabbit pancreas preparations.

cellular p a t h w a y has any physiological significance, but it may explain why no constant correlation between the increase in calcium and protein secretion is found. It may be an artifact of the isolated pancreas preparation, which may also be the case for the fact that the original calcium concentration in the secretory fluid is not restored after stimulation {Fig. 1). The absence of a 4 s Ca2÷ peak in the secreted fluid of the isolated rabbit pancreas, when 4 s Ca2+ is removed from the medium just before stimulation, seems to be contradictory to earlier findings [3,13,17] in pancreas fragments from mouse and rat. Efflux studies with these fragments, preloaded with 4 s Ca~÷ show an acetylcholine-stimulated 4 s Ca2+ efflux in an isotope-free medium. In repeating these experiments with rabbit pancreas fragments we made essentially the same observations (Fig. 6) as obtained for fragments of rat and mouse pancreas. Hence, there is no species difference involved. Rather, the difference between the isolated rabbit pancreas and the rabbit pancreas fragments must be attributed to the fact that in the latter system secretion into the stimulatory and secretory compartments together is measured. This suggests that the 4 s Ca2÷ efflux observed with pancreas fragments in our and in previous studies [3,13,17] is due to an increased efflux across the serosal membrane. This can be due either to an increase in the permeability of this membrane or to a release of calcium from intracellular stores. Both possibilities would account for the fact that the calcium released into the stimulatory compartment is easily exchangeable. We shall call this flux the stimulatory calcium flux, to distinguish it from the secretory calcium flux, which represents calcium that is poorly exchangeable. The different calcium fluxes which can be recognized in the rabbit pancreas with the aid of 4 s Ca2÷ are shown in Fig. 6. It has been proposed that calcium plays a role in the stimulus-secretion coupling in pancreatic enzyme secretion. In rabbit pancreas carbachol seems to influence the calcium fluxes at different levels: (1) it increases the permeability of the extracellular route for several components including calcium; (2) it causes a release of calcium over the apical membrane along with the proteins;

267

3) it causes an increase in the calcium flux across the serosal membrane either by an increase in the permeability of this membrane or by a release of calcium from intracellular stores. Independently of the mechanism involved, this flux seems to indicate that the cytoplasmic calcium concentration increases upon stimulation. In addition, we have recently found that pancreatic enzyme secretion can be initiated by addition of the divalent cation ionophore A-23187 [18]. Similar observations have been reported by other investigators [19,20]. This suggests that the stimulus-secretion coupling in pancreatic enzyme secretion is mediated by an increase in the cytoplasmic calcium concentration. Most likely this increase is responsible for the stimulatory calcium flux observed with pancreas fragments. The mechanism by which the cytoplasmic calcium concentration is physiologically enhanced and the molecular mechanism by which such an increased calcium concentration leads to enzyme secretion still need further elucidation.

Acknowledgements The excellent technical assistance of Mrs EUy Dekkers-Roose during the initial phase of this investigation is gratefully acknowledged. This investigation was supported in part by the Netherlands Organization for the Advancement of Basic Research (Z.W.O.), through the Foundation of Biophysics. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Argent, B.E., Case, R.M. and Scratcherd. T. (1973) J. Physiol. L o n d o n 230, 575--593 Hokin, L.E. (1966) Biochim. Biophys. Acta 115,219--221 Case, R.M. and Clausen, T. (1973) J. Physiol. L o n d o h 235, 75--102 Zimmerman, M.J., Dreillng, D.A., Rosenberg, R. and J a n o w l t z . H.D. (1967) Gastroenterology 52, 865--870 Goebell, H., Steffen, Ch: and Bode, Ch. (1972) Gut 13, 477--482 Rutten, W.J. (1974) Thesis, Nijmegen Douglas, W.W. (1974) Secretory Mechanisms of Exocrine Glands (Thorn, N.A. and Petersen, O.H., eds), pp. 1 1 6 - - 1 3 3 , Munksgaard, Copenhagen Rothman, S.S. (1964) Nature 204, 84--85 Lowry, O.H., Rosebrough, N~., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 Bernfeld, P. (1955) Methods in E n z y m o l o g y (Colowick, S.P. and Kaplan, N.O., eds), Vol. I, p. 149, A c a d e m i c Press, N e w Y o r k Wilson, I.B., Hatch, M.A. and Ginsburg, S. (1960) J. Biol. Chem. 235, 2312--2315 Wright, E.M. and Pietras, R~. (1974) J. Membrane Biol. 17,293--312 Heisler, S. (1974) Br. J. Pharmaeol. 52, 387--392 Case, R.M. and 8 c m t c h e r d , T. (1974) J. Physiol. L o n d o n 242, 415--428 Wallach, D. and 8 c h r a m m , M. (1971) Eur. J. Biochem. 21,433--437 Meldolesi, J. and Clemente, F. (1974) J. Cell Biol. 63, 222a Matthews, E.K., Petersen, O.H. and Williams, J.A. (1973) J. Physiol. L o n d o n 234, 689--701 Schreurs, V.V.A.M., de Pont, J.J.H.H.M. and Bonting, S.L. (1974) J. Cell Biol. 63, 304a Eimerl, S.. 8avion, N., Heichal, O. and Selinger, Z. (1974) J. Biol. Chem. 249, 3991--3993 Williams, J.A. and Lee, M. (1974) B i o e h e m . Biophys. Res. C o m m u n . 60, 542--548