AMERICAN JOURNAL OF PHYSIOLOGY Vol. 228, No. 6, June 1975. Printed
in
U.S.A.
Ca++ and pancreatic JOHN A. WILLIAMS Department of Physiology,
amylase
AND DOUGLAS CHANDLER University of California, San Fran&u,
WILLIAMS,JOHN A., AND DOUGLAS CHANDLER. CaS- andpancreatic amylase release. Am. J. Physiol. 228(6): 1729-l 732. 1975.-The effects of Ca++ on release of amylase by mouse pancreas in vitro was studied. The cholinergic agonist bethanechol increased release of amylase in a parallel manner in both normal and 0 Ca++ medium; however, release at all concentrations of bethanechol was depressed about 50 y0 in 0 Ca++ medium. When pancreatic fragments were superfused with 0 Ca++ medium, bethanechol still produced a normal stimulation of amylase release after half of the total tissue Ca++ was washed out. This Ca++ loss included both extracellular and some intracellular Ca? Uptake and equilibration of 45Ca++ into pancreatic fragments was multiphasic, with total equilibration with stable Ca++ still not reached after 3 h. Addition of bethanechol had no effect on the rate of 45Ca uptake and equilibration. 45Cafs- efflux was not influenced by superfusion in 0 Ca++ medium, while the stimulation of 45Ca++ efflux by bethanechol was enhanced. It is concluded that extracellular is not of major importance in triggering Ca++ and/or Ca++ influx pancreatic enzyme release, but that extracellular Ca++ may regulate the release process thus accounting for the parallel changes in unstimulated and stimulated amylase release. stimulus-secretion exocrine pancreas
coupling;
secretory
release
mechanisms;
calcium;
PANCREATIC EXOCRINE CELLS package digestive enzymes into secretory granules which may be extruded from the cell by exocytosis (7, 10, 13). Ca++ is known to be important in the control of secretion based primarily on the finding that removal of extracellular Ca++ inhibits release of pancreatic enzymes in response to cholinergic or cholecystokinin-pancreozymin stimulation (1, 3, 8, 9, 1.1, 15). This result has generally been interpreted as indicating that control of enzyme release is by a mechanism similar to the stimulus-secretion coupling model of Douglas (6). In this scheme the stimulant molecule combines with a membrane receptor leading to membrane depolarization and an influx of Ca ++ which, in an as yet unknown manner, As an alternative to an influx of promotes exocytosis. Ca+f, evidence has recently been presented that pancreatic stimulants may release intracellular stored Ca++, which could then promote exocytosis (5, 12). We have therefore reexamined the importance of extracellular Ca f+ for pancreatic amylase release to determine if entry of Ca++ into the pancreatic cell is necessary to trigger secretion.
Califurnin
9PHJ
Animals were stunned, decapitated, and the pancreas quickly removed and trimmed. The pancreas was cut into four pieces weighing 15-30 mg, and in larger pancreases the thick central portion was discarded. Each pancreatic fragment was weighed on a torsion balance and incubated in Krebs-Henseleit bicarbonate (KHB) solution which had the following composition : NaCl 118 mM, KC1 4.7 mM, CaC12 2.56 mM, MgCl2 1.13 mM, Na HCO3 25 mM, NaHgP04 1.15 mM, glucose 56 mM. All solutions were equilibrated with 95 % 02, 5 % COZ. During variation of the ionic content of the medium, Na+ was correspondingly varied to maintain isotonicity. Incubation was carried out at 37’C either in 25-ml Erlenmeyer flasks containing 3 ml of KHB or in a superfusion chamber through which KHB was passed at two chamber volumes per minute (12). Amylase released into the medium was assayed according to the method of Rinderknecht et al. (14) using amylose azure blue as the substrate. Amylase is reported in International Units, based on the reported activity of the a-amylase used as a standard (Sigma type VI). For determination of pancreatic total Ca++, and content of 45Ca++, sucroseJ4C, or inulin-3H, the pancreas was rinsed 10 s in ice-cold Ca++-free medium, blotted, and dried overnight at 80°C Ca++ was then extracted for 48 h in 0.1 N HNO3, and aliquots were either diluted for atomicabsorption spectrometry with La+++ and HCl added to prevent calcium complex formation or added to a scintilation counting mixture of Triton X-100 and toluene (1: 2) containing 4 g/liter of butyl PBD. Medium samples were also diluted with 0.1 N HNO3 and analyzed similarly. Quench correction of radioactivity was by the sample channels ratio technique. Prelabeling and efflux of 4sCa++ was as described previously (12). The fractiona efflux of 45Ca+f was calculated as the amount of 45Ca++ released during each time period divided by the amount of 45Ca+f present in the tissue at that time. Carbamyl-p-methylcholine (bethanechol) was obtained from Koch-Light Ltd., amylose azure blue from Calbiochem, 45CaCl 2 (10.8 mCi/mg) and inulin-“H from New England Nuclear Corp.; and sucroseJ4C from ICN. RESULTS
Figure 1 shows the dose-response curve for bethanechol stimulation of pancreatic amylase release in KHB and 0 Ca++ KHB. The 0 Ca++ KHB curve was shifted downward by about 50 %, but was strikingly parallel to the normal curve. Maximal response was obtained with 3 lO+ M bethanechol. In other experiments (not shown), both unstimulated and 3 low5 M bethanechol-stimulated amylase release were reduced in parallel as the concentration of l
METHODS
All studies were carried out using male white Swiss mice weighing 18-24 g, which were fasted 14-20 h prior to use.
l
1729
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 16, 2019.
1730
J. A. WILLIAMS
Ca++ in the medium was reduced from normal (2.56 mM) to zero. Table 1 shows that Sr++ could partially substitute for Ca++ in maintaining basal and stimulated amylase release, while Ba+f was only slightly effective in maintaining basal release. Ba +f also had no effect when added in the presence of Ca ++. The relative stimulation produced by bethanechol was generally independent of the presence or nature of external divalent cations, except for a small inhibition produced by Ba ++ (Table 1). Removal of Mg.++ from the medium or a lo-fold increase in its concentration were without any effect on amylase release. As Aask incubation experiments do not allow time resolution or maintenance of low-medium concentrations of Ca++ > the release of amylase and washout of tissue Ca++ was studied in a flow chamber. Superinfusion of pancreatic fragments by 0 Casf KHB (measured Ca+f = 3-5 lOA M) led to a fall in tissue Ca++ content (Fig. 2). This fall was of similar shape to that seen during 45Ca+f efflux (lZ), except that a larger percent of Ca++ remained in the tissue at longer times (90-l 20 min), indicating that slowly exchanging pools not completely labeled with J5Ca++ but included in total Ca ++ also showed a slow washout. By 90 min about 1.5 mmol/kg or half the original tissue Ca+f had been lost. Figure 3 shows examples of amylase release from pancreatic fragments superfused with KHB and 0 Ca++ KHB. Th e increase in amylase release stimulated l
y" 2 UE
AND
D. CHANDLER
3.0
1.0 ~~.~~I 0
40
80
120
TIME (Min ) FIG. 2. Effect of superfusion of mouse pancreas fragments. six values.
32
with 0 Caf4 KHB All points are mean
on Caf+ content & SE of four to
r A.
100 TIME
(Min.)
60
I
I
90
120 TIME
I 150
(Min.1
FIG. 3. Effect of bethanechol on amylase release by pancreatic fragments superfused with KHB (-----) or 0 Ca++ KHB (- - -). A and B represent two different experiments, in each of which fragments from same pancreas were superfused in parallel. Bethanechol was present at 3. 10-S M during time indicated by bar.
047
0
I 3.10~"
3.10"
I 3*1o-a
BETHANECHOL
I 3.16'
(Ml
FIG. 1. Effect of bethanechol on pancreatic amylase release by fragments of mouse pancreas incubated in vitro in KHB or 0 Ca++ KHB. Tissue was preincubated 30 min in either KHB, q I--0, or 0 Ca++ KHB, m--m, and then incubated 30 min in similar medium containing specified concentration of bethanechol. All values are mean k SE of four to six pancreases.
TABLE 1. Efects of ca++, ST++, and Ba++ on pancreatic amylase release by -fragments of mouse pancreas in vitro
Medium
~KHB
0 Ca++
Sr++ KHB Ba++ KHB
0 Ca++
LJ0 Ca++
AmyIase Unstimulated
Release,
mU/mg
per 30 min
Bethanechol,
3.103 M
0.31 0.75
zt 0.01 0.06
0.89 1.78
dz zt 0.04 0.08
0.50 0.46
zk 0.06 rt 0.06
1.43 0.77
It 0.12 xk 0.08
All values are the mean + SE of five to eight values. Pancreatic fragments were preincubated 60 min in the specified medium and then incubated 30 min in the same medium with or without bethanechol, Ca++, Sr’+, and Baff when present were at concentration of 2.56 mM.
by bethanechol was similar in all cases, and there was a general similarity in the time course between the response in KHB and 0 Ca -t-t KHB. Table 2 shows collected data for basal and bethanechol-stimulated amylase release after 30, 60, or 90 min superfusion in KHB or 0 Ca+f KHB. While there is a general increase with time in the unstimulated amylase release in KHB, the percent increase induced by bethanechol is not dependent on time or on the presence of Ca++. Since a dependence of secretion on extracellular Gaff is usually interpreted as implying an increased influx of Ca++ 9 the uptake of 45Ca++ into pancreatic fragments was studied. As shown in Fig. 4, uptake was multiphasic, with most rapid entry occurring up to 10 min, while equilibration was not yet complete at 3 h. Addition of bethanechol had no effect on 45Ca++ uptake, although total Ca++ content was slightlv decreased. Figure 5 shows uptake curves for the extracellular tracers sucrose-14C and inulin-3H. Both markers distributed in an equilibrium space of 0.32 which was not influenced by bethanechol. Figure 6 shows the effect of bethanechol on 45Ca++ efflux and the lack of any major effect of superfusion in 0 Ca+f KHB. I n six experiments, the average fractional efflux at 90 min was 0.0050 =t: 0.0009 in KHB versus OcO058 rfi 0.0009 in 0 Ca+f KHB (NS P > 0.05), while the
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 16, 2019.
AND
CA++
PANCREATIC
AMYLASE
1731
RELEASE
amj)lase release by mouse ;bancreas fragments Stimulation Time
Amylase
at
~- -- .^I
Medium
Release,
mU/mg
2
per 5 min
Bethanechol stimulated, 3.10-S M
Unstimulated
.--
30 min
KHB 0 Ca++ KHB 0 Ca++ KHB 0 Ca++
60 min 90 min
24 dz 15 + 41 h 18 =t 60 + 23 +
KHB KHB KHB
77 *
2 2 6 2 8 3
44 105 51 138 52
It & It =t +
iV 5 2 m 0.10
9 5 19 9 15 9
0.06
i-
All values are the mean + SE of five to six pancreases. Unstimulated arnylase release was measured as the mean of two 5-min samples immediately prior to stimulation and the stimulated value as the mean of six 5-min samples following stimulation. In each case two halves of the same pancreas were run simultaneously in KHB and 0 Gaff KHB as in Fig. 4.
2.50
0.20
r
0*~4L~I--~:~-0
L-.~--90
60 TIME (Min)
120
FIG. 5. Uptake of sucrose14C and inulin-W by mouse pancreatic fragments in vitro. Pancreases were preincubated 30 min in KHB and then incubated Z-120 min in KHB containing inulin-%I e---a; sucroseJ4C A---& and sucroseJ4C plus 3 lOA” M bethanechol, A--A. Radioactive isotopes were present at 0.1 &i/ml. A11 values are mean + SE of four to eight values. l
0.04
0.25
30
r
l 0
t
I I 0.10
I 0
30
60
90 TIME (Min 1
I 120
I 150
1 180
Uptake of 45Ca+f by, and total Caf+ content of, mouse pancreatic fragments in vitro. Pancreases were preincubated 30 min in KHB and then incubated from 2 to 180 min in KHB containing 0.4 PCi :‘inl 45Ca++ and 3g 10B5 M bethanechol as specified. Ca++ space (pl/mg) is defined as Ca++ content of tissue per milligram divided by Cat-+ content of medium per microliter (2.56 pmol/pl). H----m, total Ca++ in KHB; O--O, total Ca++ in KHB plus 30 10-S M bethanechol; e---e, 45Ca4f in KHB; o----o, 45Ca++ in KHB plus 3. 10-j M bethanechol. All values are mean + SE of four to eight pancreases. FIG.
4.
maximum increase induced by bethanechol, 266 & 44 70 in KHB versus 460 & 59% in 0 Ca+f KHB (P < 0.05) was significantly greater in 0 Ca+f KHB. DISCUSSION
The present experiments were designed to look in detail at eflects of alkaline earth divalent cations on pancreatic amylase release to futher understand the role of Ca++ in this process. While a number of studies have demonstrated an ef&ct of Ca++ removal on pancreatic enzyme secretion (1, 3, 5, 8, 9, 15), it has not previously been determined whether Ca++ depletion was occurring extra- or intracellularly. This is a key question since a dependence on extracellular Cat-+ is usually taken as indicating Ca++ influx as an integral part of stimulus-secreting coupling.
I
OL 80
I
90
100 TIME
110
(Min )
FXG. 6. Effect of bethanechol on fractional effiux of Wa from pancreatic fragments superfused with KHB or 0 Gaff KHB. point is fractional efflux for Z-min collection period plotted time. Fragments from a single pancreas were incubated 60 KHB containing 6 pCi/ml 4VZas-+, divided in two equal halves superfused with KHB, n , or 0 Ca $-+ KHB, q , as in amylase studies. 30 10-S M bethanechol was added after 90 min of fusion as indicated by bar.
mouse Each against min in and release super-
While the present work cannot eliminate the possibility that Ca+f entering the cell from the outside triggers secretion, two findings are certainly inconsistent with any major importance of such a mechanism. First, removal of Ca+f from the medium lowers both the unstimulated and stimulated release of amylase without aflecting the percent stimulation. These results are similar to those previously reported by Robberecht and Christophe (15). Tn our experiments this is most dramatic in the flow cell experiments, where the fall in tissue Ca++ indicates that extracellular Ca+f has been removed. Extracellular space in the mouse pancreas in vitro is 0.32 as measured from the sucrose and inulin space (Fig. 5). Thus the Ca++ content of extracellular space is 0.32 X 2.56 mM/kg or 0.83 mNI/kg, and this much Ca++ is lost by 20 min superfusion in 0 Ca++ KHB. Bethanechol still induces a normal secretory response even
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 16, 2019.
1732 after 90 min in 0 Ca -t-t KHB (Fig. 3). Second, stimulation of secretion fails to increase the rate of uptake of 45Ca+f into the pancreas (Fig. 4). These results demonstrating independence of amylase secretion from extracellular Ca++ does not eliminate a role for membrane-bound Ca++. Addition of EDTA to calcium free medium abolished amylase release from pigeon pancreas slices (9), although it was not established how much Ca++ was removed. In any case these results are clearly different from studies on the adrenal medulla, neurohypophyses and islets of Langerhans (6, 16) where simple removal of Ca+f from the medium is able to block secretion. Ca+f, however, can trigger pancreatic amylase release when intracellularly introduced by the Ca++ ionophore A23 187 (17, 18). If a rise in intracellular Ca++ activity is casually related to secretion and Ca++ does not enter from the external medium, it is likely that cellular Ca+f is in some way redistributed to increase the small fraction of cellular Ca++ existing as the free ion (2,4). The bethanecholstimulated efflux of 45Ca +f from slowly exchanging pools has been suggested as resulting from release of intracellular Ca+f (5 12). This phenomenon is unaffected or slightly increasei by removal of extracellular Ca+f. The significance of the fall in total tissue Ca++ on stimulation (Fig. 4) and how it relates to the increase in 45Ca++ efflux has not yet been established. Alteration of extracellular Ca++, however, does alter amylase release, both basal and stimulated release being affected in parallel. This could well indicate a dependence of the release process (exocytosis) on extracellular or superficially bound Ca++ as opposed to the triggering mechanism. Thus, Ca++ might function in two ways in the stimulation
J.
A.
WILLIAMS
AND
D.
CHANDLER
of amylase release. An increase in Ca++ within the pancreatic cell might trigger release of granules, while Ca++ might also participate at the outside of the membrane in the release process itself. Alternatively, the dependence of amylase release on extracellular Caf-f may indicate a continuous rise in Ca++ at subcellular sites on in vitro incubation* This increased Ca+f would then increase basal amylase release and, if available for intracellular release, on stimulation would maintain the percent stimulation similar to that seen in the absence of Ca++ loading. The lack of importance of Ca++ entry in stimulation of pancreatic amylase release could explain the difference between the effects of divalent cations on amylase release and other systems, such as release of acetylcholine at the neuromuscular junction, catecholamines from the adrenal medulla, and insulin from the islet of Langerhans. In these systems Mg++ competitively antagonizes the action of Ca++, while Ba+f can function as a stimulant (6, 16). Furthermore, removal of Ca++ from the medium completely blocks the ability of stimulators to trigger release. Secretory cells may thus vary in the source of Ca++ used for stimulus-secretion coupling, just as different types of muscle use different sources for excitation-contraction coupling. In the exocrine pancreas, in contrast to the adrenal medulla, redistribution of intracellular Ca+f would appear to be of much more importance than entry of extracellular Ca++. The technical assistance This work was supported from the National Institute Received
for
publication
of Mark Lee is gratefully acknowledged. by a research grant (L ROl GM 19998-01) of General Medical Sciences. 15 July
1974.
REFERENCES 1. ARGENT, B. E., R. M. CASE, AND T. SCRATCHERD. Amylase secretion by the perfused cat pancreas in relation to the secretion of calcium and other electrolytes and as influenced by the external ionic environment. J. Physiol., London 230: 575-593, 1973. P. F. Transport and metabolism of calcium ions in 2. BAKER, nerve. Progr. Biophys. Molecular Biol. 24 : 179-223, 1972. 3. BENZ,L., B. ECKSTEIN, E. K. MATTHEWS, AND J. A. WILLIAMS, Control of pancreatic amylase release in vitro: effects of ions, cyclic AMP and colchicine. Brit. J. Pharmacol. 46: 66-77, 1972. 4* BORLE, A. B. Calcium metabolism at the cellular level. Federation Proc. 32 : 1944-1950, 1973. 5. CASE, R. M., AND T. CLAUSEN. The relation between calcium exchange and enzyme secretion in the isolated rat pancreas. J. Physiol., London 235 : 75-102, 1973. 6. DOUGLAS, W. W. Stimulus-secretion coupling: the concept and clues from chromaffin and other cells. &it. J. Pharmacol. 34: 451-474, X968. 7. EKHOLM, R., T. ZELANDER, AND Y. EDLUND. The ultrastructural organization of the rat pancreas. I. ,4cinar cell. J. Ultrastruct. Res. 7: 61-72, 1962. 8. HEISLER, S., D. FAST, AND A. TENENHOUSE. Role of Ca2+ and cyclic AMP in protein secretion from rat pancreas. B&him. Biophys. Acta 279: 561-572, 1972. 9. HORIN, L. E. Effects of calcium omission on acetylcholine-stimulated amylase secretion and phospholipid synthesis in pigeon pancreas slices. Biochim. Biophys. Acta 115 : 219-221, 1966. 10. ICHIKAWA, A. Fine structural changes in response to hormonal stimulation of the perfused canine pancreas. J. Cell 17ioZ. 24: 369-385, 1965.
11.
12.
13.
14,
15.
16.
17.
18.
KANNO, T. Calcium-dependent physiological measurements London 226: 353-371, 1972.
in
cells
amylase of the
release pancreas,
and J.
electrophysiol.,
MATTHEWS, E. K., 0. H. PETERSEN, AND J. A. WILLIAMS. Pancreatic acinar cells : acetylcholine-induced membrane depolarization, calcium efflux and amylase release. J. Physiol., London 234: 689-701, 1973. PALADE, G. E. Functional changes in the structure of cell components. In: Subcellular Particles, edited by T. Hayashi. New York, Ronald, 1959, p. 64-80. RINDERKNECHT, J., P. WILDING, AND B. J. HAVERBACK. A new method for the determination of cY-amylase. Exparkntia 23 : 805, 1967. ROBBERECHT, P. AND J- CHRISTOPHE, Secretion of hydrolases by perfused fragments of rat pancreas: effect of calcium. Am. J. Physiol. 220: 911-917, 1971. RUBIN, R. P, The role of calcium in the release of neurotransmitter substances and hormones. Pharmacol. Rec. 22 : 389-418, 1970. SELINGER, Z., S. EIMERL, N. SAVION, AND M. SCHRAMM. A Ca++ A23 187 simulating hormone and neurotransmitter ionophore action in the rat parotid and pancreas glands. In: Secretory Mechanisms of Exocrine Glands, edited by N. A. Thorn, and 0. H. Petersen. Copenhagen: Munksgaard, 1974, p. 68-87. WILLIAMS, J. A., AND M. LEE. Pancreatic acinar cells: use of a Ca++ ionophore to separate enzyme release from the earlier steps in stimulus-secretion coupling. Biochem. Biophys. Res. Commun. 60:
542-548,
1974.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 16, 2019.
in
U.S.A.
Ca++ and pancreatic JOHN A. WILLIAMS Department of Physiology,
amylase
AND DOUGLAS CHANDLER University of California, San Fran&u,
WILLIAMS,JOHN A., AND DOUGLAS CHANDLER. CaS- andpancreatic amylase release. Am. J. Physiol. 228(6): 1729-l 732. 1975.-The effects of Ca++ on release of amylase by mouse pancreas in vitro was studied. The cholinergic agonist bethanechol increased release of amylase in a parallel manner in both normal and 0 Ca++ medium; however, release at all concentrations of bethanechol was depressed about 50 y0 in 0 Ca++ medium. When pancreatic fragments were superfused with 0 Ca++ medium, bethanechol still produced a normal stimulation of amylase release after half of the total tissue Ca++ was washed out. This Ca++ loss included both extracellular and some intracellular Ca? Uptake and equilibration of 45Ca++ into pancreatic fragments was multiphasic, with total equilibration with stable Ca++ still not reached after 3 h. Addition of bethanechol had no effect on the rate of 45Ca uptake and equilibration. 45Cafs- efflux was not influenced by superfusion in 0 Ca++ medium, while the stimulation of 45Ca++ efflux by bethanechol was enhanced. It is concluded that extracellular is not of major importance in triggering Ca++ and/or Ca++ influx pancreatic enzyme release, but that extracellular Ca++ may regulate the release process thus accounting for the parallel changes in unstimulated and stimulated amylase release. stimulus-secretion exocrine pancreas
coupling;
secretory
release
mechanisms;
calcium;
PANCREATIC EXOCRINE CELLS package digestive enzymes into secretory granules which may be extruded from the cell by exocytosis (7, 10, 13). Ca++ is known to be important in the control of secretion based primarily on the finding that removal of extracellular Ca++ inhibits release of pancreatic enzymes in response to cholinergic or cholecystokinin-pancreozymin stimulation (1, 3, 8, 9, 1.1, 15). This result has generally been interpreted as indicating that control of enzyme release is by a mechanism similar to the stimulus-secretion coupling model of Douglas (6). In this scheme the stimulant molecule combines with a membrane receptor leading to membrane depolarization and an influx of Ca ++ which, in an as yet unknown manner, As an alternative to an influx of promotes exocytosis. Ca+f, evidence has recently been presented that pancreatic stimulants may release intracellular stored Ca++, which could then promote exocytosis (5, 12). We have therefore reexamined the importance of extracellular Ca f+ for pancreatic amylase release to determine if entry of Ca++ into the pancreatic cell is necessary to trigger secretion.
Califurnin
9PHJ
Animals were stunned, decapitated, and the pancreas quickly removed and trimmed. The pancreas was cut into four pieces weighing 15-30 mg, and in larger pancreases the thick central portion was discarded. Each pancreatic fragment was weighed on a torsion balance and incubated in Krebs-Henseleit bicarbonate (KHB) solution which had the following composition : NaCl 118 mM, KC1 4.7 mM, CaC12 2.56 mM, MgCl2 1.13 mM, Na HCO3 25 mM, NaHgP04 1.15 mM, glucose 56 mM. All solutions were equilibrated with 95 % 02, 5 % COZ. During variation of the ionic content of the medium, Na+ was correspondingly varied to maintain isotonicity. Incubation was carried out at 37’C either in 25-ml Erlenmeyer flasks containing 3 ml of KHB or in a superfusion chamber through which KHB was passed at two chamber volumes per minute (12). Amylase released into the medium was assayed according to the method of Rinderknecht et al. (14) using amylose azure blue as the substrate. Amylase is reported in International Units, based on the reported activity of the a-amylase used as a standard (Sigma type VI). For determination of pancreatic total Ca++, and content of 45Ca++, sucroseJ4C, or inulin-3H, the pancreas was rinsed 10 s in ice-cold Ca++-free medium, blotted, and dried overnight at 80°C Ca++ was then extracted for 48 h in 0.1 N HNO3, and aliquots were either diluted for atomicabsorption spectrometry with La+++ and HCl added to prevent calcium complex formation or added to a scintilation counting mixture of Triton X-100 and toluene (1: 2) containing 4 g/liter of butyl PBD. Medium samples were also diluted with 0.1 N HNO3 and analyzed similarly. Quench correction of radioactivity was by the sample channels ratio technique. Prelabeling and efflux of 4sCa++ was as described previously (12). The fractiona efflux of 45Ca+f was calculated as the amount of 45Ca++ released during each time period divided by the amount of 45Ca+f present in the tissue at that time. Carbamyl-p-methylcholine (bethanechol) was obtained from Koch-Light Ltd., amylose azure blue from Calbiochem, 45CaCl 2 (10.8 mCi/mg) and inulin-“H from New England Nuclear Corp.; and sucroseJ4C from ICN. RESULTS
Figure 1 shows the dose-response curve for bethanechol stimulation of pancreatic amylase release in KHB and 0 Ca++ KHB. The 0 Ca++ KHB curve was shifted downward by about 50 %, but was strikingly parallel to the normal curve. Maximal response was obtained with 3 lO+ M bethanechol. In other experiments (not shown), both unstimulated and 3 low5 M bethanechol-stimulated amylase release were reduced in parallel as the concentration of l
METHODS
All studies were carried out using male white Swiss mice weighing 18-24 g, which were fasted 14-20 h prior to use.
l
1729
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 16, 2019.
1730
J. A. WILLIAMS
Ca++ in the medium was reduced from normal (2.56 mM) to zero. Table 1 shows that Sr++ could partially substitute for Ca++ in maintaining basal and stimulated amylase release, while Ba+f was only slightly effective in maintaining basal release. Ba +f also had no effect when added in the presence of Ca ++. The relative stimulation produced by bethanechol was generally independent of the presence or nature of external divalent cations, except for a small inhibition produced by Ba ++ (Table 1). Removal of Mg.++ from the medium or a lo-fold increase in its concentration were without any effect on amylase release. As Aask incubation experiments do not allow time resolution or maintenance of low-medium concentrations of Ca++ > the release of amylase and washout of tissue Ca++ was studied in a flow chamber. Superinfusion of pancreatic fragments by 0 Casf KHB (measured Ca+f = 3-5 lOA M) led to a fall in tissue Ca++ content (Fig. 2). This fall was of similar shape to that seen during 45Ca+f efflux (lZ), except that a larger percent of Ca++ remained in the tissue at longer times (90-l 20 min), indicating that slowly exchanging pools not completely labeled with J5Ca++ but included in total Ca ++ also showed a slow washout. By 90 min about 1.5 mmol/kg or half the original tissue Ca+f had been lost. Figure 3 shows examples of amylase release from pancreatic fragments superfused with KHB and 0 Ca++ KHB. Th e increase in amylase release stimulated l
y" 2 UE
AND
D. CHANDLER
3.0
1.0 ~~.~~I 0
40
80
120
TIME (Min ) FIG. 2. Effect of superfusion of mouse pancreas fragments. six values.
32
with 0 Caf4 KHB All points are mean
on Caf+ content & SE of four to
r A.
100 TIME
(Min.)
60
I
I
90
120 TIME
I 150
(Min.1
FIG. 3. Effect of bethanechol on amylase release by pancreatic fragments superfused with KHB (-----) or 0 Ca++ KHB (- - -). A and B represent two different experiments, in each of which fragments from same pancreas were superfused in parallel. Bethanechol was present at 3. 10-S M during time indicated by bar.
047
0
I 3.10~"
3.10"
I 3*1o-a
BETHANECHOL
I 3.16'
(Ml
FIG. 1. Effect of bethanechol on pancreatic amylase release by fragments of mouse pancreas incubated in vitro in KHB or 0 Ca++ KHB. Tissue was preincubated 30 min in either KHB, q I--0, or 0 Ca++ KHB, m--m, and then incubated 30 min in similar medium containing specified concentration of bethanechol. All values are mean k SE of four to six pancreases.
TABLE 1. Efects of ca++, ST++, and Ba++ on pancreatic amylase release by -fragments of mouse pancreas in vitro
Medium
~KHB
0 Ca++
Sr++ KHB Ba++ KHB
0 Ca++
LJ0 Ca++
AmyIase Unstimulated
Release,
mU/mg
per 30 min
Bethanechol,
3.103 M
0.31 0.75
zt 0.01 0.06
0.89 1.78
dz zt 0.04 0.08
0.50 0.46
zk 0.06 rt 0.06
1.43 0.77
It 0.12 xk 0.08
All values are the mean + SE of five to eight values. Pancreatic fragments were preincubated 60 min in the specified medium and then incubated 30 min in the same medium with or without bethanechol, Ca++, Sr’+, and Baff when present were at concentration of 2.56 mM.
by bethanechol was similar in all cases, and there was a general similarity in the time course between the response in KHB and 0 Ca -t-t KHB. Table 2 shows collected data for basal and bethanechol-stimulated amylase release after 30, 60, or 90 min superfusion in KHB or 0 Ca+f KHB. While there is a general increase with time in the unstimulated amylase release in KHB, the percent increase induced by bethanechol is not dependent on time or on the presence of Ca++. Since a dependence of secretion on extracellular Gaff is usually interpreted as implying an increased influx of Ca++ 9 the uptake of 45Ca++ into pancreatic fragments was studied. As shown in Fig. 4, uptake was multiphasic, with most rapid entry occurring up to 10 min, while equilibration was not yet complete at 3 h. Addition of bethanechol had no effect on 45Ca++ uptake, although total Ca++ content was slightlv decreased. Figure 5 shows uptake curves for the extracellular tracers sucrose-14C and inulin-3H. Both markers distributed in an equilibrium space of 0.32 which was not influenced by bethanechol. Figure 6 shows the effect of bethanechol on 45Ca++ efflux and the lack of any major effect of superfusion in 0 Ca+f KHB. I n six experiments, the average fractional efflux at 90 min was 0.0050 =t: 0.0009 in KHB versus OcO058 rfi 0.0009 in 0 Ca+f KHB (NS P > 0.05), while the
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AND
CA++
PANCREATIC
AMYLASE
1731
RELEASE
amj)lase release by mouse ;bancreas fragments Stimulation Time
Amylase
at
~- -- .^I
Medium
Release,
mU/mg
2
per 5 min
Bethanechol stimulated, 3.10-S M
Unstimulated
.--
30 min
KHB 0 Ca++ KHB 0 Ca++ KHB 0 Ca++
60 min 90 min
24 dz 15 + 41 h 18 =t 60 + 23 +
KHB KHB KHB
77 *
2 2 6 2 8 3
44 105 51 138 52
It & It =t +
iV 5 2 m 0.10
9 5 19 9 15 9
0.06
i-
All values are the mean + SE of five to six pancreases. Unstimulated arnylase release was measured as the mean of two 5-min samples immediately prior to stimulation and the stimulated value as the mean of six 5-min samples following stimulation. In each case two halves of the same pancreas were run simultaneously in KHB and 0 Gaff KHB as in Fig. 4.
2.50
0.20
r
0*~4L~I--~:~-0
L-.~--90
60 TIME (Min)
120
FIG. 5. Uptake of sucrose14C and inulin-W by mouse pancreatic fragments in vitro. Pancreases were preincubated 30 min in KHB and then incubated Z-120 min in KHB containing inulin-%I e---a; sucroseJ4C A---& and sucroseJ4C plus 3 lOA” M bethanechol, A--A. Radioactive isotopes were present at 0.1 &i/ml. A11 values are mean + SE of four to eight values. l
0.04
0.25
30
r
l 0
t
I I 0.10
I 0
30
60
90 TIME (Min 1
I 120
I 150
1 180
Uptake of 45Ca+f by, and total Caf+ content of, mouse pancreatic fragments in vitro. Pancreases were preincubated 30 min in KHB and then incubated from 2 to 180 min in KHB containing 0.4 PCi :‘inl 45Ca++ and 3g 10B5 M bethanechol as specified. Ca++ space (pl/mg) is defined as Ca++ content of tissue per milligram divided by Cat-+ content of medium per microliter (2.56 pmol/pl). H----m, total Ca++ in KHB; O--O, total Ca++ in KHB plus 30 10-S M bethanechol; e---e, 45Ca4f in KHB; o----o, 45Ca++ in KHB plus 3. 10-j M bethanechol. All values are mean + SE of four to eight pancreases. FIG.
4.
maximum increase induced by bethanechol, 266 & 44 70 in KHB versus 460 & 59% in 0 Ca+f KHB (P < 0.05) was significantly greater in 0 Ca+f KHB. DISCUSSION
The present experiments were designed to look in detail at eflects of alkaline earth divalent cations on pancreatic amylase release to futher understand the role of Ca++ in this process. While a number of studies have demonstrated an ef&ct of Ca++ removal on pancreatic enzyme secretion (1, 3, 5, 8, 9, 15), it has not previously been determined whether Ca++ depletion was occurring extra- or intracellularly. This is a key question since a dependence on extracellular Cat-+ is usually taken as indicating Ca++ influx as an integral part of stimulus-secreting coupling.
I
OL 80
I
90
100 TIME
110
(Min )
FXG. 6. Effect of bethanechol on fractional effiux of Wa from pancreatic fragments superfused with KHB or 0 Gaff KHB. point is fractional efflux for Z-min collection period plotted time. Fragments from a single pancreas were incubated 60 KHB containing 6 pCi/ml 4VZas-+, divided in two equal halves superfused with KHB, n , or 0 Ca $-+ KHB, q , as in amylase studies. 30 10-S M bethanechol was added after 90 min of fusion as indicated by bar.
mouse Each against min in and release super-
While the present work cannot eliminate the possibility that Ca+f entering the cell from the outside triggers secretion, two findings are certainly inconsistent with any major importance of such a mechanism. First, removal of Ca+f from the medium lowers both the unstimulated and stimulated release of amylase without aflecting the percent stimulation. These results are similar to those previously reported by Robberecht and Christophe (15). Tn our experiments this is most dramatic in the flow cell experiments, where the fall in tissue Ca++ indicates that extracellular Ca+f has been removed. Extracellular space in the mouse pancreas in vitro is 0.32 as measured from the sucrose and inulin space (Fig. 5). Thus the Ca++ content of extracellular space is 0.32 X 2.56 mM/kg or 0.83 mNI/kg, and this much Ca++ is lost by 20 min superfusion in 0 Ca++ KHB. Bethanechol still induces a normal secretory response even
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1732 after 90 min in 0 Ca -t-t KHB (Fig. 3). Second, stimulation of secretion fails to increase the rate of uptake of 45Ca+f into the pancreas (Fig. 4). These results demonstrating independence of amylase secretion from extracellular Ca++ does not eliminate a role for membrane-bound Ca++. Addition of EDTA to calcium free medium abolished amylase release from pigeon pancreas slices (9), although it was not established how much Ca++ was removed. In any case these results are clearly different from studies on the adrenal medulla, neurohypophyses and islets of Langerhans (6, 16) where simple removal of Ca+f from the medium is able to block secretion. Ca+f, however, can trigger pancreatic amylase release when intracellularly introduced by the Ca++ ionophore A23 187 (17, 18). If a rise in intracellular Ca++ activity is casually related to secretion and Ca++ does not enter from the external medium, it is likely that cellular Ca+f is in some way redistributed to increase the small fraction of cellular Ca++ existing as the free ion (2,4). The bethanecholstimulated efflux of 45Ca +f from slowly exchanging pools has been suggested as resulting from release of intracellular Ca+f (5 12). This phenomenon is unaffected or slightly increasei by removal of extracellular Ca+f. The significance of the fall in total tissue Ca++ on stimulation (Fig. 4) and how it relates to the increase in 45Ca++ efflux has not yet been established. Alteration of extracellular Ca++, however, does alter amylase release, both basal and stimulated release being affected in parallel. This could well indicate a dependence of the release process (exocytosis) on extracellular or superficially bound Ca++ as opposed to the triggering mechanism. Thus, Ca++ might function in two ways in the stimulation
J.
A.
WILLIAMS
AND
D.
CHANDLER
of amylase release. An increase in Ca++ within the pancreatic cell might trigger release of granules, while Ca++ might also participate at the outside of the membrane in the release process itself. Alternatively, the dependence of amylase release on extracellular Caf-f may indicate a continuous rise in Ca++ at subcellular sites on in vitro incubation* This increased Ca+f would then increase basal amylase release and, if available for intracellular release, on stimulation would maintain the percent stimulation similar to that seen in the absence of Ca++ loading. The lack of importance of Ca++ entry in stimulation of pancreatic amylase release could explain the difference between the effects of divalent cations on amylase release and other systems, such as release of acetylcholine at the neuromuscular junction, catecholamines from the adrenal medulla, and insulin from the islet of Langerhans. In these systems Mg++ competitively antagonizes the action of Ca++, while Ba+f can function as a stimulant (6, 16). Furthermore, removal of Ca++ from the medium completely blocks the ability of stimulators to trigger release. Secretory cells may thus vary in the source of Ca++ used for stimulus-secretion coupling, just as different types of muscle use different sources for excitation-contraction coupling. In the exocrine pancreas, in contrast to the adrenal medulla, redistribution of intracellular Ca+f would appear to be of much more importance than entry of extracellular Ca++. The technical assistance This work was supported from the National Institute Received
for
publication
of Mark Lee is gratefully acknowledged. by a research grant (L ROl GM 19998-01) of General Medical Sciences. 15 July
1974.
REFERENCES 1. ARGENT, B. E., R. M. CASE, AND T. SCRATCHERD. Amylase secretion by the perfused cat pancreas in relation to the secretion of calcium and other electrolytes and as influenced by the external ionic environment. J. Physiol., London 230: 575-593, 1973. P. F. Transport and metabolism of calcium ions in 2. BAKER, nerve. Progr. Biophys. Molecular Biol. 24 : 179-223, 1972. 3. BENZ,L., B. ECKSTEIN, E. K. MATTHEWS, AND J. A. WILLIAMS, Control of pancreatic amylase release in vitro: effects of ions, cyclic AMP and colchicine. Brit. J. Pharmacol. 46: 66-77, 1972. 4* BORLE, A. B. Calcium metabolism at the cellular level. Federation Proc. 32 : 1944-1950, 1973. 5. CASE, R. M., AND T. CLAUSEN. The relation between calcium exchange and enzyme secretion in the isolated rat pancreas. J. Physiol., London 235 : 75-102, 1973. 6. DOUGLAS, W. W. Stimulus-secretion coupling: the concept and clues from chromaffin and other cells. &it. J. Pharmacol. 34: 451-474, X968. 7. EKHOLM, R., T. ZELANDER, AND Y. EDLUND. The ultrastructural organization of the rat pancreas. I. ,4cinar cell. J. Ultrastruct. Res. 7: 61-72, 1962. 8. HEISLER, S., D. FAST, AND A. TENENHOUSE. Role of Ca2+ and cyclic AMP in protein secretion from rat pancreas. B&him. Biophys. Acta 279: 561-572, 1972. 9. HORIN, L. E. Effects of calcium omission on acetylcholine-stimulated amylase secretion and phospholipid synthesis in pigeon pancreas slices. Biochim. Biophys. Acta 115 : 219-221, 1966. 10. ICHIKAWA, A. Fine structural changes in response to hormonal stimulation of the perfused canine pancreas. J. Cell 17ioZ. 24: 369-385, 1965.
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MATTHEWS, E. K., 0. H. PETERSEN, AND J. A. WILLIAMS. Pancreatic acinar cells : acetylcholine-induced membrane depolarization, calcium efflux and amylase release. J. Physiol., London 234: 689-701, 1973. PALADE, G. E. Functional changes in the structure of cell components. In: Subcellular Particles, edited by T. Hayashi. New York, Ronald, 1959, p. 64-80. RINDERKNECHT, J., P. WILDING, AND B. J. HAVERBACK. A new method for the determination of cY-amylase. Exparkntia 23 : 805, 1967. ROBBERECHT, P. AND J- CHRISTOPHE, Secretion of hydrolases by perfused fragments of rat pancreas: effect of calcium. Am. J. Physiol. 220: 911-917, 1971. RUBIN, R. P, The role of calcium in the release of neurotransmitter substances and hormones. Pharmacol. Rec. 22 : 389-418, 1970. SELINGER, Z., S. EIMERL, N. SAVION, AND M. SCHRAMM. A Ca++ A23 187 simulating hormone and neurotransmitter ionophore action in the rat parotid and pancreas glands. In: Secretory Mechanisms of Exocrine Glands, edited by N. A. Thorn, and 0. H. Petersen. Copenhagen: Munksgaard, 1974, p. 68-87. WILLIAMS, J. A., AND M. LEE. Pancreatic acinar cells: use of a Ca++ ionophore to separate enzyme release from the earlier steps in stimulus-secretion coupling. Biochem. Biophys. Res. Commun. 60:
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