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The effect of isoproterenol on enzyme secretion from the isolated perfused cat pancreas
440
Biochimica et Biophysica Acta, 338 ( 1 9 7 4 ) 4 4 0 - - 4 4 6 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 2 7 3 2 0
THE EFFECT OF ISOPROTERENOL ON ENZYME SECRETION FROM THE ISOLATED PERFUSED CAT PANCREAS
R. P E D E R S O N * and I. S C H U L Z
Max Planck Institut fur Biophysik, 6 Frankfurt/M. (Germany) (Received J u l y 12th, 1973)
Summary Isoproterenol was shown to stimulate amylase secretion from the isolated perfused cat pancreas. Results suggest that isoproterenol acts on pancreatic ~-receptors and that endogenously released acetylcholine is responsible for enzyme secretion.
Introduction
Amylase secretion from parotid glands is stimulated by isoproterenol and other catecholamines [1] and to a lesser extent by cholinergic substances such as carbamylcholine and pilocarpine [5]. It is well established that in the pancreas enzyme secretion is induced by the intestinal h o r m o n e cholecystokinin-pancreozymin and acetylcholine. Whereas it has been reported that catecholamines including isoproterenol stimulate little or no amylase secretion from mouse and rat pancreas [ 4 - 6] we have observed that isoproterenol induces enzyme secretion in the saline perfused cat pancreas. Although data have accumulated which make it likely that cyclic AMP is the mediator of catecholamine action in parotid glands [2,3] and of cholecystokinin--pancreozymin in pancreas [12] it is not clear at which step of the stimulus--secretion coupling the reaction course of different stimulants runs together to yield the same response i.e. enzyme secretion. In the present paper experiments are reported which describe the influence of transmitter blockers on enzyme secretion induced by acetylcholine, cholecystokinin--pancreozymin and isoproterenol in the isolated perfused cat pancreas. It has been found that the isoproterenol effect is inhibited only by the ~-receptor blocker, propranolol, and that atropine inhibits the acetylcholine as well as the isoproterenol effect. Both blockers, however, are without influence on the effect of cholecystokinin--pancreozymin. We conclude that in the action of isoproterenol, a fl-receptor is involved and that an interaction of the * Present address: Physiology Dept., University of B.C., Vancouver 8, B.C., Canada.
441 isoproterenol and acetylcholine effects takes place at or before the cell membrane. Results further suggest that the stimulatory action of isoproterenol is mediated by the release of endogenous acetylcholine from cholinergic nerve endings. Methods Experiments were carried out on cats of either sex, fasted for 18 h prior to an experiment. Cats were anaesthetized with pentobarbital (60 mg/kg intraperitoneal). The pancreas was surgically isolated according to the m e t h o d of Case et al. [7]. Perfusion fluid, maintained at 36 °C by a heating coil, was infused from a reservoir into the glands arterial supply (coeliac and superior mesenteric arteries) by means of a roller pump, and effluent from the gland collected from the superior mesenteric vein. The main pancreatic duct was cannulated and secretion was collected in graduated pipettes (10-min samples). The composition of the standard perfusion fluid in mmoles/1 was as follows: NaC1, 104; NaHCO3, 26; KC1, 4.8; MgCl:, 1.2; CaC12, 2.5; and D-(+)-glucose, 15. The perfusion fluid was gassed continuously with 02 and CO2 (95:5; v/v)yielding an equilibrated perfusion fluid of pH 7.4. In order to guarantee a complete washout of any secreted enzymes, t h r o u g h o u t all experiments the pancreatic fluid secretion was stimulated maximally by a constant infusion of 0.2 units/min synthetic secretin (gift from Professor Wtinsch, Munich) delivered by a Braun infusion pump into the arterial cannula. The volume of pancreatic juice was recorded at 10-min inter° vals and amylase content determined by the m e t h o d of Street and Close [8]. Amylase is expressed as total o u t p u t in I.U. per 10 min. As a second indicator of enzyme secretion, total protein (mg) was estimated in all samples by the method of Lowry et al. [9]. Enzyme secretion was stimulated by single rapid injections of isoproterenol (Serva), acetylcholine chloride (Serva) and cholec y s t o k i n i n - p a n c r e o z y m i n (G.I.H., Stockholm), administered via the arterial cannula. Other agents when required in experimental procedure; D-(-)propranolol and L-(+)-propranolol (Imperial Chemical Industries Ltd), choline chloride (Merck), phentolamine (Regitin-CIBA) and eserine sulphate (Merck), were added to the perfusion fluid. Results
The effect of isoproterenol on pancreatic amylase secretion In 25 out of 30 experiments, single rapid injections of 1 0 - 5 0 pg of isoproterenol stimulated amylase from the isolated cat pancreas (Fig. 1). In five experiments isoproterenol produced no increase in amylase secretion over basal levels. As reported by Argent et al. [ 1 0 ] , we found that the amylase secretory response to small doses of either acetylcholine or c h o l e c y s t o k i n i n - p a n c r e o zymin was repeatable over several hours (Fig. 1). The enzyme secreted in response to isoproterenol decreased u p o n repeated stimulation much more rapidly than the response to cholecystokinin--pancreozymin or acetylcholine (Fig. 1). Variation was observed in the number of responses elicited by isoproterenol in a single experiment and a relationship between dose of iso-
442 ,~.,h, I ~
I ,J,c.h I Ach I #oh [ ~ .
1 k:h
120. .~ 100-
w 60-
u. Z0-
TIME(hi) Fig. 1. Secretion of amylase and protein by the perfused eat pancreas in response to single rapid injections o f 30 #g isoproterenol ( l ) alternating w i t h single injections o f 100 ng acetylehotine (Aeh). Considering t h e space o f distribution o f both substances in the pancreas, one can estimate intrapanereatie concentrations in the range o f 10--4M f o r isoproterenol and 3 • 10--TM f o r aeetyleholine. Electrolyte secretion was stimulated m a x i m a l l y throughout by infusing synthetic seeretin at a dose o f 0.2 unit/rain. In this and subsequent figures, total columns (hatched plus black bars) represent amylase secretion and hatehed bars alone total protein output.
proterenol and enzyme response was difficult to achieve because of the rapid falling off of the response with repeated stimulation. The dose of isoproterenol used in most experiments (30 pg) was found to yield amylase responses in the same range as 0.25 unit (Ivy Dog Units) cholecystokinin--pancreozymin and 100 ng acetylcholine (doses of acetylcholine and cholecystokinin--pancreozymin which yielded repeatable responses). In contrast to isoproterenol, the catecholamines, epinephrine and norepinephrine, when injected in amounts up to 1 mg elicited no enzyme response.
The effect of the a-blocking agent phentolamine on pancreatic amylase secretion To determine if an enzyme-stimulating action of epinephrine could be revealed in the absence of a-receptor stimulation, epinephrine was given in single injections of 1, 10 and 50 pg, before and subsequent to perfusion of the pancreas with 1 0 - 5 M of the a-blocking agent phentolamine. The mean values of two experiments presented in the table indicate t h a t epinephrine failed t o TABLE I A m y l a s e s e c r e t i o n in r e s p o n s e t o single r a p i d i n j e c t i o n s o f a c e t y l c h o H n e , cholecystokinin -- pancreozymin, isoproterenol and epinephrine b e f o r e a n d a f t e r p e r f u s i o n o f t h e p a n c r e a s w i t h 10 -5 M of t h e ~blocking agent phentolamine. Treatment
Acetylcholine
Isoproterenol Epinephrine Epinephrine Epinephrine Cholecystokinin-pancreozymin
Amount
100 30 1 10 50 0.25
ng /~g /~g ~g ~zg Unit
A m y l a s e r e s p o n s e ( I . U . / I O rain) Normal per fusate
P h e n t o l a m i n e (10-5M)
21 11 0 0.45 0.15 26
20 9 0.15 0.3 0 24
443 Ach
O(-)PROPRANOLOL10-~M 1001 so
Ii
6O
c
0
z
2o
~
~
,o ~ 0
0
TIME(hr) i Ach
120c
Je~:h
Ach
L(*)PROPRANOLOL10-5M
100-
sow
6040.
o u.
20
i I
o
~
~g 4z~
TIME(hr) Fig. 2.
T h e e f f e c t of p e r f u s l o n w i t h
(A) 10-5M D-(--).propranolol
and
(B) 10-5M
L - ( + ) - p r o p r a n o ] o ] on
a m y l a s e and p r o t e i n s t i m u l a t e d in r e s p o n s e t o single i n j e c t i o n s o f i s o p r o t e r e n o l ( 3 0 # g ) and a c e t y l c h o l i n e ( 1 0 0 n g ) . T h e p e r f u s i o n fluid c o n t a i n e d D - ( - - ) - or L - ( + ) - p r o p r a n o l o l for the l e n g t h o f the h o r i z o n t a l b a r .
stimulate amylase secretion in the presence or absence of phentolamine. The data presented in Table I indicate that phentolamine in the concentration used had no observable effect on the amylase response to cholecystokinin--pancreozymin, acetylcholine or isoproterenol.
The effect of D-(--)-propranolol, and L-(+)-propranolol on amylase secretion stimulated by isoproterenol In five experiments isoproterenol stimulation of amylase secretion was reversibly inhibited by perfusion of the pancreas with 10 - s M D-(-)propranolol (Fig. 2A) but not by L-(+)-propranolol (Fig. 2B). The degree of inhibition by D-(--)-propranolol varied from 50 - 85% with a mean of 68.6%. Propranolol D-(- -)- or L-(+)-infusion was initiated 30 min prior to isoproterenol injection. These agents had no observable effect on basal amylase secretion or on the amylase sec,:etory response to acetylcholine or cholecystokinin--pancreozymin.
The effect of atropine on amylase secretion stimulated by isoproterenol In order to determine whether isoproterenol was acting directly on the
444 Pz Ach I Pz
I
Ach
120"
Ach. l PZ
ATROPINE 1.4mlO-~M
~tO0_c E 80~. 60,
=- 20"
o-
I-,o
L-O
0
TIME (hr) Fig. 3. T h e e f f e c t o f P e r f u s i o n o f t h e cat pancreas w i t h a t r o p i n e sulphate, o n a m y l a s e and p r o t e i n secretion s t i m u l a t e d b y single i n j e c t i o n s o f 3 0 /Jg i s o p r o t e r e n o ] (I), 1 0 0 ng a e e t y ] e h o l i n e ( A e h . ) a n d 0 . 2 5 u n i t p a n c r e o z y m i n (Pz.). F o r t h e d u r a t i o n o f t h e h o r i z o n t a l b a r hot-real p e r ~ s a t e was replaced b y one c o n t a i n i n g 1.4 • 1 0 - - 7 M a t r o p i n e .
acinar cell, or stimulating amylase secretion secondarily by releasing acetylcholine, atropine was used. In the experiments atropine sulphate when added to the perfusion fluid to give a final concentration of 1.4.10 -TM completely inhibited the enzyme response to 30 pg isoproterenol, as well as inhibiting the response to exogenous acetylcholine. The response to cholecystokinin-pancreozymin was unaffected by atropine. 1 h after cessation of atropine infusion, the gland again responded to acetylcholine and to isoproterenol stimulation (Fig. 3). The effect of eserine sulphate and choline chloride infusion on the e n z y m e secretory response to isoproterenol Further evidence implicating actylcholine as a mediator of isoproterenol5O)OO Ach
IO0-
I
I
I
I
l
I
[
CHOLINE
I
Cl'÷ E,C~ERINE
~6o
I,o LO
0
TIME (hr) Fig. 4. T h e e f f e c t o f 1-h p e r f u s i o n w i t h ei',oline c h l o r i d e (3 rag/l) plus eseri~e s u l p h a t e (5 r a g / l ) o n t h e amylase s e c r e t o r y response t o single i n j e c t i o n s o f i s o p r o t e r e n o l (1). Doses o f i s o p r o t e r e n o l given w e r e 3 0 /Jg e x c e p t w h e r e i n d i c a t e d .
445 stimulated amylase secretion was provided by three experiments in which choline chloride, the precursor of acetylcholine and eserine sulphate, a cholinesterase inhibitor were added to the perfusion fluid after the amylase response to isoproterenol had been exhausted. After repeated doses of isoproterenol failed to elicit an amylase response, 1 h infusion of 3 mg/1 choline chloride and 5 mg/1 eserine sulphate effected a return in responsiveness to an injection of 30 pg isoproterenol (Fig. 4). It was found that neither choline chloride nor eserine sulphate alone achieved a return of the isoproterenol response (data n o t shown). Discussion
The data presented show that in addition to the well known enzyme stimulatory action of cholecystokinin--pancreozymin and acetylcholine, amylase secretion from the saline perfused cat pancreas can be stimulated by isoproterenol. As has been reported for the parotid [ 1 ] , the amylase-stimulating action of isoproterenol on the pancreas has been demonstrated by us to be abolished by the ~-adrenergic blocker propranolol. Furthermore we have shown that only the D-(--)-form, which has been shown to be 50 - 100 times more p o t e n t than the L-(+)-form in blocking ~3-adrenergic stimulants [ 11 ], inhibited the secretory response to isoproterenol. The latter finding provides evidence that isoproterenol is in fact acting via receptors and n o t via a nonspecific effect. Inhibition of isoproterenol-stimulated amylase secretion from the perfused cat pancreas by atropine suggests that isoproterenol in this case is acting via the release of acetylcholine from cholinergic nerve endings in the pancreas. Further indirect evidence for this conclusion was provided by data from experiments in which the amylase response to isoproterenol was exhausted, then returned after perfusion of the gland with eserine sulphate and choline chloride. These results suggest that the exhausted glandular supply of acetylcholine was sufficiently replenished by choline chloride perfusion and that the newly synthesized acetylcholine could be sufficiently protected from cholinesterase hydrolysis by eserine to permit a return in responsiveness to isoproterenol stimulation. If the action of isoproterenol on amylase secretion is in fact mediated by acetylcholine release, then the a m o u n t of endogenously stored acetylcholine in any experiment determines the efficiency of isoproterenol as a stimulant of amylase secretion, rather than the absolute amounts of stored enzyme. This would explain the differences in enzyme response elicited by isoproterenol as compared with acetylcholine or cholecystokinin--pancreozymin. When interpreting our results and discussing a possible mechanism of action of isoproterenol on the pancreas, the lack of effect of norepinephrine and particularly epinephrine must be considered. Therefore the data presented support a mechanism of action of isoproterenol involving stimulation of 13-receptors which lead to amylase secretion via the release of acetylcholine. The absence of a similar action by naturally occurring catecholamines, epinephrine and norepinephrine, however, indicates that the mechanism involved is not a physiological one.
446
Acknowledgements We would like to)express our gratitude to Professor Dr K.J. Ullrich for his advice and Professor Dr D. Palm and Professor Dr H. Grobecker from the Institute of Pharmacology, University of Frankfurt, for helpful discussion. We thank Mrs A.L. Christian and Mrs J. Wiechmann for their excellent technical assistance. We a/so express our thanks to Professor Dr E. Wtinsch for the gift of synthetic secretin and Mr Braun from Rhein-Pharma for the gift of D-(--)- and L-(+)-propranolol. References 1 2 3 4 5 6 7 8 9 10 11 12
B y r t , P. ( 1 9 6 6 ) N a t u r e 2 1 2 , 1 2 1 2 - 1 2 1 5 . B a b a d H., B e n - Z v i , R., B d o l a h , A, a n d S c h r a m m , M. ( 1 9 6 7 ) Eur. J. B i o c h e r a . 1, 96 - 101. B d o l a h , A. a n d S c h r a m m , M. ( 1 9 6 5 ) B i o e h e m . B i o p h y s . Res. C o m m u n . 1 8 , 4 5 2 - 4 5 4 . K u l k a , R . G . a n d S t e r n l i c h t , E. ( 1 9 6 8 ) P r o c . Natl. A c a d . Sei. U.S. 6 1 , 1 1 2 3 - 1 1 2 8 . S c h r a m m , M. ( 1 9 6 7 ) A n n u . R e v . B i o c h e m . 36, 3 0 7 - 3 2 0 . M a l a m u d , D. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 7 9 , 3 7 3 - 3 7 6 . Case. R.M., H a r p e r , A . A . a n d S e r a t c h e r d , T. ( 1 9 6 8 ) J. P h y s i o l . 1 9 6 , 1 3 3 - 1 4 9 . S t r e e t , H . V . a n d Close, J . R . ( 1 9 5 6 ) Clin. C h i m . A c t a 1, 2 5 6 - 261. L o w r y , O.H., R o s e b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - 2 7 5 . A r g e n t , B.E., Case, R.M. a n d S c r a t c h e r d , T. ( 1 9 7 3 ) J. P h y s i o l . 2 3 0 , 5 7 5 - 593. F i t z g e r a l d , J . D . ( 1 9 6 9 ) , Clin. P h a r m a c o l . T h e r . 10, 2 9 2 - 3 0 6 . R u t t e n , W.J., De Pont, J . J . H . H . M . a n d B o n t i n g , S.L. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 7 4 , 201 - 213.
Biochimica et Biophysica Acta, 338 ( 1 9 7 4 ) 4 4 0 - - 4 4 6 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 2 7 3 2 0
THE EFFECT OF ISOPROTERENOL ON ENZYME SECRETION FROM THE ISOLATED PERFUSED CAT PANCREAS
R. P E D E R S O N * and I. S C H U L Z
Max Planck Institut fur Biophysik, 6 Frankfurt/M. (Germany) (Received J u l y 12th, 1973)
Summary Isoproterenol was shown to stimulate amylase secretion from the isolated perfused cat pancreas. Results suggest that isoproterenol acts on pancreatic ~-receptors and that endogenously released acetylcholine is responsible for enzyme secretion.
Introduction
Amylase secretion from parotid glands is stimulated by isoproterenol and other catecholamines [1] and to a lesser extent by cholinergic substances such as carbamylcholine and pilocarpine [5]. It is well established that in the pancreas enzyme secretion is induced by the intestinal h o r m o n e cholecystokinin-pancreozymin and acetylcholine. Whereas it has been reported that catecholamines including isoproterenol stimulate little or no amylase secretion from mouse and rat pancreas [ 4 - 6] we have observed that isoproterenol induces enzyme secretion in the saline perfused cat pancreas. Although data have accumulated which make it likely that cyclic AMP is the mediator of catecholamine action in parotid glands [2,3] and of cholecystokinin--pancreozymin in pancreas [12] it is not clear at which step of the stimulus--secretion coupling the reaction course of different stimulants runs together to yield the same response i.e. enzyme secretion. In the present paper experiments are reported which describe the influence of transmitter blockers on enzyme secretion induced by acetylcholine, cholecystokinin--pancreozymin and isoproterenol in the isolated perfused cat pancreas. It has been found that the isoproterenol effect is inhibited only by the ~-receptor blocker, propranolol, and that atropine inhibits the acetylcholine as well as the isoproterenol effect. Both blockers, however, are without influence on the effect of cholecystokinin--pancreozymin. We conclude that in the action of isoproterenol, a fl-receptor is involved and that an interaction of the * Present address: Physiology Dept., University of B.C., Vancouver 8, B.C., Canada.
441 isoproterenol and acetylcholine effects takes place at or before the cell membrane. Results further suggest that the stimulatory action of isoproterenol is mediated by the release of endogenous acetylcholine from cholinergic nerve endings. Methods Experiments were carried out on cats of either sex, fasted for 18 h prior to an experiment. Cats were anaesthetized with pentobarbital (60 mg/kg intraperitoneal). The pancreas was surgically isolated according to the m e t h o d of Case et al. [7]. Perfusion fluid, maintained at 36 °C by a heating coil, was infused from a reservoir into the glands arterial supply (coeliac and superior mesenteric arteries) by means of a roller pump, and effluent from the gland collected from the superior mesenteric vein. The main pancreatic duct was cannulated and secretion was collected in graduated pipettes (10-min samples). The composition of the standard perfusion fluid in mmoles/1 was as follows: NaC1, 104; NaHCO3, 26; KC1, 4.8; MgCl:, 1.2; CaC12, 2.5; and D-(+)-glucose, 15. The perfusion fluid was gassed continuously with 02 and CO2 (95:5; v/v)yielding an equilibrated perfusion fluid of pH 7.4. In order to guarantee a complete washout of any secreted enzymes, t h r o u g h o u t all experiments the pancreatic fluid secretion was stimulated maximally by a constant infusion of 0.2 units/min synthetic secretin (gift from Professor Wtinsch, Munich) delivered by a Braun infusion pump into the arterial cannula. The volume of pancreatic juice was recorded at 10-min inter° vals and amylase content determined by the m e t h o d of Street and Close [8]. Amylase is expressed as total o u t p u t in I.U. per 10 min. As a second indicator of enzyme secretion, total protein (mg) was estimated in all samples by the method of Lowry et al. [9]. Enzyme secretion was stimulated by single rapid injections of isoproterenol (Serva), acetylcholine chloride (Serva) and cholec y s t o k i n i n - p a n c r e o z y m i n (G.I.H., Stockholm), administered via the arterial cannula. Other agents when required in experimental procedure; D-(-)propranolol and L-(+)-propranolol (Imperial Chemical Industries Ltd), choline chloride (Merck), phentolamine (Regitin-CIBA) and eserine sulphate (Merck), were added to the perfusion fluid. Results
The effect of isoproterenol on pancreatic amylase secretion In 25 out of 30 experiments, single rapid injections of 1 0 - 5 0 pg of isoproterenol stimulated amylase from the isolated cat pancreas (Fig. 1). In five experiments isoproterenol produced no increase in amylase secretion over basal levels. As reported by Argent et al. [ 1 0 ] , we found that the amylase secretory response to small doses of either acetylcholine or c h o l e c y s t o k i n i n - p a n c r e o zymin was repeatable over several hours (Fig. 1). The enzyme secreted in response to isoproterenol decreased u p o n repeated stimulation much more rapidly than the response to cholecystokinin--pancreozymin or acetylcholine (Fig. 1). Variation was observed in the number of responses elicited by isoproterenol in a single experiment and a relationship between dose of iso-
442 ,~.,h, I ~
I ,J,c.h I Ach I #oh [ ~ .
1 k:h
120. .~ 100-
w 60-
u. Z0-
TIME(hi) Fig. 1. Secretion of amylase and protein by the perfused eat pancreas in response to single rapid injections o f 30 #g isoproterenol ( l ) alternating w i t h single injections o f 100 ng acetylehotine (Aeh). Considering t h e space o f distribution o f both substances in the pancreas, one can estimate intrapanereatie concentrations in the range o f 10--4M f o r isoproterenol and 3 • 10--TM f o r aeetyleholine. Electrolyte secretion was stimulated m a x i m a l l y throughout by infusing synthetic seeretin at a dose o f 0.2 unit/rain. In this and subsequent figures, total columns (hatched plus black bars) represent amylase secretion and hatehed bars alone total protein output.
proterenol and enzyme response was difficult to achieve because of the rapid falling off of the response with repeated stimulation. The dose of isoproterenol used in most experiments (30 pg) was found to yield amylase responses in the same range as 0.25 unit (Ivy Dog Units) cholecystokinin--pancreozymin and 100 ng acetylcholine (doses of acetylcholine and cholecystokinin--pancreozymin which yielded repeatable responses). In contrast to isoproterenol, the catecholamines, epinephrine and norepinephrine, when injected in amounts up to 1 mg elicited no enzyme response.
The effect of the a-blocking agent phentolamine on pancreatic amylase secretion To determine if an enzyme-stimulating action of epinephrine could be revealed in the absence of a-receptor stimulation, epinephrine was given in single injections of 1, 10 and 50 pg, before and subsequent to perfusion of the pancreas with 1 0 - 5 M of the a-blocking agent phentolamine. The mean values of two experiments presented in the table indicate t h a t epinephrine failed t o TABLE I A m y l a s e s e c r e t i o n in r e s p o n s e t o single r a p i d i n j e c t i o n s o f a c e t y l c h o H n e , cholecystokinin -- pancreozymin, isoproterenol and epinephrine b e f o r e a n d a f t e r p e r f u s i o n o f t h e p a n c r e a s w i t h 10 -5 M of t h e ~blocking agent phentolamine. Treatment
Acetylcholine
Isoproterenol Epinephrine Epinephrine Epinephrine Cholecystokinin-pancreozymin
Amount
100 30 1 10 50 0.25
ng /~g /~g ~g ~zg Unit
A m y l a s e r e s p o n s e ( I . U . / I O rain) Normal per fusate
P h e n t o l a m i n e (10-5M)
21 11 0 0.45 0.15 26
20 9 0.15 0.3 0 24
443 Ach
O(-)PROPRANOLOL10-~M 1001 so
Ii
6O
c
0
z
2o
~
~
,o ~ 0
0
TIME(hr) i Ach
120c
Je~:h
Ach
L(*)PROPRANOLOL10-5M
100-
sow
6040.
o u.
20
i I
o
~
~g 4z~
TIME(hr) Fig. 2.
T h e e f f e c t of p e r f u s l o n w i t h
(A) 10-5M D-(--).propranolol
and
(B) 10-5M
L - ( + ) - p r o p r a n o ] o ] on
a m y l a s e and p r o t e i n s t i m u l a t e d in r e s p o n s e t o single i n j e c t i o n s o f i s o p r o t e r e n o l ( 3 0 # g ) and a c e t y l c h o l i n e ( 1 0 0 n g ) . T h e p e r f u s i o n fluid c o n t a i n e d D - ( - - ) - or L - ( + ) - p r o p r a n o l o l for the l e n g t h o f the h o r i z o n t a l b a r .
stimulate amylase secretion in the presence or absence of phentolamine. The data presented in Table I indicate that phentolamine in the concentration used had no observable effect on the amylase response to cholecystokinin--pancreozymin, acetylcholine or isoproterenol.
The effect of D-(--)-propranolol, and L-(+)-propranolol on amylase secretion stimulated by isoproterenol In five experiments isoproterenol stimulation of amylase secretion was reversibly inhibited by perfusion of the pancreas with 10 - s M D-(-)propranolol (Fig. 2A) but not by L-(+)-propranolol (Fig. 2B). The degree of inhibition by D-(--)-propranolol varied from 50 - 85% with a mean of 68.6%. Propranolol D-(- -)- or L-(+)-infusion was initiated 30 min prior to isoproterenol injection. These agents had no observable effect on basal amylase secretion or on the amylase sec,:etory response to acetylcholine or cholecystokinin--pancreozymin.
The effect of atropine on amylase secretion stimulated by isoproterenol In order to determine whether isoproterenol was acting directly on the
444 Pz Ach I Pz
I
Ach
120"
Ach. l PZ
ATROPINE 1.4mlO-~M
~tO0_c E 80~. 60,
=- 20"
o-
I-,o
L-O
0
TIME (hr) Fig. 3. T h e e f f e c t o f P e r f u s i o n o f t h e cat pancreas w i t h a t r o p i n e sulphate, o n a m y l a s e and p r o t e i n secretion s t i m u l a t e d b y single i n j e c t i o n s o f 3 0 /Jg i s o p r o t e r e n o ] (I), 1 0 0 ng a e e t y ] e h o l i n e ( A e h . ) a n d 0 . 2 5 u n i t p a n c r e o z y m i n (Pz.). F o r t h e d u r a t i o n o f t h e h o r i z o n t a l b a r hot-real p e r ~ s a t e was replaced b y one c o n t a i n i n g 1.4 • 1 0 - - 7 M a t r o p i n e .
acinar cell, or stimulating amylase secretion secondarily by releasing acetylcholine, atropine was used. In the experiments atropine sulphate when added to the perfusion fluid to give a final concentration of 1.4.10 -TM completely inhibited the enzyme response to 30 pg isoproterenol, as well as inhibiting the response to exogenous acetylcholine. The response to cholecystokinin-pancreozymin was unaffected by atropine. 1 h after cessation of atropine infusion, the gland again responded to acetylcholine and to isoproterenol stimulation (Fig. 3). The effect of eserine sulphate and choline chloride infusion on the e n z y m e secretory response to isoproterenol Further evidence implicating actylcholine as a mediator of isoproterenol5O)OO Ach
IO0-
I
I
I
I
l
I
[
CHOLINE
I
Cl'÷ E,C~ERINE
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I,o LO
0
TIME (hr) Fig. 4. T h e e f f e c t o f 1-h p e r f u s i o n w i t h ei',oline c h l o r i d e (3 rag/l) plus eseri~e s u l p h a t e (5 r a g / l ) o n t h e amylase s e c r e t o r y response t o single i n j e c t i o n s o f i s o p r o t e r e n o l (1). Doses o f i s o p r o t e r e n o l given w e r e 3 0 /Jg e x c e p t w h e r e i n d i c a t e d .
445 stimulated amylase secretion was provided by three experiments in which choline chloride, the precursor of acetylcholine and eserine sulphate, a cholinesterase inhibitor were added to the perfusion fluid after the amylase response to isoproterenol had been exhausted. After repeated doses of isoproterenol failed to elicit an amylase response, 1 h infusion of 3 mg/1 choline chloride and 5 mg/1 eserine sulphate effected a return in responsiveness to an injection of 30 pg isoproterenol (Fig. 4). It was found that neither choline chloride nor eserine sulphate alone achieved a return of the isoproterenol response (data n o t shown). Discussion
The data presented show that in addition to the well known enzyme stimulatory action of cholecystokinin--pancreozymin and acetylcholine, amylase secretion from the saline perfused cat pancreas can be stimulated by isoproterenol. As has been reported for the parotid [ 1 ] , the amylase-stimulating action of isoproterenol on the pancreas has been demonstrated by us to be abolished by the ~-adrenergic blocker propranolol. Furthermore we have shown that only the D-(--)-form, which has been shown to be 50 - 100 times more p o t e n t than the L-(+)-form in blocking ~3-adrenergic stimulants [ 11 ], inhibited the secretory response to isoproterenol. The latter finding provides evidence that isoproterenol is in fact acting via receptors and n o t via a nonspecific effect. Inhibition of isoproterenol-stimulated amylase secretion from the perfused cat pancreas by atropine suggests that isoproterenol in this case is acting via the release of acetylcholine from cholinergic nerve endings in the pancreas. Further indirect evidence for this conclusion was provided by data from experiments in which the amylase response to isoproterenol was exhausted, then returned after perfusion of the gland with eserine sulphate and choline chloride. These results suggest that the exhausted glandular supply of acetylcholine was sufficiently replenished by choline chloride perfusion and that the newly synthesized acetylcholine could be sufficiently protected from cholinesterase hydrolysis by eserine to permit a return in responsiveness to isoproterenol stimulation. If the action of isoproterenol on amylase secretion is in fact mediated by acetylcholine release, then the a m o u n t of endogenously stored acetylcholine in any experiment determines the efficiency of isoproterenol as a stimulant of amylase secretion, rather than the absolute amounts of stored enzyme. This would explain the differences in enzyme response elicited by isoproterenol as compared with acetylcholine or cholecystokinin--pancreozymin. When interpreting our results and discussing a possible mechanism of action of isoproterenol on the pancreas, the lack of effect of norepinephrine and particularly epinephrine must be considered. Therefore the data presented support a mechanism of action of isoproterenol involving stimulation of 13-receptors which lead to amylase secretion via the release of acetylcholine. The absence of a similar action by naturally occurring catecholamines, epinephrine and norepinephrine, however, indicates that the mechanism involved is not a physiological one.
446
Acknowledgements We would like to)express our gratitude to Professor Dr K.J. Ullrich for his advice and Professor Dr D. Palm and Professor Dr H. Grobecker from the Institute of Pharmacology, University of Frankfurt, for helpful discussion. We thank Mrs A.L. Christian and Mrs J. Wiechmann for their excellent technical assistance. We a/so express our thanks to Professor Dr E. Wtinsch for the gift of synthetic secretin and Mr Braun from Rhein-Pharma for the gift of D-(--)- and L-(+)-propranolol. References 1 2 3 4 5 6 7 8 9 10 11 12
B y r t , P. ( 1 9 6 6 ) N a t u r e 2 1 2 , 1 2 1 2 - 1 2 1 5 . B a b a d H., B e n - Z v i , R., B d o l a h , A, a n d S c h r a m m , M. ( 1 9 6 7 ) Eur. J. B i o c h e r a . 1, 96 - 101. B d o l a h , A. a n d S c h r a m m , M. ( 1 9 6 5 ) B i o e h e m . B i o p h y s . Res. C o m m u n . 1 8 , 4 5 2 - 4 5 4 . K u l k a , R . G . a n d S t e r n l i c h t , E. ( 1 9 6 8 ) P r o c . Natl. A c a d . Sei. U.S. 6 1 , 1 1 2 3 - 1 1 2 8 . S c h r a m m , M. ( 1 9 6 7 ) A n n u . R e v . B i o c h e m . 36, 3 0 7 - 3 2 0 . M a l a m u d , D. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 7 9 , 3 7 3 - 3 7 6 . Case. R.M., H a r p e r , A . A . a n d S e r a t c h e r d , T. ( 1 9 6 8 ) J. P h y s i o l . 1 9 6 , 1 3 3 - 1 4 9 . S t r e e t , H . V . a n d Close, J . R . ( 1 9 5 6 ) Clin. C h i m . A c t a 1, 2 5 6 - 261. L o w r y , O.H., R o s e b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - 2 7 5 . A r g e n t , B.E., Case, R.M. a n d S c r a t c h e r d , T. ( 1 9 7 3 ) J. P h y s i o l . 2 3 0 , 5 7 5 - 593. F i t z g e r a l d , J . D . ( 1 9 6 9 ) , Clin. P h a r m a c o l . T h e r . 10, 2 9 2 - 3 0 6 . R u t t e n , W.J., De Pont, J . J . H . H . M . a n d B o n t i n g , S.L. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 7 4 , 201 - 213.