J. Phyeiol. (1977), 264, pp. 323-339 WBith 7 text -figure8 Printed in Great Britain
323
EFFECTS OF THE CALCIUM IONOPHORE A23187 ON PANCREATIC ACINAR CELL MEMBRANE POTENTIALS AND AMYLASE RELEASE
BY JORGEN HEDEMARK POULSEN* AND JOHN A. WILLIAMS From the Department of Physiology, University of California, San Francisco, California 94143 U.S.A.
(Received 13 April 1976) SUMMARY
1. The effects of the Ca2+-ionophore A23187 and the non-metabolizable cholinergic agonist bethanechol on acinar cell membrane potentials and amylase release from the superfused mouse pancreas were studied. 2. In the presence of extracellular Ca2+ (2.56 mM), A23187 (10-5M) and bethanechol (3 x 10-, M) caused an equal increase in the release of amylase. Both stimulants depolarized the acinar cells, A23187 by 60 mV and bethanechol by 12-3 mV. 3. When Ca2+ and Mg2+ were removed from the superfusate, the ability of A23187 to increase the rate of amylase release was virtually abolished, while the effect of bethanechol remained unaltered. Similarly, in the absence of these divalent cations, A23187 did not cause depolarization of the acinar cells, while depolarization in response to bethanechol was largely normal. Consequently it is unlikely that cholinergic agonists initiate secretion by activating a Ca2+-ionophore-like mechanism in the cell membrane. 4. When the concentration of Ca2+ in the medium was raised to 10 mM, as the only divalent cation present, the depolarization in response to A23187 was increased to 11-8 mV. When Mg2+ in a concentration of 10 mM was the only extracellular divalent cation, the depolarization was only 2-1 mV. 5. The Ca2+ dependent, A23187-induced depolarization was abolished in the absence of Na+ (Tris substitution). Addition of Na+ to the superfusate caused an immediate depolarization. 6. It is concluded that the Ca2+ dependent depolarization of pancreatic acinar cells induced by A23187 is not directly due to an increased divalent * Permanent address: Institute of Medical Physiology A, University of Copenhagen, Denmark.
J. H. POULSEN AND J. A. WILLIAMS 324 cation conductance. Our findings are consistent with the view that the depolarization is due to an increased influx of Na+ resulting from a Ca2+ mediated increase in Na+ permeability. INTRODUCTION
Secretion of protein by the exocrine pancreas is known to be dependent on Ca2+ (Hokin, 1966; Robberecht & Christophe, 1971; Benz, Eckstein, Matthews & Williams, 1972; Heisler, Fast & Tenenhouse, 1972; Argent, Case & Scratcherd, 1973; Schultz, 1975). Recent observations indicate that the Ca2+, important in the initiation of enzyme secretion by pancreatic acinar cells, is released from intracellular stores upon stimulation with the physiological secretagogues, ACh and pancreozymin (Case & Clausen, 1973; Matthews, Petersen & Williams 1973; Chandler & Williams, 1974; Williams & Chandler, 1975; Gardner, Conlon, Klaeveman, Adams & Ondetti, 1975). This is somewhat different from the original model of stimulus-secretion coupling, in which the stimulation-induced increase in the intracellular concentration of free Ca2+ is due to an influx of extracellular Ca2+ (Douglas, 1968; Rubin, 1970). That an increased concentration of intracellular free Ca2+ is a crucial step in stimulus-secretion coupling has gained substantial support by the recent finding that Ca2+ ionophores, which are able to increase the permeability of membranes to Ca2+ (Reed & Lardy, 1972), can release secretary products stored in granules from a number of tissues including mast cells (Foreman, Mongar & Gomperts, 1973; Cochrane & Douglas, 1974), exocrine pancreas (Selinger, Eimerl, Savion & Schramm, 1974; Williams & Lee, 1974), neurohypophysis (Russel, Hansen & Thorn, 1974) and adrenal medulla (Cochrane, Douglas, Mouri & Nakazato, 1975). A23187-induced pancreatic amylase release is dependent on the presence of extracellular Ca2+, and is associated with an enhanced uptake of extracellular Ca2+ (Williams & Lee, 1974). This is in contrast to cholinergic agonists, which do not increase Ca2+ influx or require extracellular Ca2+ for their action (Chandler & Williams, 1974; Williams & Chandler, 1975). Electrophysiological techniques have recently been applied to pancreatic acinar cells as a means of studying the earliest membrane-related steps in stimulus-secretion coupling (Dean & Matthews, 1972; Kanno, 1972; Matthews & Petersen, 1973; Greenwell, 1975). Nishiyama & Petersen (1974, 1975) have shown that depolarization induced by cholinergic agonists can be explained as due to an increased membrane permeability to Na+, possibly associated with a smaller increase in the permeability to K+. No evidence was found in support of a secretagogue induced increase in Ca2+ permeability. The present study was undertaken to investigate the
PANCREATIC MEMBRANE POTENTIALS 325 effects of A23187 on the pancreatic acinar cell membrane potential and to compare simultaneous measurements of membrane potentials and amylase release in response to A23187 and the cholinergic agonist bethanechol. A preliminary report of some of the observations described in the present work has been given (Poulsen & Williams, 1976). METUODS Superfu8ion The studies were carried out using male white Swiss mice weighing 18-30 g. After decapitation the pancreas was removed and secured to a Perspex platform, which was mounted in a superfusion chamber as described by Dean & Matthews (1972). Superfusion fluid heated to 370 C was pumped through the chamber, which had a volume of 4 ml., at a constant rate of 3.5 ml./min. When the release of amylase was studied, samples of the effluent pumped out of the chamber were collected at 2° C at 5 min intervals. The basic Krebs-Henseleit-bicarbonate (KHB) solution used for superfusion had the following composition (mm): NaCl 103, NaHCO8 25, KCl 4 7, CaClg 2-56, MgCl2 1-3, NaH2PO4 1-15, D-glucose 5-6, Na pyruvate 4-9, Na fumarate 2-7, Na glutamate 4- 9. It was gassed with 95 % 02 and 5 % CO, and adjusted when necessary to pH 7-4. (Ca2+, Mg2+)-free solution was prepared by omission of Ca2+ and Mg2+ and osmolality was maintained by appropriate increase in the concentration of NaCl. The (Ca2+, Mg&+)-free solution had a residual Ca2+ content of 5 x 10^ M as measured by atomic absorption spectrophotometry. When solutions having potassium concentrations of 23-5 or 47 mm were used, the Na concentration was reduced accordingly. In some cases a Tris Ringer solution was used which was similar to KHB but contained 10 mm Tris Cl, pH 7-4, and no bicarbonate or phosphate. This solution which was equilibrated with 100% 02, allowed the Ca2+ or Mg2+ concentration to be increased to 10 mim. Na+-free Tris Ringer was made by substituting Tris Cl for NaCl and using the free acids of the substrates with the exception of fumnarate which was present as the K salt, in which case KCI was omitted.
Electrical recording Cell membrane potentials were recorded by use of glass micro-electrodes. Electrodes were filled with 5 M K acetate using the glass fibre method and had resistances of about 100 MC. A Winston Model 1090 preamplifier (input resistance: 1013 a) was used, and the signal was displayed both on an oscilloscope and a pen recorder. The micro-electrodes were advanced vertically with a hydraulic microdrive, although impalement of cells was obtained by tapping on the micromanipulator. Impalements were judged satisfactory if an abrupt negative deflexion was observed which was maintained for at least 30 sec. Stable potentials could generally be maintained for much longer periods up to 1 hr. The value obtained immediately before withdrawal of the micro-electrode was taken to represent the membrane potential. The tissue was allowed to equilibrate in the superfusion chamber for 30 min before membrane potentials were recorded. Stimulation of the pancreas was achieved by superfusing the pancreas with solutions containing acetylcholine chloride (ACh), bethanechol or A23187 in the concentrations stated in the Results section. ACh and bethanechol were dissolved directly in the solutions, while A23187 was kept as a stock solution in ethanol and diluted 200 times when used. Control experiments showed that this concentration of ethanol (0-5 %) was without any effect on the membrane potentials or amylase release. A23187 was kindly provided as a generous gift from Dr R. Hamill of Eli Lilly and Co.
326
J. H. POULSEN AND J. A. WILLIAMS
Amyla8e determination The release of amylase was determined by measuring the amylase concentration of the effluent using the method described by Rinderknecht, Wilding & Haverback (1967) with amylose azure (Cal Biochem) as the substrate. Amylase is expressed in International Units based on the reported activity of the a-amylase (Sigma type VI) used as a standard.
Presentation of data In most experiments serial micro-electrode impalements were made and each potential plotted against time from the start of the experiment. During the initial superfusion with KHB or standard Tris-Ringer, the mean value of all successful measurements was calculated (Em). During stimulation with cholinergic agonists or A23187 the mean value was also calculated, the induced potential change A Em was calculated by subtraction. When studying the effect of drugs in the absence of extracellular Ca and Mg, where the membrane potentials exhibited a steady decline, the mean value for the last 15 min preceding the addition of the drug was taken as a reference, and compared to the mean value obtained during the 5th to the 15th min in the presence of the drug. Numbers given in text and tables are means of mean values from individual experiments + s.E. of mean, unless otherwise indicated. Amylase release is indicated in the figures as concentration (mu./ml.) in the effluent pumped out of the chamber. Since the flow was constant, this concentration was proportional to the rate of the release. In the tables and the text amylase release caused by stimulation is expressed in % of basal (unstimulated) release, using the mean value of the two last samples preceding the stimulation as the reference.
RESULTS
Resting membrane Potentials The mean resting membrane potential measured in fifty-nine experiments over the period August 1975 to January 1976 during superfusion with the standard KHB solution was -43-1 + 0-5 mV. This value is similar to although slightly more negative than those reported previously (Dean & Matthews, 1972; Matthews & Petersen, 1973; Nishiyama & Petersen, 1974). Measured potentials exhibited a tendency to become more negative throughout the period during which the experiments were carried out and in most recent experiments have been in the range of -45 to -50 mV. We attribute this to improvement in technique. The mean resting membrane potential measured in twenty experiments during superfusion with standard Tris buffered Ringer solution was - 43-8 + 0-8 mV, which is similar to that found using KHB and more negative than the value reported by Matthews & Petersen (1973) of -35 mV. Effect of cholinergic agonists on membrane potentials and amylase release As shown in Table 1 the non-metabolizable, cholinergic agonist bethanechol, is able to depolarize the pancreatic acinar cells in a dose dependent manner. The effect is qualitatively similar to that of the acetyl-
327 PANCREATIC MEMBRANE POTENTIALS choline, although the relative potency of bethanechol expressed on a molar basis is approximately 30 times less. These relative potencies are similar to those found for stimulation of amylase release from mouse pancreatic fragments, where maximal release was observed at 3 x 10-5 M bethanechol and 10-6M ACh (Williams, 1975). The magnitude of the depolarization in response to maximal concentrations of both agonists is similar to that reported previously (Dean & Matthews, 1972; Matthews &
Petersen, 1973; Matthews et al. 1973). The effect of bethanechol on acinar TABLE 1. Depolarization of mouse pancreas cells superfused with KHB by addition of bethanechol and acetylcholine Membrane potential (mV)
Agent Bethanechol (M) 3x104 3X 10-5 3x 10-4 Acetylcholine (M) 10-6
10-5
Control Em
Depolarization
n
-44-4+1-7
2-7±0.6
-42.3+±1P2 -42.0 + 2.2
12.3+±11 14*3±+ 15
(6) (10) (6)
-39.7+ 1.1 -41.5±165
9.3+ 1.7 159+ 1-1
(6) (6)
All values are the mean + S.E. of mean for the number of experiments listed under n.
cell membrane potentials and amylase release during superfusion with standard KHB in an individual experiment is shown in Fig. 1 A. Bethanechol depolarized the cells fairly immediately by 10-15 mV, and the cells remained depolarized throughout the period of superfusion with the cholinergic agonist. The process was reversible, but full recovery required superfusion with KHB for 30-60 min. The bethanechol-induced release of amylase was slow in comparison to the depolarization as previously found by Matthews et atl. (1973). The apparent discrepancy between the time course of the two phenomena is at least in part a consequence of the recording chamber, which was designed mainly for electrophysiological studies with a flow rate of about one chamber volume per minute as well as a relatively small effective surface of the tissue with consequent long diffusion distances from the deeper parts. In studies using superfused pancreatic fragments maximal amylase release was observed between 5 and 10 min after stimulation and upon withdrawal of the secretagogue amylase release returned to basal rate within 10 min (Matthews et al. 1973; Williams & Chandler, 1975). Fig. 1B demonstrates that the presence of extracellular Ca is not required for either stimulation-induced depolarization or amylase release. Summarized measurement of amylase release
J. H. POULSEN AND J. A. WILLIAMS 328 from a number of experiments is included in Table 2. That the further bethanechol-induced depolarization seen during superfusion with (Ca2+ Mg&+)-free medium is real and does not merely reflect the effect of prolonged exposure to divalent cation free solution is demonstrated by Fig. 2, Bethanechol (3 x 1O-5M)
A
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60
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1 40
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10
20
0 30 40
I
50 60 70 Time (min)
80
90 100 180 190
Fig. 1. Effect of bethanechol on membrane potentials and amylase release from mouse pancreas superfused with KHB or (Ca2+, Mg2+)-free KHB. Bethanechol (3 x 10- M) was present in the bathing medium during the period indicated by the hatched horizontal bar. Each point represents the membrane potential recorded from a single cell plotted as a function of time from the start of recording. The continuous lines represent the amylase concentration in the effluent pumped out of the superfusion chamber at a constant flow rate of 3-5 ml./min and collected as 5 min samples. A, bethanechol added during superfusion with KHB (Ca2+, Mg2+ present). B, bethanechol added during superfusion with (Ca2+, Mg2+)-free KHB.
which shows while recording from a single cell that the depolarizing effect of bethanechol is prompt and very similar to that obtained when divalent cations are present in the superfusion fluid. The data in Fig. 3 shows that the effect on the membrane potential of further exposure to (Ca2+, Mg2+)-
PANCREA TIC MEMBRANE POTENTIALS 329 free solution is small, However. both Fig. IB and Fig. 3 show that the absence of extracellular Ca2+ and Mg2+ leads to a gradual depolarization to about - 30 mV. Readmission of Ca2+, either alone or together with Mg2+, causes a rapid and full recovery of the membrane potentials (Fig. 3), again with little effect on amylase release. A
Bethanechol
50 mV
KHB
1 min
B
Bethanechol
50 mV
(Ca2, Mg2+ -free)
Fig. 2. The effect of bethanechol on the membrane potential of single pancreatic cells during superfusion with KHB or (Ca2+, Mg2+)-free KHB. The sharp downward and upward deflexion represent impalement of the cell and withdrawal of the micro-electrode, respectively. Bethanechol (3 x 10-6 M) was present in the bathing medium from the time indicated by the arrow. A, bethanechol added directly to KHB. B, bethanechol added following superfusion with a (Ca2+, Mg2+)-free KHB solution for 40 min.
(Ca2+, Mg2+)-free I~ F** -30t -;,- -40
*
-
-
-40
16° °
_2O
0
E
0
-30
60
720
-20
140
.0~~~~~~~~~~~~~~~~~ E 20
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Fi.. Thefeto 0
10
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wi
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uefiofr60 mi wit a (C(, g+)f 30
40
50
60
70
90
80
100
Time (min)
Fig. 3. The effect of superfusion for 60 min with
a
(Ca2+, Mg2+)-free
solution followed by a return to KEB on membrane potentials and amylase release from mouse pancreas. Symbols as in Fig. 1: points, membrane potential; continuous line, amylase release.
J. H. POULSEN AND J. A. WILLIAMS
330
Effect of Ca2+ ionophore A23187 on membrane potentials and amylase release The effect of adding the Ca2+ ionophore A23187 to the superfusion medium on membrane potentials and of amylase release by mouse pancreas superfused with KHB is shown in Fig. 4A. Qualitatively a response A23187 (10-M )
A -50
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30 40 50 60 70 80 90 100 Time (min) Fig. 4. Effect of A23187 on membrane potentials and amylase release from mouse pancreas superfused with KHB or (Ca2+, Mg2+)-free KHB. A23187 (10-5 M) was present in the superfusion fluid during the period indicated by the shaded horizontal bar. A, A23187 added during superfusion with KHB. B, A23187 added during superfusion with (Ca2+, Mg2+)-free KHB. Symbols as in Fig. 1: filled circles, membrane potential; continuous line, amylase release. 0
similar to that induced by bethanechol is observed; a maintained depolarization of the acinar cells and an increase release of amylase. A23187induced depolarization, however, was considerably smaller than that induced by bethanechol, being 6-0 + 0 9 mV(n = 8), for a concentration of
PANCREATIC MEMBRANE POTENTIALS
331 the ionophore (10-5 M) which increases amylase release comparable to that maximally induced by bethanechol (Table 2). Following removal of A23187 from the superfusate the membrane potentials partially recovered. In five experiments in which A23187 lowered the membrane potential from -43'4 + 1.1 mV to - 376 + 1-6 mV the potential increased to - 40-4 + 14 during the initial 20 min after return to KHB. Amylase release remained elevated during this period. The apparent difference TABLE 2. Effect of bethanechol and A23187 on amylase release by mouse pancreas superfused with KHB or (Ca2+, Mg2+)-free KHB
Amylase release (% increase) A
Agent
KHB
(Ca2+, Mg2+)-free KHB
Bethanechol (3 x 10-5M) A23187
146 ± 26 (6)
150±17 (6)
138± 35 (6)
23± 7 (5)
(10-5 M)
All values are the mean + S.E. for the number of experiments shown in parentheses.
between changes in membrane potential and amylase release in the recovery period may not be real, however, due to the delay in amylase washout from the tissue in the preparation used. It should also be noted that the concentration of the ionophore is the total, but that 70-90 % of the A23187 is present in particulate form in the presence of physiological concentrations of divalent cations (D. E. Chandler & J. A. Williams, unpublished observations), and in the present study particulates could be seen through the stereomicroscope when medium containing A23187 entered the superfusion chamber. Identical results, depolarization and amylase release, in response to A23187 were observed when atropine was added to a concentration of 3 x 106 M, sufficient to block the response to ACh. This result indicates that the ionophore is not acting indirectly through release of endogenous ACh from nerve terminals, as is the case when the K+ concentration of the medium is increased (Argent, Case & Scratcherd, 1971). In contrast to studies using KHB, addition of A23187 failed to influence either membrane potential or amylase release (Fig. 4B) when divalent cations were omitted from the medium. The lack of effect is not a consequence of the depolarization caused by the absence of divalent cations, since when a similar depolarization was induced by raising the K+ concentration five or tenfold, A23187 produced its characteristic effects on both membrane potentials and amylase release in four experiments. That A23187 undoubtedly is incorporated in the cell membranes even in the
332 J. H. POULSEN AND J. A. WILLIAMS absence of extracellular Ca2+ and Mg2+, is indicated by the fact that readmission of both divalent cations (or Ca2+ alone) after superfusion with A23187 caused a rapid and very substantial increase in amylase release (Fig. 4B). In five experiments amylase release increased 231 + 27 % upon reintroduction of Ca2+. In contrast to the rapid repolarization seen upon readmission of Ca2+ without previous superfusion with A23187 (Fig. 3), the cells did not repolarize after exposure to the ionophore (Fig. 4B), i.e. the cells remained depolarized during the period of increased amylase release. [K+]=47 mmI -50 _ > -40 E C
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323
EFFECTS OF THE CALCIUM IONOPHORE A23187 ON PANCREATIC ACINAR CELL MEMBRANE POTENTIALS AND AMYLASE RELEASE
BY JORGEN HEDEMARK POULSEN* AND JOHN A. WILLIAMS From the Department of Physiology, University of California, San Francisco, California 94143 U.S.A.
(Received 13 April 1976) SUMMARY
1. The effects of the Ca2+-ionophore A23187 and the non-metabolizable cholinergic agonist bethanechol on acinar cell membrane potentials and amylase release from the superfused mouse pancreas were studied. 2. In the presence of extracellular Ca2+ (2.56 mM), A23187 (10-5M) and bethanechol (3 x 10-, M) caused an equal increase in the release of amylase. Both stimulants depolarized the acinar cells, A23187 by 60 mV and bethanechol by 12-3 mV. 3. When Ca2+ and Mg2+ were removed from the superfusate, the ability of A23187 to increase the rate of amylase release was virtually abolished, while the effect of bethanechol remained unaltered. Similarly, in the absence of these divalent cations, A23187 did not cause depolarization of the acinar cells, while depolarization in response to bethanechol was largely normal. Consequently it is unlikely that cholinergic agonists initiate secretion by activating a Ca2+-ionophore-like mechanism in the cell membrane. 4. When the concentration of Ca2+ in the medium was raised to 10 mM, as the only divalent cation present, the depolarization in response to A23187 was increased to 11-8 mV. When Mg2+ in a concentration of 10 mM was the only extracellular divalent cation, the depolarization was only 2-1 mV. 5. The Ca2+ dependent, A23187-induced depolarization was abolished in the absence of Na+ (Tris substitution). Addition of Na+ to the superfusate caused an immediate depolarization. 6. It is concluded that the Ca2+ dependent depolarization of pancreatic acinar cells induced by A23187 is not directly due to an increased divalent * Permanent address: Institute of Medical Physiology A, University of Copenhagen, Denmark.
J. H. POULSEN AND J. A. WILLIAMS 324 cation conductance. Our findings are consistent with the view that the depolarization is due to an increased influx of Na+ resulting from a Ca2+ mediated increase in Na+ permeability. INTRODUCTION
Secretion of protein by the exocrine pancreas is known to be dependent on Ca2+ (Hokin, 1966; Robberecht & Christophe, 1971; Benz, Eckstein, Matthews & Williams, 1972; Heisler, Fast & Tenenhouse, 1972; Argent, Case & Scratcherd, 1973; Schultz, 1975). Recent observations indicate that the Ca2+, important in the initiation of enzyme secretion by pancreatic acinar cells, is released from intracellular stores upon stimulation with the physiological secretagogues, ACh and pancreozymin (Case & Clausen, 1973; Matthews, Petersen & Williams 1973; Chandler & Williams, 1974; Williams & Chandler, 1975; Gardner, Conlon, Klaeveman, Adams & Ondetti, 1975). This is somewhat different from the original model of stimulus-secretion coupling, in which the stimulation-induced increase in the intracellular concentration of free Ca2+ is due to an influx of extracellular Ca2+ (Douglas, 1968; Rubin, 1970). That an increased concentration of intracellular free Ca2+ is a crucial step in stimulus-secretion coupling has gained substantial support by the recent finding that Ca2+ ionophores, which are able to increase the permeability of membranes to Ca2+ (Reed & Lardy, 1972), can release secretary products stored in granules from a number of tissues including mast cells (Foreman, Mongar & Gomperts, 1973; Cochrane & Douglas, 1974), exocrine pancreas (Selinger, Eimerl, Savion & Schramm, 1974; Williams & Lee, 1974), neurohypophysis (Russel, Hansen & Thorn, 1974) and adrenal medulla (Cochrane, Douglas, Mouri & Nakazato, 1975). A23187-induced pancreatic amylase release is dependent on the presence of extracellular Ca2+, and is associated with an enhanced uptake of extracellular Ca2+ (Williams & Lee, 1974). This is in contrast to cholinergic agonists, which do not increase Ca2+ influx or require extracellular Ca2+ for their action (Chandler & Williams, 1974; Williams & Chandler, 1975). Electrophysiological techniques have recently been applied to pancreatic acinar cells as a means of studying the earliest membrane-related steps in stimulus-secretion coupling (Dean & Matthews, 1972; Kanno, 1972; Matthews & Petersen, 1973; Greenwell, 1975). Nishiyama & Petersen (1974, 1975) have shown that depolarization induced by cholinergic agonists can be explained as due to an increased membrane permeability to Na+, possibly associated with a smaller increase in the permeability to K+. No evidence was found in support of a secretagogue induced increase in Ca2+ permeability. The present study was undertaken to investigate the
PANCREATIC MEMBRANE POTENTIALS 325 effects of A23187 on the pancreatic acinar cell membrane potential and to compare simultaneous measurements of membrane potentials and amylase release in response to A23187 and the cholinergic agonist bethanechol. A preliminary report of some of the observations described in the present work has been given (Poulsen & Williams, 1976). METUODS Superfu8ion The studies were carried out using male white Swiss mice weighing 18-30 g. After decapitation the pancreas was removed and secured to a Perspex platform, which was mounted in a superfusion chamber as described by Dean & Matthews (1972). Superfusion fluid heated to 370 C was pumped through the chamber, which had a volume of 4 ml., at a constant rate of 3.5 ml./min. When the release of amylase was studied, samples of the effluent pumped out of the chamber were collected at 2° C at 5 min intervals. The basic Krebs-Henseleit-bicarbonate (KHB) solution used for superfusion had the following composition (mm): NaCl 103, NaHCO8 25, KCl 4 7, CaClg 2-56, MgCl2 1-3, NaH2PO4 1-15, D-glucose 5-6, Na pyruvate 4-9, Na fumarate 2-7, Na glutamate 4- 9. It was gassed with 95 % 02 and 5 % CO, and adjusted when necessary to pH 7-4. (Ca2+, Mg2+)-free solution was prepared by omission of Ca2+ and Mg2+ and osmolality was maintained by appropriate increase in the concentration of NaCl. The (Ca2+, Mg&+)-free solution had a residual Ca2+ content of 5 x 10^ M as measured by atomic absorption spectrophotometry. When solutions having potassium concentrations of 23-5 or 47 mm were used, the Na concentration was reduced accordingly. In some cases a Tris Ringer solution was used which was similar to KHB but contained 10 mm Tris Cl, pH 7-4, and no bicarbonate or phosphate. This solution which was equilibrated with 100% 02, allowed the Ca2+ or Mg2+ concentration to be increased to 10 mim. Na+-free Tris Ringer was made by substituting Tris Cl for NaCl and using the free acids of the substrates with the exception of fumnarate which was present as the K salt, in which case KCI was omitted.
Electrical recording Cell membrane potentials were recorded by use of glass micro-electrodes. Electrodes were filled with 5 M K acetate using the glass fibre method and had resistances of about 100 MC. A Winston Model 1090 preamplifier (input resistance: 1013 a) was used, and the signal was displayed both on an oscilloscope and a pen recorder. The micro-electrodes were advanced vertically with a hydraulic microdrive, although impalement of cells was obtained by tapping on the micromanipulator. Impalements were judged satisfactory if an abrupt negative deflexion was observed which was maintained for at least 30 sec. Stable potentials could generally be maintained for much longer periods up to 1 hr. The value obtained immediately before withdrawal of the micro-electrode was taken to represent the membrane potential. The tissue was allowed to equilibrate in the superfusion chamber for 30 min before membrane potentials were recorded. Stimulation of the pancreas was achieved by superfusing the pancreas with solutions containing acetylcholine chloride (ACh), bethanechol or A23187 in the concentrations stated in the Results section. ACh and bethanechol were dissolved directly in the solutions, while A23187 was kept as a stock solution in ethanol and diluted 200 times when used. Control experiments showed that this concentration of ethanol (0-5 %) was without any effect on the membrane potentials or amylase release. A23187 was kindly provided as a generous gift from Dr R. Hamill of Eli Lilly and Co.
326
J. H. POULSEN AND J. A. WILLIAMS
Amyla8e determination The release of amylase was determined by measuring the amylase concentration of the effluent using the method described by Rinderknecht, Wilding & Haverback (1967) with amylose azure (Cal Biochem) as the substrate. Amylase is expressed in International Units based on the reported activity of the a-amylase (Sigma type VI) used as a standard.
Presentation of data In most experiments serial micro-electrode impalements were made and each potential plotted against time from the start of the experiment. During the initial superfusion with KHB or standard Tris-Ringer, the mean value of all successful measurements was calculated (Em). During stimulation with cholinergic agonists or A23187 the mean value was also calculated, the induced potential change A Em was calculated by subtraction. When studying the effect of drugs in the absence of extracellular Ca and Mg, where the membrane potentials exhibited a steady decline, the mean value for the last 15 min preceding the addition of the drug was taken as a reference, and compared to the mean value obtained during the 5th to the 15th min in the presence of the drug. Numbers given in text and tables are means of mean values from individual experiments + s.E. of mean, unless otherwise indicated. Amylase release is indicated in the figures as concentration (mu./ml.) in the effluent pumped out of the chamber. Since the flow was constant, this concentration was proportional to the rate of the release. In the tables and the text amylase release caused by stimulation is expressed in % of basal (unstimulated) release, using the mean value of the two last samples preceding the stimulation as the reference.
RESULTS
Resting membrane Potentials The mean resting membrane potential measured in fifty-nine experiments over the period August 1975 to January 1976 during superfusion with the standard KHB solution was -43-1 + 0-5 mV. This value is similar to although slightly more negative than those reported previously (Dean & Matthews, 1972; Matthews & Petersen, 1973; Nishiyama & Petersen, 1974). Measured potentials exhibited a tendency to become more negative throughout the period during which the experiments were carried out and in most recent experiments have been in the range of -45 to -50 mV. We attribute this to improvement in technique. The mean resting membrane potential measured in twenty experiments during superfusion with standard Tris buffered Ringer solution was - 43-8 + 0-8 mV, which is similar to that found using KHB and more negative than the value reported by Matthews & Petersen (1973) of -35 mV. Effect of cholinergic agonists on membrane potentials and amylase release As shown in Table 1 the non-metabolizable, cholinergic agonist bethanechol, is able to depolarize the pancreatic acinar cells in a dose dependent manner. The effect is qualitatively similar to that of the acetyl-
327 PANCREATIC MEMBRANE POTENTIALS choline, although the relative potency of bethanechol expressed on a molar basis is approximately 30 times less. These relative potencies are similar to those found for stimulation of amylase release from mouse pancreatic fragments, where maximal release was observed at 3 x 10-5 M bethanechol and 10-6M ACh (Williams, 1975). The magnitude of the depolarization in response to maximal concentrations of both agonists is similar to that reported previously (Dean & Matthews, 1972; Matthews &
Petersen, 1973; Matthews et al. 1973). The effect of bethanechol on acinar TABLE 1. Depolarization of mouse pancreas cells superfused with KHB by addition of bethanechol and acetylcholine Membrane potential (mV)
Agent Bethanechol (M) 3x104 3X 10-5 3x 10-4 Acetylcholine (M) 10-6
10-5
Control Em
Depolarization
n
-44-4+1-7
2-7±0.6
-42.3+±1P2 -42.0 + 2.2
12.3+±11 14*3±+ 15
(6) (10) (6)
-39.7+ 1.1 -41.5±165
9.3+ 1.7 159+ 1-1
(6) (6)
All values are the mean + S.E. of mean for the number of experiments listed under n.
cell membrane potentials and amylase release during superfusion with standard KHB in an individual experiment is shown in Fig. 1 A. Bethanechol depolarized the cells fairly immediately by 10-15 mV, and the cells remained depolarized throughout the period of superfusion with the cholinergic agonist. The process was reversible, but full recovery required superfusion with KHB for 30-60 min. The bethanechol-induced release of amylase was slow in comparison to the depolarization as previously found by Matthews et atl. (1973). The apparent discrepancy between the time course of the two phenomena is at least in part a consequence of the recording chamber, which was designed mainly for electrophysiological studies with a flow rate of about one chamber volume per minute as well as a relatively small effective surface of the tissue with consequent long diffusion distances from the deeper parts. In studies using superfused pancreatic fragments maximal amylase release was observed between 5 and 10 min after stimulation and upon withdrawal of the secretagogue amylase release returned to basal rate within 10 min (Matthews et al. 1973; Williams & Chandler, 1975). Fig. 1B demonstrates that the presence of extracellular Ca is not required for either stimulation-induced depolarization or amylase release. Summarized measurement of amylase release
J. H. POULSEN AND J. A. WILLIAMS 328 from a number of experiments is included in Table 2. That the further bethanechol-induced depolarization seen during superfusion with (Ca2+ Mg&+)-free medium is real and does not merely reflect the effect of prolonged exposure to divalent cation free solution is demonstrated by Fig. 2, Bethanechol (3 x 1O-5M)
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Fig. 1. Effect of bethanechol on membrane potentials and amylase release from mouse pancreas superfused with KHB or (Ca2+, Mg2+)-free KHB. Bethanechol (3 x 10- M) was present in the bathing medium during the period indicated by the hatched horizontal bar. Each point represents the membrane potential recorded from a single cell plotted as a function of time from the start of recording. The continuous lines represent the amylase concentration in the effluent pumped out of the superfusion chamber at a constant flow rate of 3-5 ml./min and collected as 5 min samples. A, bethanechol added during superfusion with KHB (Ca2+, Mg2+ present). B, bethanechol added during superfusion with (Ca2+, Mg2+)-free KHB.
which shows while recording from a single cell that the depolarizing effect of bethanechol is prompt and very similar to that obtained when divalent cations are present in the superfusion fluid. The data in Fig. 3 shows that the effect on the membrane potential of further exposure to (Ca2+, Mg2+)-
PANCREA TIC MEMBRANE POTENTIALS 329 free solution is small, However. both Fig. IB and Fig. 3 show that the absence of extracellular Ca2+ and Mg2+ leads to a gradual depolarization to about - 30 mV. Readmission of Ca2+, either alone or together with Mg2+, causes a rapid and full recovery of the membrane potentials (Fig. 3), again with little effect on amylase release. A
Bethanechol
50 mV
KHB
1 min
B
Bethanechol
50 mV
(Ca2, Mg2+ -free)
Fig. 2. The effect of bethanechol on the membrane potential of single pancreatic cells during superfusion with KHB or (Ca2+, Mg2+)-free KHB. The sharp downward and upward deflexion represent impalement of the cell and withdrawal of the micro-electrode, respectively. Bethanechol (3 x 10-6 M) was present in the bathing medium from the time indicated by the arrow. A, bethanechol added directly to KHB. B, bethanechol added following superfusion with a (Ca2+, Mg2+)-free KHB solution for 40 min.
(Ca2+, Mg2+)-free I~ F** -30t -;,- -40
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Time (min)
Fig. 3. The effect of superfusion for 60 min with
a
(Ca2+, Mg2+)-free
solution followed by a return to KEB on membrane potentials and amylase release from mouse pancreas. Symbols as in Fig. 1: points, membrane potential; continuous line, amylase release.
J. H. POULSEN AND J. A. WILLIAMS
330
Effect of Ca2+ ionophore A23187 on membrane potentials and amylase release The effect of adding the Ca2+ ionophore A23187 to the superfusion medium on membrane potentials and of amylase release by mouse pancreas superfused with KHB is shown in Fig. 4A. Qualitatively a response A23187 (10-M )
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30 40 50 60 70 80 90 100 Time (min) Fig. 4. Effect of A23187 on membrane potentials and amylase release from mouse pancreas superfused with KHB or (Ca2+, Mg2+)-free KHB. A23187 (10-5 M) was present in the superfusion fluid during the period indicated by the shaded horizontal bar. A, A23187 added during superfusion with KHB. B, A23187 added during superfusion with (Ca2+, Mg2+)-free KHB. Symbols as in Fig. 1: filled circles, membrane potential; continuous line, amylase release. 0
similar to that induced by bethanechol is observed; a maintained depolarization of the acinar cells and an increase release of amylase. A23187induced depolarization, however, was considerably smaller than that induced by bethanechol, being 6-0 + 0 9 mV(n = 8), for a concentration of
PANCREATIC MEMBRANE POTENTIALS
331 the ionophore (10-5 M) which increases amylase release comparable to that maximally induced by bethanechol (Table 2). Following removal of A23187 from the superfusate the membrane potentials partially recovered. In five experiments in which A23187 lowered the membrane potential from -43'4 + 1.1 mV to - 376 + 1-6 mV the potential increased to - 40-4 + 14 during the initial 20 min after return to KHB. Amylase release remained elevated during this period. The apparent difference TABLE 2. Effect of bethanechol and A23187 on amylase release by mouse pancreas superfused with KHB or (Ca2+, Mg2+)-free KHB
Amylase release (% increase) A
Agent
KHB
(Ca2+, Mg2+)-free KHB
Bethanechol (3 x 10-5M) A23187
146 ± 26 (6)
150±17 (6)
138± 35 (6)
23± 7 (5)
(10-5 M)
All values are the mean + S.E. for the number of experiments shown in parentheses.
between changes in membrane potential and amylase release in the recovery period may not be real, however, due to the delay in amylase washout from the tissue in the preparation used. It should also be noted that the concentration of the ionophore is the total, but that 70-90 % of the A23187 is present in particulate form in the presence of physiological concentrations of divalent cations (D. E. Chandler & J. A. Williams, unpublished observations), and in the present study particulates could be seen through the stereomicroscope when medium containing A23187 entered the superfusion chamber. Identical results, depolarization and amylase release, in response to A23187 were observed when atropine was added to a concentration of 3 x 106 M, sufficient to block the response to ACh. This result indicates that the ionophore is not acting indirectly through release of endogenous ACh from nerve terminals, as is the case when the K+ concentration of the medium is increased (Argent, Case & Scratcherd, 1971). In contrast to studies using KHB, addition of A23187 failed to influence either membrane potential or amylase release (Fig. 4B) when divalent cations were omitted from the medium. The lack of effect is not a consequence of the depolarization caused by the absence of divalent cations, since when a similar depolarization was induced by raising the K+ concentration five or tenfold, A23187 produced its characteristic effects on both membrane potentials and amylase release in four experiments. That A23187 undoubtedly is incorporated in the cell membranes even in the
332 J. H. POULSEN AND J. A. WILLIAMS absence of extracellular Ca2+ and Mg2+, is indicated by the fact that readmission of both divalent cations (or Ca2+ alone) after superfusion with A23187 caused a rapid and very substantial increase in amylase release (Fig. 4B). In five experiments amylase release increased 231 + 27 % upon reintroduction of Ca2+. In contrast to the rapid repolarization seen upon readmission of Ca2+ without previous superfusion with A23187 (Fig. 3), the cells did not repolarize after exposure to the ionophore (Fig. 4B), i.e. the cells remained depolarized during the period of increased amylase release. [K+]=47 mmI -50 _ > -40 E C
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