413

J. Phyeiol. (1979), 290, p. 413419 With 1 text-figure Printed in Great Britain

EFFECT OF ACETYLCHOLINE STIMULATION ON CALCIUM AND SODIUM UPTAKE BY THE ISOLATED RAT PANCREAS

BY H. BOBINSKI AND JULIE A. KELLY From the Department of Physiology, University of Manchester, Manchester M13 9PT

(Received 25 July 1978) SUMMARY

1. The role of Cal+ and Na+ ions in 'stimulus-secretion' coupling in the isolated uncinate pancreas of 4-week-old rats has been examined using radioisotope tracer techniques. The amount of "6Ca2+ and 22Na+ (,umole/g wet wt.) taken up by unstimulated glands was compared to that of glands in which amylase release was stimulated by ACh(105 M) at various incubation times. 2. The amount of "6Ca taken up by the glands within 60 min of incubation was not found to be increased by the presence of ACh(10-5M) In fact, during short incubation periods the 4"Ca uptake was significantly less in the stimulated glands than the unstimulated glands. 3. Presence of ACh(10-5 M) did cause a significant initial increase in 22Na uptake lasting up to 20 min from onset of stimulation. 4. These results indicate that the rise in intracellular Ca2+ concentration which is suggested by an increase in 45Ca efflux during the action of pancreatic secretagogues, is not a consequence of increased 4"Ca uptake by the pancreas; but they indicate that an initial action of ACh could be to elicit an increase in Na+ influx and that Na+ ions are likely to be involved in the action of ACh on pancreatic acinar cells. INTRODUCTION

It is generally accepted that Ca2+ ions play an essential role in 'stimulus-secretion' coupling (Douglas, 1968; Rubin, 1970) and that in the pancreas, pancreatic secretagogues bring about a rise in intracellular Ca2+ concentration (Kanno, Cochrane & Douglas, 1973; Case & Clausen, 1973; Iwatsuki & Petersen, 1977). There is conflicting evidence, however, concerning the mechanism by which the secretagogues cause this rise, resulting in the emergence of two main hypotheses to explain how it is initiated. One proposes that it is caused by an increase in the uptake of Ca2+ from the extracellular medium (Kanno, 1972; Kondo & Schultz, 1976). A second suggests that the primary action of the secretagogues is to cause an increase in Na+ influx which elicits a release of Ca2+ from intracellular stores (Case & Clausen, 1973). But Nishiyama & Petersen (1975) have suggested that since the most important effect of acetylcholine (ACh) on the acinar cell membrane is to increase Na+ conductance, the ACh/membrane-receptor interaction evokes release of membranebound Ca2+, causing an increase in Na+ conductance, and that the membrane-bound 0022-3751/79/3500-0633 $01.50 © 1979 The Physiological Society

H. BOBINSKI AND JULIE A. KELLY Ca2+ is in some form of equilibrium with that in the medium. In the light of this conflicting evidence the aim of the present study was to measure '5Ca2+ and Na22+ uptake in the isolated uncinate rat pancreas under ACh stimulation to clarify the role of Ca2+ and Na+ ions in 'stimulus-secretion' coupling in the exocrine pancreas. 414

METHODS Animal. Male and female rats of the Sprague-Dawley strain, about 4 weeks old and weighing 60-70 g, were used. They were maintained under standard conditions but were denied food the night before the experiment. Ti8sue preparation. The pancreata were removed according to the method described by Case & Clausen (1973). After removal, the tissue was washed and weighed, by difference, in KrebsRinger bicarbonate buffer, and was found to weigh between 70 and 100 mg. In all experiments the pancreata were transferred, after weighing, to vials containing 3 ml. Krebs-Ringer bicarbonate buffer at 37 0C and shaken slowly for 60 min in a waterbath at 37 'C. The buffer was gassed with 5 % C02 and 95% 02 immediately prior to transfer of the glands. This stage was introduced to ensure that steady-state conditions prevail before treatment of the glands with chemicals or hormones. The glands were transferred from one vial to another using a polyethylene hook to avoid tissue damage during manipulations. Incubation conditions. Prior to use Krebs-Ringer bicarbonate buffer containing 1 % albumin was equilibrated with a mixture of 95% 02 and 5 % C02 to maintain pH at 7-4. This solution is referred to as the 'incubation solution'. When Ca2+ was entirely removed from the incubation solution, iso-osmolarity was maintained by an appropriate change in the concentration of Na+, and 0-5 mM-ethyleneglycol-bis-(fl-aminoethylether)-N,N'-tetracetic acid (EGTA) was added. The 'load-solution' was made up of Krebs-Ringer bicarbonate buffer containing 1 % albumin and either "CaCI2 or 22NaCl (0-4 /esCi/ml.). All incubations were carried out in 20 ml. polyethylene vials (New England Nuclear) which were shaken in a water-bath maintained at 37 'C. The vials contained 3 ml. of incubation or load solution. In experiments where the effect of ACh on "Ca uptake was being investigated, 30 1l. acetylcholine chloride solution (10-3 M) was added to 3 ml. load solution immediately before transfer of the gland into the load solution, giving a final concentration of 10- M-acetylcholine. This concentration was presumed to be maintained throughout the whole of the incubation period (these pancreata seem to have little cholinesterase activity as judged by sustained amylase output during the washout period after exposure of the glands to ACh. Radioactive isotope8 and counting. 1 1Ci "CaCl2 and 1 1sCi 22NaCl was purchased from the Radiochemical Centre, Amersham. Counting was performed in a liquid scintillation spectrophotometer (Intertechnique Model SL30). To count 4Ca activity, 0 5 ml. test solution was added to 10 ml. scintillator in a polyethylene vial and counted in the liquid scintillation counter. Scintillator was composed of equal volumes of toluene and PCS solubilizer (Amersham/ Searle). To count 22Na activity, 0-5 ml. test solution was placed in a gamma counting vial (Koch-Light Laboratories Ltd.) and counted in the liquid scintillation counter. Uptake 8tudie8. The glands were incubated in the appropriate load solution for a predetermined time (see Results), after which they were removed from the load solution and washed twice in either ice-cold iso-osmolar Ca2+-free buffer containing EGTA, if investigating '"Ca uptake, or ice-cold choline solution (155 mM) if investigating 22Na uptake. The glands were then blotted on filter paper, weighed and boiled with 2 ml. 0-1 N-HNO, solution. After boiling, the glands were broken up in the hot solution using a vortex mixer and the resultant solution centrifuged for 5 min at 2500 rev/min using an MSE Mistral 6L centrifuge. 0-5 ml. aliquotes of the supernatant and 0-5 ml. aliquotes of load solution were withdrawn for determination of "Ca or 22Na activity. Amylase activity in the remaining load solution was determined using a method adapted from that of Bernfield (1955) and expressed in i.u./g. mink. Assuming that all the calcium exists as the free ion, the 4"Ca and 22Na activity of the tissue was expressed as a percentage of that in the medium and, from the specific activity of the "Ca or 22Na in the medium, this was converted to /czmole "Ca or 22Na per g wet wt. of tissue.

415

CATION UPTAKE IN STIMULATED PANCREAS

The statistical significance of the results was calculated using Student's t test on unpaired samples (Snedecor & Cochran, 1974). Materials. All chemicals were of analytical grade. Bovine serum albumin and acetylcholine chloride were both products of Sigma Chemicals.

Acetylcholine 50 r Acetylcholine -

Q(3

260 r

40 1 -T

cm Ca

0

30 1

.

0

20

F

220

(U 0

I

E

0

a-

180

co -

0

0.

10

z

F

140

- - -

0

I

a

.

$ I I

L-----:

100 I

-1 I

E I

10 20 30 0 40 Duration of incubation (min)

0 30 40 10 20 Duration of incubation (min)

Fig. 1. Effectof acetylcholine on uptake of 22Na. The glands were incubated at 37 'C in either Krebs buffer containing "2NaCl (0 4 ,sCi/ml.) (Ad - -) or Krebs buffer+ACh (10- M) containing 22NaCl (0 4 #uCi/ml.) (a-) for the time indicated on the graph. The 22Na uptake by the tissue is expressed in /smole Na+/g wet wt. of tissue. TABLE 1. Effect of ACh on 22Na uptake and amylase release by pancreas. The glands were incubated for the time shown in either Krebs buffer containing 22NaCl (04 ,uCi/ml.) or Krebs buffer containing 22NaCl (0-4 #sCi/ml.) and ACh 10-b M. The activity of the tissue was calculated as a percentage of that in the medium and converted into jsmole 22Na+/g. wet wt. on the basis of the specific activity of 22Na in the medium. Each result shown represents the mean ± S.E. of six observations Time of incubaIncubation tion medium (min.) 10 Krebs buffer + 2"Na 10 Krebs buffer + 2"Na + ACh 20 Krebs buffer + 2"Na 20 Krebs buffer + 2N a + ACh 40 Krebs buffer + 22Na 40 Krebs buffer + 22Na + ACh

22Na uptake ,umole/g wet wt. (mean ± s.E.) 22'3618 + 0-8982 25*1686 ± 0*9000

27*6745 ± 0-5483 30*0203 ± 0-4682 32*5455 ± 0-2331 33-3553 ± 0'2262

Amylase release Statistical significance P < 0*05

i.u/g. min-' Statistical (mean ± s.E.) significance P < 0*01 8.55 ± 0-36 10*92 + 0-44

P




0*05

2*03 ± 0*19 5 90 ± 0*47

P < 0.001

RESULTS

22Na uptake. The study of cellular influx of an ion such as Na+ is complicated by the presence of the extracellular space which makes it difficult to change rapidly and to know precisely the ionic concentration of the bathing media. A pilot experiment was performed to see whether any changes in 22Na uptake could be detected during stimulation of the isolated rat pancreas by ACh(10- M).

H. BOBINSKI AND JULIE A. KELLY Fig. 1 shows that in this experiment an initial rise in 22Na uptake occurred on incubation of the gland with ACh, and that this subsequently declined. On the basis of the transient increase in 22Na uptake demonstrated in this experiment further similar studies were performed. The data from these studies are summarized in Table 1. 416

TABLE 2. Effect of ACh on "Ca uptake by pancreas. The glands were incubated for the time show in either Krebs buffer containing "CaCl2 (0.4 #sCi/ml.) or Krebs buffer containing "CaCi2 (0 4isCi/ml.) and ACh (10-i M). The activity of the tissue was calculated as a percentage of that in the medium and then converted into moles "Ca2+/g wet wt. on the basis of the specific activity of "Ca in the medium. Each result shown represents the mean + S.E. of six to ten observations

45Ca uptake Time of incubation (min) 10 10 20 20 40 40 60 60

jumole/g. min

Statistical significance

Incubation medium

(mean + S.E.)

Krebs buffer+ "Ca Krebs buffer+'"Ca+ACh Krebsbuffer+4Ca Krebs buffer +45Ca + ACh Krebsbuffer+ "Ca Krebs buffer + "Ca + ACh

0*2021 + 0*0097 0-1632 + 0-0084

P < 0 05

0-3193±0-0160

P < 0-05

Krebsbuffer+45Ca Krebs buffer+ "Ca+ACh

0-2716 0-0012

0-4198±0-0186

P < 005

0-3639+ 00179 0*4740+0-0513 0*3989 + 0-0392

P > 0.05

ACh(105 M) caused a significant increase in 22Na uptake in the 10 and 20 min incubation periods, whereas by -40 min any difference between stimulated and unstimulated glands was no longer significant (Fig. 1; Table 1). In contrast, ACh caused significantly higher amylase release over all the periods of incubation (Fig. 1; Table 1). These experiments indicate that ACh induces a transient increase in Na+ influx, which may be involved in triggering a more sustained increase in pancreatic amylase secretion. PiCa uptake. The changes in '5Ca2+ uptake during stimulation of the glands with ACh(10- M) are shown in Table 2. Analagous to the results of Case & Clausen (1973), which showed that cholecystokinin-pancreozymin (0.5 u./ml.) tended to diminish the 45Ca uptake as measured after 60 min incubation, the present data show that ACh similarly did not increase influx of 45Ca2+ within a period of 60 min incubation. Indeed, for short periods of incubation (10, 20 and 40 min) there was a significant decrease in 45Ca2+ taken up by the glands (Table 2). Amylase output by the glands was increased by the presence of ACh in a manner similar to that shown in Fig. 1. Thus, after 60 min incubation the amylase released into the incubation vials by the stimulated glands, 3-23 + 0'46 i.u./g.min-'. (s.E. of mean), was significantly greater (P < 0.05) than that released by the unstimulated glands, 2-15 + 0-13 i.u./g.min-'.

CATION UPTAKE IN STIMULATED PANCREAS

417

DISCUSSION

The main findings of the present study are that in experimental conditions where ACh caused a prolonged (i.e. at least 60 min) increase in amylase release from the isolated pancreas, a more transient (less than 40 min) increase in Na+ influx occurred, together with a transient (more than 40 min) decrease in Ca2+ influx. An increase in cytosol Ca concentration during the action of ACh on pancreatic acinar cells is suggested by the finding that ACh elicits an increase in '6Ca.efflux from the pancreas with a concomitant rise in amylase output (Case & Clausen, 1973; Matthews, Petersen & Williams, 1973; Kelly, 1977). This is also suggested by the observation that application of Ca in pancreatic acinar cells results in membrane depolarization similar to that evoked by ACh (Iwatsuki & Petersen, 1977). How ACh, which is known to act on cell membranes from the extracellular side, increases the cytosol Ca concentration is a point of controversy. Using the isolated and perfused rat pancreas, Kanno & Nishimura (1976) reported that Mn2+ competed with Ca2+ in the secretary effect of pancreozymin and inferred that the influx of Ca into the acinar cells is the major contributor to the rise in intracellular Ca2+ concentration and that this influx of extracellular Ca2+ is mediated by a carrier in the plasma membrane. Kondo & Schultz (1976) have shown that 45Ca uptake in isolated pancreatic cells is stimulated by pancreatic secretagogues (pancreozymin and carbamylcholine). In contrast, Case & Clausen (1973) failed to observe any increase in Ca2+ uptake during stimulation of the isolated rat pancreas by ACh. This result is analogous to the findings of Chandler & Williams (1974) that in mouse pancreatic fragments the presence of bethanecol does not increase Ca2+ uptake into intracellular compartments, and to other observations of Williams & Chandler (1975) showing that bethanecol does not stimulate total 45Ca uptake. The present results show that the presence of ACh actually decreased 45Ca uptake by the isolated rat pancreas for an incubation period of up to 40 min and that within a 60 min incubation period there was no significant difference in 45Ca uptake between stimulated and unstimulated glands. These results, then, are not in agreement with the hypothesis that the ACh induced rise in intracellular Ca2+ concentration is caused by increased uptake in Ca2+ from the extracellular medium. Furthermore, from studies on the ionic dependence of membrane potential and resistance, Nishiyama & Petersen (1975) concluded that there was no electrophysiological evidence for an effect of ACh on membrane conductance to Ca2+. In contrast, Nishiyama & Petersen (1975) were able to show an ACh-induced change in membrane conductance to Na+. Using segments of mouse or rat pancreas, Petersen & Ueda (1976) showed that replacement of Na+ in superfusion fluid by TRIS abolished ACh-evoked amylase release. These findings suggest involvement of Na+ in the action of ACh on pancreatic acinar cells, but until this present investigation, the effect of pancreatic secretagogues on Na+ influx in the rat pancreas had not been examined directly. The present results show an increase in 22Nat uptake by the isolated rat pancreas in the presence of ACh (10- M), which suggests that an initial action of ACh is to elicit an increase in Na+ influx. This is in accord with the electrophysiological findings (Matthews & Petersen, 1973; Nishiyama & Petersen, 1975; Petersen & Ueda, 1976). 14

PHY 290

H. BOBINSKI AND JULIE A. KELLY Thus, the present results show an involvement of both Na+ and Ca2+ in the action of ACh on pancreatic acinar cells, but although they exclude the hypothesis suggesting that ACh causes an increase in Ca2+ from the extracellular medium, they do not disclose the relationship between Na+ influx and increased intracellular Ca2+ concentration. Petersen & Ueda (1976) have suggested that interaction between ACh and membrane receptor evokes release of membrane-bound Ca2+, causing an increase in Na+ conductance; and that the membrane-bound Ca2+ (in some sort of equilibrium with the extracellular Ca2+) determines the membrane resistance to ions (refer to fig. 14 in Petersen & Ueda, 1976). In conclusion, the results from this present investigation suggest that the increase in 'Ca2+ efflux, which has been observed (see references above) when amylase release from the isolated rat pancreas was stimulated by presence of ACh (104 and 10- M), does not arise from an increase in calcium influx; and that Na+ ions are involved in the action of ACh on the acinar cell membrane. Furthermore, the sequence of events is compatible with the model of Petersen & Ueda (1976): the increase in Na+ influx observed during the action of ACh(10-5 M) supports the proposal that a primary action of ACh on the acinar cell membrane is to increase Na+ conductance, and the absence of an increase in the uptake of Ca2W from the extracellular medium in the present investigation is compatible with the view that increased intracellular Ca2+ concentration arises from release of membrane-bound calcium.

418

We wish to thank the Medical Research Council for financial support to J. A. K., and Dr B. Argent for his kind assistance.

REFERENCES

BxRNF!nLD, P. (1955). In Methog in Enzymology, ed. COLOWIcK, S. P. & KAPLAN, N. O., vol. 1, pp. 149-158. New York: Academic Press. CAsE, R. M. & CLAusER, T. (1973). The relationship between calcium exchange and enzyme secretion in the isolated rat pancreas. J. Physiol. 235, 75-102. CHANDLER, D. E. & WILLIAMS, J. A. (1974). Pancreatic acinor cells: effects of Ln3+ ions on amylase release and calcium ion fluxes. J. Physiol. 243, 831-846. DouGLAs, W. W. (1968). Stimulus-secretion coupling: the concept and clues from chromaffin and other cells. Br. J. Pharmac. 34, 451-474. IwATsuKI, N. & PETmRsEN, 0. H. (1977). Acetylcholine-like effects of intracellular calcium application in pancreatic acinar cells. Nature, Lond. 268, 147-149. KANNo, T. (1972). Calcium-dependent amylase release and electrophysiological measurements in cells of the pancreas. J. Phy&iol. 226, 353-371. KAINwo, T., CocHRAEN, D. E. & DOUGLAS, W. W. (1973). Exocytosis induced by injection of calcium into mast cells. Can. J. Physiol. Pharmac. 51, 1001-1004. KANR o, T. & NIsmmURA, 0. (1976). Stimulus secretion coupling in pancreatic acinar cells: inhibitory effects of calcium removal and manganese addition on pancreozymin-induced amylase release. J. Physiol. 257, 309-324. KrnLLY, J. A. (1977). Some factors affecting enzyme secretion and ion movements in the exocrine pancreas. MSc. thesis, Manchester University. KoNDo, S. & SCHULTZ, I. (1976). Calcium ion uptake in isolated pancreas cells induced by secretagogues. Biochim. biophys. Acta 419, 76-92. MATTHEWS, E. K. & PrrxsiEN, 0. H. (1973). Pancreatic acinar cells: ionic dependence of the membrane potential and acetylcholine-induced depolarization. J. Physiol. 231, 283-295.

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MA~nmtws, E. K., PrrTEREN, 0. H. & WILLIAMS, J. A. (1973). Pancreatic acinar cells: acetylcholine-induced membrane depolarization, calcium efflux and amylase release. J. Phy8siol. 234, 689-701. NISHIYAMA, A. & PEThRSEN, 0. H. (1975). Pancreatic acinar cells: ionic dependence of acetylcholine-induced membrane depolarization and resistance change. J. Phy8iol. 244, 431-465. PETERSEN, 0. H. & UEDA, N. (1976). Pancreatic acinar cells: the role of calcium in stimulussecretion coupling. J. Physiol. 254, 583-606. Prmrmsx, 0. H. & UEDA, N. (1977). Secretion of fluid and amylase in the perfused rat pancreas. J. Physiol. 264, 819-837. RuBIN, R. P. (1970). The role of calcium in the release of neurotransmitter substances and hormones. Pharmac. Rev. 22, 389-428. SKEDECOR, G. W. & CocNAw, W. G. (1974). In Statidtical Metho&, sixth edition. Ames, Iowa, U.S.A.: Iowa State University Press. Wm.IAms, J. A. & CHANDLER, D. E. (1975). The effects of calcium on release of amylase by mouse pancreas, in vitro. Am. J. Phy8iol. 228, 1729-1732.

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