Cell Tiss. Res. 186, 287 295 (1978)
Cell and Tissue Research (~ by Springer-Verlag 1978
The Effect of the Ionophore A23187 on Amylase Release, Cellular Integrity and Ultrastructure of Mouse Pancreatic Acini* John A. Williams** Department of Physiology, University of California, San Francisco, California, USA
Summary. The effects of the Ca 2 + ionophore A 2317 on pancreatic amylase and lactate dehydrogenase ( L D H ) release, cellular electrolyte balance and ultrastructure were studied with the use of incubated pancreatic fragments. A 23187 (0.3 gM) in the presence of Ca 2+, increased amylase release but at higher concentrations (1-10 gM) also increased L D H release and increased uptake of 14C-sucrose with concomitant loss of tissue K + and gain in N a +. The ultrastructure of the majority of acini appeared normal and showed depletion of zymogen granules. Microtubules and microfilaments which have been implicated in the release process were normal or increased in numberl In the absence of Ca + the ionophore had no effect on secretion, cellular integrity or ultrastructure. It is concluded that A 23187 in the presence of C a 2 + increases amylase release by a mechanism comparable to the terminal steps in stimulussecretion coupling induced by physiological secretagogues. This provides further evidence that amylase release is mediated by a rise in cell Ca 2 + although the mechanisms of the ionophore- and physiological secretagogue-induced rise in Ca + are probably different. High concentrations o f i o n o p h o r e ( > 1 gM) also induce Ca 2 + dependent damage in a fraction of the cells. Key words: Pancreas - Calcium - Ionophore. The ionophore A23187 facilitates the movement of divalent cations across biological membranes including both plasma and intracellular membranes (Reed and Lardy, 1972; Babcock et al., 1976; Reed, 1976), It has been shown to affect a large number of cellular processes involving Ca 2 +, particularly those whose rate is thought to be increased by a rise in cytoplasmic Ca 2 +. As a result of this property, A 23187 has been used as a tool to study stimulus-secretion coupling in pancreatic Send offprint requests to: Dr. John A. Williams,Department of Physiology,Universityof California, San
Francisco, California 94143, USA * Supported by grants from the NIH (GM 19998) and the Cystic Fibrosis Foundation ** I am indebted to Drs. Douglas Chandler and John Heuser for discussion and advice and to M. Lee and E. Roach for technical assistance
0302-766X/78/0186/0287/$1.80
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a c i n a r cells. T h e i o n o p h o r e i n c r e a s e s t h e r a t e o f u p t a k e o f C a 2 + i n t o o r e f f i u x f r o m a c i n a r cells, a n d i n t h e p r e s e n c e o f e x t r a c e l l u l a r C a 2 + i n c r e a s e s r e l e a s e o f a m y l a s e ( W i l l i a m s a n d Lee, 1 9 7 4 ; S e l i n g e r et al., 1974; S c h r e u r s e t al., 1976; C h r i s t o p h e e t al., 1976). A l t h o u g h A 2 3 1 8 7 e n t e r s i n t o a c i n a r cells ( C h a n d l e r a n d W i l l i a m s , 1977), its s t i m u l a t o r y e f f e c t o n s e c r e t i o n a p p e a r s t o b e d u e t o t h e i o n o p h o r e a c t i n g a t t h e level o f t h e p l a s m a m e m b r a n e t o p r o m o t e C a z + i n f l u x , w i t h C a z + t h e n p r o m o t i n g s e c r e t i o n . I n i s o l a t e d p a n c r e a t i c a c i n a r cells t h e s e r e s u l t s a r e c o m p l i c a t e d b y C a 2 +m e d i a t e d cell d a m a g e as d e m o n s t r a t e d b o t h b y l a c t a t e d e h y r o g e n a s e ( L D H ) r e l e a s e a n d b y m o r p h o l o g i c a l e v i d e n c e o f cell d a m a g e ( C h a n d l e r a n d W i l l i a m s , 1977). Since the ultrastructural correlates of A23187-induced enzyme release have not been examined in intact pancreatic acini nor much consideration has been given to the role of damage in enzyme release, the present report concerns the effects of A 2 3 1 8 7 o n a m y l a s e a n d L D H r e l e a s e , w a t e r a n d e l e c t r o l y t e c o n t e n t , as well as the ultrastructure of mouse pancreatic fragments. While some evidence of damage was found, most acini were intact and showed degranulation comparable to that induced by cholinergic analogs or caerulein.
Materials and Methods Tissue Preparation. All studies were carried out on pancreas obtained from White Swiss mice weighing 18-22 gm which had been fasted overnight before use. The pancreas was removed and divided into four or five 20-25 mg fragments which were incubated in Krebs-Henseleit bicarbonate (KHB) medium equilibrated with 95 % 02-5 % CO2 (Williams and Chandler, 1975). Incubation was carried out in 25 ml Erlenmeyer flasks at 37 ~C. Normally a 30 min preincubation was carried out with KHB from which Ca 2 + had been omitted (Ca 2 +-free KHB) with or without A 23187 added. This was followed by an incubation for 30-90 min in fresh medium with C a 2+ present as specified. In experiments with pancreatic fragments, such preincubation with A 23187 followed by readdition of Ca 2 + gives better stimulation of amylase release than does addition of ionophore to complete medium (Williams and Lee, 1974), probably due to the low amount of soluble A 23187 and the slow rate of uptake of the ionophore in the presence of Ca 2 + (Chandler and Williams, 1977). After incubation, the fragments were removed from the medium and, when required, fixed for electron microscopy or homogenized in ice cold sodium phosphate buffer (10 mM, pH 7.0). Medium and homogenate were saved at 2 ~C for subsequent assay of amylase or LDH. lonophore Addition. A 23187 was dissolved in ethanol and added to the KHB at 37~C; the final ethanol concentration was 0.33 % in most experiments. Ethanol in the same concentration was present in all control flasks and had no effect on amylase or LDH release or cellular ultrastructure. The A 23187 used was a gift from Dr. Rober Hamill of Eli Lilly Co. Analytical Procedures. Amylase activity of medium or homogenate was assayed by the method of Rinderknecht et al. (1967) by use of the dye-coupled substrate, amylose azure. Amylase is expressed in Units based on the activity of the amylase (Sigma Type VI) used for the standard curve. LDH activity was measured spectrophotometrically by the method of Wacker et al. 1956). For determination of tissue water and electrolytes, incubation medium included 0.2 gCi/ml of 14Csucrose which served to estimate the extraceUular fluid compartment (Williams and Chandler, 1975). After incubation the tissue was blotted dry and placed in a preweighed polypropylene vial. Tissue water was determined gravimetrically and the electrolytes extracted with 0.1 M HNO3, as previously described (Williams, 1975). The 14C content of tissue extract and medium was determined by liquid scintillation counting under conditions of similar quenching as checked by an internal standard. The Na + and K + content were determined by use of a flame photometer (Instrumentation Laboratories) with Li + present as an internal standard.
Ionophore A23187 and Pancreatic Acinar Cells
289
Electron Microscopic Procedures. Fixation was carried out at 37~
in 1.5 % glutaraldehyde - 1% paraformaldehyde in 0.08 M sodium cacodylate, p H 7.4, with postfixation in 2 % OsO 4 as in previous studies (Williams and Lee, 1976; Williams, 1977). Thin sections were doubly stained with uranyl acetate and lead citrate and examined in a J E M 100B electron microscope at 60 KV. For estimation o f the a m o u n t of microtubules and o f luminal structure, electron micrographs were prepared at a magnification of 12,000 and enlarged to a magnification o f 30,000. For each experimental condition, at least eight prints were examined from each of four pancreases. In all cases, control as well as ionophore treated pancreatic fragments from the same m o u s e were processed and examined.
Results
Effects of A 23187 on Amylase and LDH Release. In the presence of Ca 2+, A 23187
at a concentration of 0.3 ~tM significantly increased amylase release; a further increase occurred when the concentration o f ionophore was raised to 10 ~tM, the highest concentration studied (Fig. l). In the absence o f Ca e+, A23187 at concentrations of 0.1-10 ~tM had no effect. In the presence o f Ca 2 +, L D H release was not affected by 0.3 ~tM A 23187 but was increased by 1-10 ~tM ionophore in a dose-dependent manner. Similar to the effect on amylase release, there was no effect o f A 23187 on L D H release in the absence o f Ca 2 +. In the pancreases studied, the total content o f amylase was 1.83 • 18 U / m g (n = 5) so that release increased from a basal level o f about 12 %/30 rain to a maximum o f about 35 %/30 min. Total
0.8 0
0.5 0.4
g
o3
,.~/.-+/ 0.2
,4/--,
,
~,
+.
+
0.1
oi ~%,., 7
03
I i O - 3'.0
i~.O
6 I
E
o
5 4
g
3 2 I 0
// i
o'.3
,'o
3'.o
AZ3187 (JAM)
,%o
Fig. 1. Effect ofA23187 on release of amylase and L D H by fragments o f m o u s e pancreas in vitro. Fragments were preincubated 30 rain in Ca z+ free K H B with the specified concentration ofA23187 followed by a 30 min incubation with a similar concentration o f A23187 but with Ca 2+ added as specified. 9 . . . . o , Ca 2+ (2.56 m M ) added; 9 . . . . . 9 Ca 2+ free (no added Ca 2 +). All points are the m e a n • S.E. of 5-10 pancreases
290
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c o n t e n t o f L D H was 5 4 . 7 • U / r a g with basal release o f a b o u t 1~o/30 min increasing to a b o u t 11 ~ / 3 0 m i n with 10 g M A 23187 plus Ca 2 +. As in o t h e r studies ( C h a n d l e r a n d Williams, 1977) the cholinergic a n a l o g b e t h a n e c h o l h a d no effect on L D H release.
Effects of A 23187 on Pancreatic Water and Electrolyte Content. The effects o f A 2 3 1 8 7 on p a n c r e a t i c water a n d electrolyte c o n t e n t were studied b o t h to gain insight into h o w the i o n o p h o r e m i g h t affect cellular m e t a b o l i s m a n d as a way o f assessing cellular d a m a g e . T h e latter is possible, since r e t e n t i o n o f t r a n s m e m b r a n e N a + a n d K + gradients requires b o t h a supply o f m e t a b o l i c energy a n d n o r m a l structure a n d function o f the p l a s m a m e m b r a n e . In the presence o f C a 2 +, high c o n c e n t r a t i o n s o f A 23187 (1 a n d 3 laM) caused an increase in t o t a l w a t e r c o n t e n t a n d extracellular space e s t i m a t e d by use o f 14C-sucrose (Table 1). T o t a l N a + a n d C a + c o n t e n t s were increased while t h a t o f K + was decreased. H o w e v e r , when the e x t r a c e l l u l a r ion c o n t e n t is s u b t r a c t e d o u t a n d the m e a n intracellular c o n c e n t r a t i o n calculated, there is no change in the N a + a n d K + c o n c e n t r a t i o n s ( N a +, 48 m M o l e / 1 , K +, 151, m M o l e / 1 cell water). I n o t h e r words, the change in t o t a l N a + a n d K + can be a c c o u n t e d for by a small fraction ( a b o u t 10 ~ ) o f cells which are l e a k y to 14C_sucros e a n d p r e s u m a b l y have lost their ion gradients. By contrast, the c a l c u l a t e d cellular c o n c e n t r a t i o n o f C a 2 + was increased by A 23187 f r o m 4.1 to 6.9 m M o l e s / 1 cell water. T h e lower c o n c e n t r a t i o n o f A 23187 (0.3 g M ) h a d no effect on w a t e r c o n t e n t , extracellular space o r electrolyte content. I n c u b a t i o n in C a 2 +-free K H B also increased the extracellular space a n d total N a +, decreased the total K + a n d C a +, b u t the a d d i t i o n o f the high c o n c e n t r a t i o n o f A 2 3 1 8 7 h a d no further effect. T h e fall in total C a 2 + reflected b o t h the absence o f extracellular calcium a n d a fall in c a l c u l a t e d cellular Ca 2+ to 2.8 m M o l e s / 1 cell water. These results are consistent with the higher c o n c e n t r a t i o n s o f A 23187 leading to a C a 2 + - d e p e n d e n t d a m a g e o f a similar extent as t h a t e s t i m a t e d f r o m L D H release. The lower c o n c e n t r a t i o n (0.3 g M ) , however, released a m y l a s e w i t h o u t affecting either L D H release or cellular w a t e r a n d electrolyte balance, including the calculated cellular C a 2 + content. Table h Effect of A23187 on water and electrolyte content of incubated pancreatic fragments Medium
Water content (~o)
Extracellular space (~o)
Na + (nMoles/ mg wet wt)
K+ (nMoles/ mg wet wt)
Ca 2+ (nMoles/ mg wet wt)
80.73-• 82.63• 82.83i0.65 80.51i0.67
24.15:1.8 32.35:0.6 36.75:1.7 26.25:1.9
47.85:2.7 56.35:0.8 62.6• 51.1il.3
86.8• 77.35:2.6 74.05:2.6 85.45:1.8
2.915:0.06 3.275:0.23 4.145:0.10 2.895:0,10
81.92i0.87 82.205:0.68
39.05:6.2 36.4t- 3.8
64.4• 62.2•
68.75:6.9 69.8•
h18• 1.00•
KHB
+Ethanol +A23187
3 laM 1 I-tM 0.3 laM Ca 2+ Free KHB +Ethanol +A23187 3 /aM
All values are the mean + S.E. of 4 to 5 pancreatic fragments from 5 mice. All fragments were preincubated 30 min in Ca 2+ free KHB with ionophore or ethanol added and then incubated 60 min with similar medium containing Ca 2+ unless noted and 0.2 ~tCi/ml 14C-sucrose which was used to estimate extracellular space
lonophore A23187 and Pancreatic Acinar Cells
291
Fig. 2. Mouse pancreas preincubated 30 min and then incubated 60 min in Ca 2 +free K H B containing 3 ~M A 23187. L lumen; Z zymogen granule, x 5400 Fig. 3. Mouse pancreas preincubated 30 min in Ca 2 + free K H B containing 3 ~tM A 23187 and incubated 60 min in similar medium but with Ca 2 + (2.56 m M ) added. • 5400
292
J.A. Williams
Fig. 4. Mouse pancreas incubated as in Figure 3 but with a 90 min incubation in medium containing both A 23187 and Ca 2+. L lumen; microtubules (arrows); microfilaments (broad arrows), x 35,200
Effects o f A 23187 on Pancreatic Acinar Cell Structure. E l e c t r o n m i c r o s c o p y was used to e v a l u a t e A 2 3 1 8 7 - i n d u c e d secretion m o r p h o l o g i c a l l y a n d to estimate c o n c o m i t a n t structural d a m a g e . W h i l e it is o u r i m p r e s s i o n t h a t A 2 3 1 8 7 at 3 or 10 laM increased the n u m b e r o f d i s r u p t e d cells, this was n o t easy to evaluate due to the presence o f small b u t v a r y i n g n u m b e r s o f d a m a g e d cells in c o n t r o l specimens. In a n y case, the m a j o r i t y o f cells in pancreases treated with A 23187 were structurally
lonophore A23187 and PancreaticAcinar Cells
293
intact and the observations reported pertain to these cells. After preincubation and incubation in A23187 (3 ~M) without Ca 2+ (Fig. 2) acini contained numerous zymogen granules and were ultrastructurally similar to fragments from the same pancreas incubated in either complete or Ca2+-free KHB. If Ca 2+ was added during the incubation period, however, the ultrastructural pattern was similar to that after cholinergic stimulation. After 30 and 60 min, lumens were filled with secretory material, and after 60 min there was a consistent depletion of zymogen granules (Fig. 3). After 90 min mature zymogen granules were totally absent from at least half the acini, although a few acinar cells still contained a modest number of granules. After stimulation with A23187 plus Ca + for 90 min (Fig. 4) the microtubular and microfilamentous components are especially prominent. Mitochondrial ultrastructure is normal. Qualitatively similar tendencies were seen with 0.3 pM A 23187 although effects were not as striking or as consistent from cell to cell. It could not be established that ionophore induced release by exocytosis as the number of exocytic images in this preparation is low and not grossly affected by any pancreatic secretagogue. While the morphological approach used is not suitable for quantitation, the majority of acinar cells seem intact ultrastructurally, and A23187 plus Ca 2+ induces an ultrastructural picture of secretion comparable to normal secretagogues.
Discussion
The purpose of this study was to evaluate the effects of the C a 2 + ionophore A 23187 on secretion of amylase as to physiological versus pathological mechanisms of enzyme release. Evidence for a pathological action of A 23187 which will lead to amylase release includes the following: A23187 at concentrations above 1 pM increased L D H release, tissue penetration of sucrose and loss of cellular K + and gain of Na +. All of these effects of ionophore were dependent on the presence of Ca z + in the medium. These results confirm our earlier studies on isolated acinar cells (Chandler and Williams, 1977) and eliminate the possiblity that the damage seen in isolated cells was in part due to the isolation procedure. In fact, the dose response curves for ionophore-induced amylase and L D H release are very similar in the two preparations. In one other study of pancreatic secretion, L D H release was increased by A 23187, although the effect was small relative to the effect on amylase and was not considered significant (Selinger et al., 1974). A 23187 has also been found to damage other types of cells including lymphocytes (Kaiser and Edelman, 1977) and granulocytes (Wright et al., 1977). That A 23187 induced release of pancreatic amylase is largely by physiologic mechanisms is confirmed by our ultrastructural studies showing degranulation and accumulation of luminal secretory material in otherwise intact acini. The presence of a large number of intact cells with a small fraction of cells grossly damaged by the ionophore rather than of many cells being partially compromised is consistent with the water and electrolyte data (Table 1) which indicate that cells remaining impermeable to sucrose had normal Na + and K + contents. Furthermore, the ultrastructural aspects of ionophore-induced secretion are similar to those seen when cholinergic agonists or caerulein are used as stimulants. Little is currently
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k n o w n o f the mechanism o f Ca 2 +-induced enzyme release by exocytosis or other means. Since recent studies have suggested the possible involvement o f microtubules (Williams and Lee, 1977) and microfilaments (Bauduin et al., 1975; Williams, 1977) the effects o f Ca 2 + on these cytoskeletal elements is o f interest. Since Ca 2 + is k n o w n to prevent polymerization o f microtubules from subunits (Weisenberg, 1972) and to depolymerize microtubules so formed (Gaskin et al., 1975), it has been suggested that Ca 2 + might control the assembly and disassembly o f microtubules in the living cell. However, our study showed a normal or increased n u m b e r o f microtubules in the apical region o f the acinar cells under conditions o f C a : + stimulated secretion. The subapical m e s h w o r k o f microfilaments was also quite prominent. In summary, the present study provides further evidence for the usefulness o f calcium i o n o p h o r e stimulated pancreatic secretion as assessed both by release o f amylase and by morphological evaluation. Since A 2 3 1 8 7 also increase Ca 2+ uptake into pancreatic fragments or cells (Williams and Lee, 1974; Christophe et al., 1976; K o n d o and Shultz, 1976), it has been generally assumed that ionophore allows a rise in cytoplasmic Ca z + activity which then activates some mechanism culminating in exocytosis o f z y m o g e n granule contents. This implies that the later steps in ionophore-induced secretion should be similar to those induced by physiological secretagogues whereas the earlier steps leading up to a rise in Ca 2 + m a y be, and p r o b a b l y are, different as indicated by the differing dependency on extracellular Ca 2+ (Chandler and Williams, 1977). The present study further substantiates that Ca 2 + is the key intracellular control in regulation o f acinar cell secretion. However, the results also emphasize that the ionophore must be used cautiously to avoid Ca z +-mediated cell damage. This can be done either by use o f appropriate low concentrations o f the ionophore or by evaluating secretion only in intact cells.
References
Babcock, D.F., First, N.L., Lardy, H.A. : Action ofionophore A 23187at the cellular level. Separation of effects at the plasma and mitochondrial membranes. J. biol. Chem. 251, 3881 3886 (1976) Bauduin, H., Stock, C., Vincent, D., Grenier, J.F.: Microfilamentous system and secretion of enzyme in the exocrine pancreas. Effect of cytochalasin B. J. Cell Biol. 66, 165-181 (1975) Chandler, D.E., Williams, J.A. : Intracellular uptake and e-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A 23187 in dissociated pancreatic acinar cells. J. Membrane Biol. 32, 201-230 (1977) Christophe, J,P., Frandsen, E.K., Conlon, T.P., Krishna, G., Gardner, J.D. :Action of cholecystokinin, cholinergic agents, and A 23187 on accumulation of guanosine 3' : 5'-monophosphate in dispersed guinea pig pancreatic acinar cells. J. biol. Chem. 251, 4640-4645 (1976) Gaskin, F., Cantor, C.R., Shelanski, M.L.: Biochemicalstudies on the in vitro assembly and disassembly of microtubules. Ann. N.Y. Acad. Sci. 253, 133 (1975) Kaiser, N., Edelman, I.S.: Calcium dependence of glucocorticoid-induced lymphocytolysis. Proc. nat. Acad. Sci. (Wash.) 74, 638-642 (1977) Reed, P.W.: Effects of the divalent cation ionophore A 23187 on potassium permeability of rat erythrocytes. J. biol. Chem. 251, 3489-3494 (1976) Reed, P.W., Lardy, H.A.: A23187: A divalent cation ionophore. J. biol. Chem. 247, 6970-6977 (1972) Rinderknecht, J., Wilding, P., Haverback, BJ.: A new method for the determination of e-amylase. Experientia (Basel) 23, 805 (1967)
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Schreurs, V.V.A.M., Swarts, H.G.P., dePont, J.J.H.H.M., Bonting, S.L. : Role of calcium in exocrine pancreatic secretion. II. Comparison of the effects of carbachol and the ionophore A23187 on enzyme secretion and calcium movements in rabbit pancreas. Biochem. biophys. Acta (Amst.) 419, 320-330 (1976) Selinger, Z., Eimerl, S., Savion, N., Schramm, M.: A Ca ++ ionophore (A23187) simulating hormone and neurotransmitter action in the rat parotid and pancreas glands. In: Secretory mechanisms of exocrine glands (N.A. Thorn and O.H. Petersen, eds.), pp. 68-78. Copenhagen: Munksgaard 1974 Wacker, W.E.C., Ulmer, D.D., Vallee, B.L.: Metaloenzymes and myocardial infarction. II. Malic and lactic dehydrogenase activities and zinc concentrations in serum. New Engl. J. Med. 255, 449-456 (1956) Weisenberg, R.C.: Microtubule formation in vitro in solutions containing low calcium concentrations. Science 166, 1104-1105 (1972) Williams, J.A. : Na § dependence of in vitro pancreatic amylase release. Amer. J. Physiol. 229, 1023 1026 (1975) Williams, J.A. : Effects of cytochalasin B on pancreatic acinar cell structure and secretion. Cell Tiss. Res. 179, 453-466 (1977) Williams, J.A., Chandler, D.: Ca § § and pancreatic amylase release. Amer. J. Physiol. 228, 172%1723 (1975) Williams, J.A., Lee, M. : 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) Williams, J.A., Lee, M.: Microtubules and pancreatic amylase release by mouse pancreas in vitro. J. Cell Biol. 71, 795-806 (1976) Wright, D.G., Bralove, D.A. Gallin, J.I.: The differential mobilization of human neutrophil granules. Amer. J. Path. 87, 273~84 (1977)
Accepted September 27, 1977
Cell and Tissue Research (~ by Springer-Verlag 1978
The Effect of the Ionophore A23187 on Amylase Release, Cellular Integrity and Ultrastructure of Mouse Pancreatic Acini* John A. Williams** Department of Physiology, University of California, San Francisco, California, USA
Summary. The effects of the Ca 2 + ionophore A 2317 on pancreatic amylase and lactate dehydrogenase ( L D H ) release, cellular electrolyte balance and ultrastructure were studied with the use of incubated pancreatic fragments. A 23187 (0.3 gM) in the presence of Ca 2+, increased amylase release but at higher concentrations (1-10 gM) also increased L D H release and increased uptake of 14C-sucrose with concomitant loss of tissue K + and gain in N a +. The ultrastructure of the majority of acini appeared normal and showed depletion of zymogen granules. Microtubules and microfilaments which have been implicated in the release process were normal or increased in numberl In the absence of Ca + the ionophore had no effect on secretion, cellular integrity or ultrastructure. It is concluded that A 23187 in the presence of C a 2 + increases amylase release by a mechanism comparable to the terminal steps in stimulussecretion coupling induced by physiological secretagogues. This provides further evidence that amylase release is mediated by a rise in cell Ca 2 + although the mechanisms of the ionophore- and physiological secretagogue-induced rise in Ca + are probably different. High concentrations o f i o n o p h o r e ( > 1 gM) also induce Ca 2 + dependent damage in a fraction of the cells. Key words: Pancreas - Calcium - Ionophore. The ionophore A23187 facilitates the movement of divalent cations across biological membranes including both plasma and intracellular membranes (Reed and Lardy, 1972; Babcock et al., 1976; Reed, 1976), It has been shown to affect a large number of cellular processes involving Ca 2 +, particularly those whose rate is thought to be increased by a rise in cytoplasmic Ca 2 +. As a result of this property, A 23187 has been used as a tool to study stimulus-secretion coupling in pancreatic Send offprint requests to: Dr. John A. Williams,Department of Physiology,Universityof California, San
Francisco, California 94143, USA * Supported by grants from the NIH (GM 19998) and the Cystic Fibrosis Foundation ** I am indebted to Drs. Douglas Chandler and John Heuser for discussion and advice and to M. Lee and E. Roach for technical assistance
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J.A. Williams
a c i n a r cells. T h e i o n o p h o r e i n c r e a s e s t h e r a t e o f u p t a k e o f C a 2 + i n t o o r e f f i u x f r o m a c i n a r cells, a n d i n t h e p r e s e n c e o f e x t r a c e l l u l a r C a 2 + i n c r e a s e s r e l e a s e o f a m y l a s e ( W i l l i a m s a n d Lee, 1 9 7 4 ; S e l i n g e r et al., 1974; S c h r e u r s e t al., 1976; C h r i s t o p h e e t al., 1976). A l t h o u g h A 2 3 1 8 7 e n t e r s i n t o a c i n a r cells ( C h a n d l e r a n d W i l l i a m s , 1977), its s t i m u l a t o r y e f f e c t o n s e c r e t i o n a p p e a r s t o b e d u e t o t h e i o n o p h o r e a c t i n g a t t h e level o f t h e p l a s m a m e m b r a n e t o p r o m o t e C a z + i n f l u x , w i t h C a z + t h e n p r o m o t i n g s e c r e t i o n . I n i s o l a t e d p a n c r e a t i c a c i n a r cells t h e s e r e s u l t s a r e c o m p l i c a t e d b y C a 2 +m e d i a t e d cell d a m a g e as d e m o n s t r a t e d b o t h b y l a c t a t e d e h y r o g e n a s e ( L D H ) r e l e a s e a n d b y m o r p h o l o g i c a l e v i d e n c e o f cell d a m a g e ( C h a n d l e r a n d W i l l i a m s , 1977). Since the ultrastructural correlates of A23187-induced enzyme release have not been examined in intact pancreatic acini nor much consideration has been given to the role of damage in enzyme release, the present report concerns the effects of A 2 3 1 8 7 o n a m y l a s e a n d L D H r e l e a s e , w a t e r a n d e l e c t r o l y t e c o n t e n t , as well as the ultrastructure of mouse pancreatic fragments. While some evidence of damage was found, most acini were intact and showed degranulation comparable to that induced by cholinergic analogs or caerulein.
Materials and Methods Tissue Preparation. All studies were carried out on pancreas obtained from White Swiss mice weighing 18-22 gm which had been fasted overnight before use. The pancreas was removed and divided into four or five 20-25 mg fragments which were incubated in Krebs-Henseleit bicarbonate (KHB) medium equilibrated with 95 % 02-5 % CO2 (Williams and Chandler, 1975). Incubation was carried out in 25 ml Erlenmeyer flasks at 37 ~C. Normally a 30 min preincubation was carried out with KHB from which Ca 2 + had been omitted (Ca 2 +-free KHB) with or without A 23187 added. This was followed by an incubation for 30-90 min in fresh medium with C a 2+ present as specified. In experiments with pancreatic fragments, such preincubation with A 23187 followed by readdition of Ca 2 + gives better stimulation of amylase release than does addition of ionophore to complete medium (Williams and Lee, 1974), probably due to the low amount of soluble A 23187 and the slow rate of uptake of the ionophore in the presence of Ca 2 + (Chandler and Williams, 1977). After incubation, the fragments were removed from the medium and, when required, fixed for electron microscopy or homogenized in ice cold sodium phosphate buffer (10 mM, pH 7.0). Medium and homogenate were saved at 2 ~C for subsequent assay of amylase or LDH. lonophore Addition. A 23187 was dissolved in ethanol and added to the KHB at 37~C; the final ethanol concentration was 0.33 % in most experiments. Ethanol in the same concentration was present in all control flasks and had no effect on amylase or LDH release or cellular ultrastructure. The A 23187 used was a gift from Dr. Rober Hamill of Eli Lilly Co. Analytical Procedures. Amylase activity of medium or homogenate was assayed by the method of Rinderknecht et al. (1967) by use of the dye-coupled substrate, amylose azure. Amylase is expressed in Units based on the activity of the amylase (Sigma Type VI) used for the standard curve. LDH activity was measured spectrophotometrically by the method of Wacker et al. 1956). For determination of tissue water and electrolytes, incubation medium included 0.2 gCi/ml of 14Csucrose which served to estimate the extraceUular fluid compartment (Williams and Chandler, 1975). After incubation the tissue was blotted dry and placed in a preweighed polypropylene vial. Tissue water was determined gravimetrically and the electrolytes extracted with 0.1 M HNO3, as previously described (Williams, 1975). The 14C content of tissue extract and medium was determined by liquid scintillation counting under conditions of similar quenching as checked by an internal standard. The Na + and K + content were determined by use of a flame photometer (Instrumentation Laboratories) with Li + present as an internal standard.
Ionophore A23187 and Pancreatic Acinar Cells
289
Electron Microscopic Procedures. Fixation was carried out at 37~
in 1.5 % glutaraldehyde - 1% paraformaldehyde in 0.08 M sodium cacodylate, p H 7.4, with postfixation in 2 % OsO 4 as in previous studies (Williams and Lee, 1976; Williams, 1977). Thin sections were doubly stained with uranyl acetate and lead citrate and examined in a J E M 100B electron microscope at 60 KV. For estimation o f the a m o u n t of microtubules and o f luminal structure, electron micrographs were prepared at a magnification of 12,000 and enlarged to a magnification o f 30,000. For each experimental condition, at least eight prints were examined from each of four pancreases. In all cases, control as well as ionophore treated pancreatic fragments from the same m o u s e were processed and examined.
Results
Effects of A 23187 on Amylase and LDH Release. In the presence of Ca 2+, A 23187
at a concentration of 0.3 ~tM significantly increased amylase release; a further increase occurred when the concentration o f ionophore was raised to 10 ~tM, the highest concentration studied (Fig. l). In the absence o f Ca e+, A23187 at concentrations of 0.1-10 ~tM had no effect. In the presence o f Ca 2 +, L D H release was not affected by 0.3 ~tM A 23187 but was increased by 1-10 ~tM ionophore in a dose-dependent manner. Similar to the effect on amylase release, there was no effect o f A 23187 on L D H release in the absence o f Ca 2 +. In the pancreases studied, the total content o f amylase was 1.83 • 18 U / m g (n = 5) so that release increased from a basal level o f about 12 %/30 rain to a maximum o f about 35 %/30 min. Total
0.8 0
0.5 0.4
g
o3
,.~/.-+/ 0.2
,4/--,
,
~,
+.
+
0.1
oi ~%,., 7
03
I i O - 3'.0
i~.O
6 I
E
o
5 4
g
3 2 I 0
// i
o'.3
,'o
3'.o
AZ3187 (JAM)
,%o
Fig. 1. Effect ofA23187 on release of amylase and L D H by fragments o f m o u s e pancreas in vitro. Fragments were preincubated 30 rain in Ca z+ free K H B with the specified concentration ofA23187 followed by a 30 min incubation with a similar concentration o f A23187 but with Ca 2+ added as specified. 9 . . . . o , Ca 2+ (2.56 m M ) added; 9 . . . . . 9 Ca 2+ free (no added Ca 2 +). All points are the m e a n • S.E. of 5-10 pancreases
290
J.A~ Williams
c o n t e n t o f L D H was 5 4 . 7 • U / r a g with basal release o f a b o u t 1~o/30 min increasing to a b o u t 11 ~ / 3 0 m i n with 10 g M A 23187 plus Ca 2 +. As in o t h e r studies ( C h a n d l e r a n d Williams, 1977) the cholinergic a n a l o g b e t h a n e c h o l h a d no effect on L D H release.
Effects of A 23187 on Pancreatic Water and Electrolyte Content. The effects o f A 2 3 1 8 7 on p a n c r e a t i c water a n d electrolyte c o n t e n t were studied b o t h to gain insight into h o w the i o n o p h o r e m i g h t affect cellular m e t a b o l i s m a n d as a way o f assessing cellular d a m a g e . T h e latter is possible, since r e t e n t i o n o f t r a n s m e m b r a n e N a + a n d K + gradients requires b o t h a supply o f m e t a b o l i c energy a n d n o r m a l structure a n d function o f the p l a s m a m e m b r a n e . In the presence o f C a 2 +, high c o n c e n t r a t i o n s o f A 23187 (1 a n d 3 laM) caused an increase in t o t a l w a t e r c o n t e n t a n d extracellular space e s t i m a t e d by use o f 14C-sucrose (Table 1). T o t a l N a + a n d C a + c o n t e n t s were increased while t h a t o f K + was decreased. H o w e v e r , when the e x t r a c e l l u l a r ion c o n t e n t is s u b t r a c t e d o u t a n d the m e a n intracellular c o n c e n t r a t i o n calculated, there is no change in the N a + a n d K + c o n c e n t r a t i o n s ( N a +, 48 m M o l e / 1 , K +, 151, m M o l e / 1 cell water). I n o t h e r words, the change in t o t a l N a + a n d K + can be a c c o u n t e d for by a small fraction ( a b o u t 10 ~ ) o f cells which are l e a k y to 14C_sucros e a n d p r e s u m a b l y have lost their ion gradients. By contrast, the c a l c u l a t e d cellular c o n c e n t r a t i o n o f C a 2 + was increased by A 23187 f r o m 4.1 to 6.9 m M o l e s / 1 cell water. T h e lower c o n c e n t r a t i o n o f A 23187 (0.3 g M ) h a d no effect on w a t e r c o n t e n t , extracellular space o r electrolyte content. I n c u b a t i o n in C a 2 +-free K H B also increased the extracellular space a n d total N a +, decreased the total K + a n d C a +, b u t the a d d i t i o n o f the high c o n c e n t r a t i o n o f A 2 3 1 8 7 h a d no further effect. T h e fall in total C a 2 + reflected b o t h the absence o f extracellular calcium a n d a fall in c a l c u l a t e d cellular Ca 2+ to 2.8 m M o l e s / 1 cell water. These results are consistent with the higher c o n c e n t r a t i o n s o f A 23187 leading to a C a 2 + - d e p e n d e n t d a m a g e o f a similar extent as t h a t e s t i m a t e d f r o m L D H release. The lower c o n c e n t r a t i o n (0.3 g M ) , however, released a m y l a s e w i t h o u t affecting either L D H release or cellular w a t e r a n d electrolyte balance, including the calculated cellular C a 2 + content. Table h Effect of A23187 on water and electrolyte content of incubated pancreatic fragments Medium
Water content (~o)
Extracellular space (~o)
Na + (nMoles/ mg wet wt)
K+ (nMoles/ mg wet wt)
Ca 2+ (nMoles/ mg wet wt)
80.73-• 82.63• 82.83i0.65 80.51i0.67
24.15:1.8 32.35:0.6 36.75:1.7 26.25:1.9
47.85:2.7 56.35:0.8 62.6• 51.1il.3
86.8• 77.35:2.6 74.05:2.6 85.45:1.8
2.915:0.06 3.275:0.23 4.145:0.10 2.895:0,10
81.92i0.87 82.205:0.68
39.05:6.2 36.4t- 3.8
64.4• 62.2•
68.75:6.9 69.8•
h18• 1.00•
KHB
+Ethanol +A23187
3 laM 1 I-tM 0.3 laM Ca 2+ Free KHB +Ethanol +A23187 3 /aM
All values are the mean + S.E. of 4 to 5 pancreatic fragments from 5 mice. All fragments were preincubated 30 min in Ca 2+ free KHB with ionophore or ethanol added and then incubated 60 min with similar medium containing Ca 2+ unless noted and 0.2 ~tCi/ml 14C-sucrose which was used to estimate extracellular space
lonophore A23187 and Pancreatic Acinar Cells
291
Fig. 2. Mouse pancreas preincubated 30 min and then incubated 60 min in Ca 2 +free K H B containing 3 ~M A 23187. L lumen; Z zymogen granule, x 5400 Fig. 3. Mouse pancreas preincubated 30 min in Ca 2 + free K H B containing 3 ~tM A 23187 and incubated 60 min in similar medium but with Ca 2 + (2.56 m M ) added. • 5400
292
J.A. Williams
Fig. 4. Mouse pancreas incubated as in Figure 3 but with a 90 min incubation in medium containing both A 23187 and Ca 2+. L lumen; microtubules (arrows); microfilaments (broad arrows), x 35,200
Effects o f A 23187 on Pancreatic Acinar Cell Structure. E l e c t r o n m i c r o s c o p y was used to e v a l u a t e A 2 3 1 8 7 - i n d u c e d secretion m o r p h o l o g i c a l l y a n d to estimate c o n c o m i t a n t structural d a m a g e . W h i l e it is o u r i m p r e s s i o n t h a t A 2 3 1 8 7 at 3 or 10 laM increased the n u m b e r o f d i s r u p t e d cells, this was n o t easy to evaluate due to the presence o f small b u t v a r y i n g n u m b e r s o f d a m a g e d cells in c o n t r o l specimens. In a n y case, the m a j o r i t y o f cells in pancreases treated with A 23187 were structurally
lonophore A23187 and PancreaticAcinar Cells
293
intact and the observations reported pertain to these cells. After preincubation and incubation in A23187 (3 ~M) without Ca 2+ (Fig. 2) acini contained numerous zymogen granules and were ultrastructurally similar to fragments from the same pancreas incubated in either complete or Ca2+-free KHB. If Ca 2+ was added during the incubation period, however, the ultrastructural pattern was similar to that after cholinergic stimulation. After 30 and 60 min, lumens were filled with secretory material, and after 60 min there was a consistent depletion of zymogen granules (Fig. 3). After 90 min mature zymogen granules were totally absent from at least half the acini, although a few acinar cells still contained a modest number of granules. After stimulation with A23187 plus Ca + for 90 min (Fig. 4) the microtubular and microfilamentous components are especially prominent. Mitochondrial ultrastructure is normal. Qualitatively similar tendencies were seen with 0.3 pM A 23187 although effects were not as striking or as consistent from cell to cell. It could not be established that ionophore induced release by exocytosis as the number of exocytic images in this preparation is low and not grossly affected by any pancreatic secretagogue. While the morphological approach used is not suitable for quantitation, the majority of acinar cells seem intact ultrastructurally, and A23187 plus Ca 2+ induces an ultrastructural picture of secretion comparable to normal secretagogues.
Discussion
The purpose of this study was to evaluate the effects of the C a 2 + ionophore A 23187 on secretion of amylase as to physiological versus pathological mechanisms of enzyme release. Evidence for a pathological action of A 23187 which will lead to amylase release includes the following: A23187 at concentrations above 1 pM increased L D H release, tissue penetration of sucrose and loss of cellular K + and gain of Na +. All of these effects of ionophore were dependent on the presence of Ca z + in the medium. These results confirm our earlier studies on isolated acinar cells (Chandler and Williams, 1977) and eliminate the possiblity that the damage seen in isolated cells was in part due to the isolation procedure. In fact, the dose response curves for ionophore-induced amylase and L D H release are very similar in the two preparations. In one other study of pancreatic secretion, L D H release was increased by A 23187, although the effect was small relative to the effect on amylase and was not considered significant (Selinger et al., 1974). A 23187 has also been found to damage other types of cells including lymphocytes (Kaiser and Edelman, 1977) and granulocytes (Wright et al., 1977). That A 23187 induced release of pancreatic amylase is largely by physiologic mechanisms is confirmed by our ultrastructural studies showing degranulation and accumulation of luminal secretory material in otherwise intact acini. The presence of a large number of intact cells with a small fraction of cells grossly damaged by the ionophore rather than of many cells being partially compromised is consistent with the water and electrolyte data (Table 1) which indicate that cells remaining impermeable to sucrose had normal Na + and K + contents. Furthermore, the ultrastructural aspects of ionophore-induced secretion are similar to those seen when cholinergic agonists or caerulein are used as stimulants. Little is currently
294
J.A. Williams
k n o w n o f the mechanism o f Ca 2 +-induced enzyme release by exocytosis or other means. Since recent studies have suggested the possible involvement o f microtubules (Williams and Lee, 1977) and microfilaments (Bauduin et al., 1975; Williams, 1977) the effects o f Ca 2 + on these cytoskeletal elements is o f interest. Since Ca 2 + is k n o w n to prevent polymerization o f microtubules from subunits (Weisenberg, 1972) and to depolymerize microtubules so formed (Gaskin et al., 1975), it has been suggested that Ca 2 + might control the assembly and disassembly o f microtubules in the living cell. However, our study showed a normal or increased n u m b e r o f microtubules in the apical region o f the acinar cells under conditions o f C a : + stimulated secretion. The subapical m e s h w o r k o f microfilaments was also quite prominent. In summary, the present study provides further evidence for the usefulness o f calcium i o n o p h o r e stimulated pancreatic secretion as assessed both by release o f amylase and by morphological evaluation. Since A 2 3 1 8 7 also increase Ca 2+ uptake into pancreatic fragments or cells (Williams and Lee, 1974; Christophe et al., 1976; K o n d o and Shultz, 1976), it has been generally assumed that ionophore allows a rise in cytoplasmic Ca z + activity which then activates some mechanism culminating in exocytosis o f z y m o g e n granule contents. This implies that the later steps in ionophore-induced secretion should be similar to those induced by physiological secretagogues whereas the earlier steps leading up to a rise in Ca 2 + m a y be, and p r o b a b l y are, different as indicated by the differing dependency on extracellular Ca 2+ (Chandler and Williams, 1977). The present study further substantiates that Ca 2 + is the key intracellular control in regulation o f acinar cell secretion. However, the results also emphasize that the ionophore must be used cautiously to avoid Ca z +-mediated cell damage. This can be done either by use o f appropriate low concentrations o f the ionophore or by evaluating secretion only in intact cells.
References
Babcock, D.F., First, N.L., Lardy, H.A. : Action ofionophore A 23187at the cellular level. Separation of effects at the plasma and mitochondrial membranes. J. biol. Chem. 251, 3881 3886 (1976) Bauduin, H., Stock, C., Vincent, D., Grenier, J.F.: Microfilamentous system and secretion of enzyme in the exocrine pancreas. Effect of cytochalasin B. J. Cell Biol. 66, 165-181 (1975) Chandler, D.E., Williams, J.A. : Intracellular uptake and e-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A 23187 in dissociated pancreatic acinar cells. J. Membrane Biol. 32, 201-230 (1977) Christophe, J,P., Frandsen, E.K., Conlon, T.P., Krishna, G., Gardner, J.D. :Action of cholecystokinin, cholinergic agents, and A 23187 on accumulation of guanosine 3' : 5'-monophosphate in dispersed guinea pig pancreatic acinar cells. J. biol. Chem. 251, 4640-4645 (1976) Gaskin, F., Cantor, C.R., Shelanski, M.L.: Biochemicalstudies on the in vitro assembly and disassembly of microtubules. Ann. N.Y. Acad. Sci. 253, 133 (1975) Kaiser, N., Edelman, I.S.: Calcium dependence of glucocorticoid-induced lymphocytolysis. Proc. nat. Acad. Sci. (Wash.) 74, 638-642 (1977) Reed, P.W.: Effects of the divalent cation ionophore A 23187 on potassium permeability of rat erythrocytes. J. biol. Chem. 251, 3489-3494 (1976) Reed, P.W., Lardy, H.A.: A23187: A divalent cation ionophore. J. biol. Chem. 247, 6970-6977 (1972) Rinderknecht, J., Wilding, P., Haverback, BJ.: A new method for the determination of e-amylase. Experientia (Basel) 23, 805 (1967)
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Schreurs, V.V.A.M., Swarts, H.G.P., dePont, J.J.H.H.M., Bonting, S.L. : Role of calcium in exocrine pancreatic secretion. II. Comparison of the effects of carbachol and the ionophore A23187 on enzyme secretion and calcium movements in rabbit pancreas. Biochem. biophys. Acta (Amst.) 419, 320-330 (1976) Selinger, Z., Eimerl, S., Savion, N., Schramm, M.: A Ca ++ ionophore (A23187) simulating hormone and neurotransmitter action in the rat parotid and pancreas glands. In: Secretory mechanisms of exocrine glands (N.A. Thorn and O.H. Petersen, eds.), pp. 68-78. Copenhagen: Munksgaard 1974 Wacker, W.E.C., Ulmer, D.D., Vallee, B.L.: Metaloenzymes and myocardial infarction. II. Malic and lactic dehydrogenase activities and zinc concentrations in serum. New Engl. J. Med. 255, 449-456 (1956) Weisenberg, R.C.: Microtubule formation in vitro in solutions containing low calcium concentrations. Science 166, 1104-1105 (1972) Williams, J.A. : Na § dependence of in vitro pancreatic amylase release. Amer. J. Physiol. 229, 1023 1026 (1975) Williams, J.A. : Effects of cytochalasin B on pancreatic acinar cell structure and secretion. Cell Tiss. Res. 179, 453-466 (1977) Williams, J.A., Chandler, D.: Ca § § and pancreatic amylase release. Amer. J. Physiol. 228, 172%1723 (1975) Williams, J.A., Lee, M. : 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) Williams, J.A., Lee, M.: Microtubules and pancreatic amylase release by mouse pancreas in vitro. J. Cell Biol. 71, 795-806 (1976) Wright, D.G., Bralove, D.A. Gallin, J.I.: The differential mobilization of human neutrophil granules. Amer. J. Path. 87, 273~84 (1977)
Accepted September 27, 1977
