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Alcohol, pancreatic muscarinic receptors and acute pancreatitis
Exp Toxic Pathol 1993/94; 45: 503-505 Gustav Fischer Verlag Jena
Departments of Surgery, Pharmacology and Pathology, The University of Turku, Turku, Finland
Alcohol, pancreatic muscarinic receptors and acute pancreatitis JUHA M. GRONROOS, TIMO KArLA, and ANTII J. HIETARANTA With 1 figure Received: February 9,1993; Accepted: March 2, 1993
Address for correspondence: JUHA M. GRONROOS, M. D., Department of Surgery, The University Central Hospital of Turku, Kiinamyllynkatu 4-8, 20520 Turku, Finland .
Key words: Muscarinic receptors; Receptors, muscarinic; Pancreatitis, acute; Alcohol, pancreatitis; Cholinergic stimulation; Acetylcholine.
Summary Recently, a new theory about the pathogenesis of acute alcoholic pancreatitis was proposed. The aim of the present work was to further study the basis of this cholinergic theory about the pathogenesis of acute alcoholic pancreatitis. The results indicated that already a short-term alcohol consumption induces in some rats a dramatic decrease in the number of pancreatic muscarinic receptors. This decrease may predispose to acute alcoholic pancreatitis by increasing the cholinergic tone, since excessive cholinergic tone invariably leads to acute pancreatitis both in experimental animals and in man. Thus, the pathogenetic mechanism triggering acute alcoholic pancreatitis might be similar to the mechanism triggering acute pancreatitis caused by the scorpion sting, intoxication with an antiacetylcholine-esterase-containing insecticides or after excessive cholinergic stimulation.
riably to acute pancreatitis both in animals (1, 3, 6, 11) and in man (2, 5). Since the effects of short-term alcohol intake on pancreatic muscarinic receptors are unknown, the question still remains, how the theory accounts for those attacks of acute alcoholic pancreatitis after only a few hours or days of alcohol consumption without preceding chronic alcohol exposure. Although this group consisting mostly of young patients is clearly a minority among those suffering from acute alcoholic pancreatitis, we all meet these patients every now and then in our clinical practice. Accordingly, the aim of the present investigation was to characterize the effects of short-term alcohol intake on pancreatic muscarinic receptors in our rat model of alcoholism.
Introduction
Material and methods
The pathogenesis of acute alcoholic pancreatitis is still poorly understood. Research into the pathogenesis of acute alcoholic pancreatitis has progressed from a "big duct" to a "small duct" approach and finally, in recent times, to studies of the effects of alcohol on pancreatic acinar cells themselves (15-17) .
Twenty male Wistar rats were kept on a standard laboratory diet (Ewos R3, Ewos Ab, SOdertalje, Sweden) under controlled conditions (22 ± 1°C, 60 % humidity, lights on from 7.00 to J9.()(). At the age of two months the animals were randomly divided into two groups. Ten rats were offered alcohol only as 15 % (w/V) solution of ethanol in water for four days. Ten control animals were identical to the test animals in all respects but were offered water instead of alcohol. The animals were housed in individual cages and had free access to food. The daily fluid consumption per rat (x ± SD) was 18.5 ± 4.8 ml (range 11.3-23.8 ml) in alcoholic group and 36.6 ± 6.0 ml (range 27.5-46.2 ml) in control group. After the four days test period the animals were decapitated and the pancreas was removed, trimmed free of fat and weighed. The number and affinity of pancreatic muscarinic receptors were measured by using N-3H-methylscopolamine (NMS , 73.8 Ci/mmol), New England Nuclear) as a radioligand. Preparation of pancreatic plasma membranes, the time course
Recently, a new theory about the pathogenesis of acute alcoholic pancreatitis was proposed (7, 8). This theory is based on the studies indicating that chronic alcohol consumption decreases by 60 % the number of rat pancreatic muscarinic receptors (9) and, by feedback mechanism, increases manifoldly the ratio of pancreatic acetylcholine concentration/muscarinic receptor density (4, 7, 8, 9, 12). These changes may playa crucial part in the pathogenesis of acute alcoholic pancreatitis, since excessive cholinergic stimulation of pancreatic muscarinic receptors leads inva-
Exp Toxic Pathol 4S (1993/94) 8
503
fmol/m~
I)rO'. n
80
•
70
&0
•
50
•
• •
•
It
• • ••
10
•
I
•
A
c
Fig. 1. The maximal binding capacity to NMS of rat pancreatic muscarinic receptors/mg protein after four days of alcohol/water intake. A = alcoholic rats (n = 10); C = control rats (n
= 10).
studies of NMS binding, saturation analysis and protein determinations were performed as described earlier (9). Scatchard analyses were performed for saturation binding studies and LIGAND-program (Elsevier-BIO-SOFT, Cambridge, UK) was utilized in non-linear regression analysis. Mann-Whitney U-test was used in statistical analysis.
Results The effect of four days of alcohol intake on the number of rat pancreatic muscarinic receptors is shown in fig. 1. Although the difference between the two groups was statistically suggestive only (P = 0,09), it is of interest to notice that there was a most conspicuous decrease in the number of pancreatic muscarinic receptors in three alcoholic animals out of ten. In the alcoholic group, no correlation was found between the decrease in the number of muscarinic receptors and the amount of alcohol consumed. There were no differences in the receptor affinities for NMS between the alcohol-exposed and control animals, indicating that the function of the remaining muscarinic receptors in the alcoholic animals is normal.
Discussion The present study indicated that already a short-term alcohol consumption induces in some rats a dramatic decrease in the number of pancreatic muscarinic receptors. The lowest number of pancreatic muscarinic receptors in control animals was lO-fold higher than the lowest number 504
Exp Toxic Pathol 45 (1993/94) 8
in alcoholic animals. As we know that the decrease in the number of muscarinic receptors increases, by feedback mechanism, the cholinergic tone (10, 13, 14) and that excessive cholinergic tone (lO-fold higher than the tone causing maximum secretory response) affecting pancreatic acinar cells leads invariably to acute pancreatitis both in laboratory animals (1, 3, 6, 11) and in man (2, 5), it is fascinating to propose that those having the most dramatic decrease in the number of pancreatic muscarinic receptors after alcohol consumption are predisposed to an attack of acute alcoholic pancreatitis. In conclusion, the results of our present and former studies indicate that short-term alcohol consumption in some rats and long-term alcohol consumption in all rats decreases the number of pancreatic muscarinic receptors, which may predispose those with downregulated muscarinic receptors to acute alcoholic pancreatitis by increasing the cholinergic tone bombarding pancreatic acinar cells. The results are in accordance with the clinical finding that those with acute alcoholic pancreatitis after a short-term alcohol consumption without preceding chronic alcohol exposure are clearly a minority among patients suffering from acute alcoholic pancreatitis. Our results suggest that the pathogenetic mechanism triggering acute alcoholic pancreatitis would be similar to the mechanism triggering acute pancreatitis caused by the scorpion sting (2, 6), intoxication with an antiacetylcholinesterase-containing insecticides (5), or after excessive cholinergic stimulation (1, 3, 11). In future, it is essential to study the effects of alcohol consumption on human pancreatic muscarinic receptors and cholinergic tone and, thus, clarify their possible role in the pathogenesis of acute alcoholic pancreatitis. Acknowledgements: This study was supported by the Finnish Foundation for Alcohol studies.
References 1. ADLER G, GERHARDS G, SCHICK J, et al.: Effects of in vivo
2. 3.
4.
5.
6.
7.
cholinergic stimulation of rat exocrine pancreas. Am J Physiol 1983; 244: G624-G629. BARTHOLOMEW C: Acute scorpion pancreatitis in Trinidad. Br Med J 1970; 1: 666-668. BILCHIK AJ, ZUCKER KA, ADRIAN TE et al.: Amelioration of cholinergic-induced pancreatitis with a selective cholecystokinin receptor antagonist. Arch Surg 1990; 125: 1546-1549. CELENER P, DE LA PORTE P, TISCORNIA OM, et al.: Histochemical study of cholinergic activities in exocrine pancreas of dogs: modifications related to chronic alcoholism. Biomed (Express) 1977; 27: 161-165. DRESSEL TD, GOODALE RL, ARNESON MA, et al.: Pancreatitis as a complication of anticholinesterase insecticide intoxication. Ann Surg 1979; 189: 199-204. GALLAGHER S, HARIHARAN S, WILLIAMS JA: Mechanism of scorpion toxin-induced enzyme secretion in rat pancreas. Gastroenterology 1981; 80: 970-973. GRONROOS JM: Pathogenesis of acute alcoholic pancreatitis. Lancet 1990; 335: 1046.
8. GRONROOS JM, AHO HJ, NEVALAlNEN TJ: Cholinergic hypothesis of alcoholic pancreatitis. Dig Dis 1992; 10: 38-45. 9. GRONROOS JM, KAlLA T, AHO HJ, et al.: Decrease in the number of muscarinic receptors in rat pancreas after chronic alcohol intake. Pharmacol Toxicol1989; 64: 356-359. 10. KILBINGER H, NAFZIGER M: Two types of neuronal muscarine receptors modulating acetylcholine release from guinea-pig myenteric plexus. Naunyn Schmiedeberg's Arch PharmacoI1985;328:304-309. 11. LAMPEL M, KERN HF: Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue. Virchows Arch A 1977; 373: 97-117. 12. PEREc CJ, CELENER D, TISCORNJA OM, et al.: Effects of chronic ethanol administration on the autonomic innervation of salivary glands, pancreas and heart. Am J Gastroenterol1979; 72: 46-59.
13. SCHMIDT DN: Does ethanol exert its effect on the canine pancreas by mediation of cholinergic nerves? Scand J Gastroenterol1986; 126: 69-74. 14. SCHOEFFELMEER AN, VAN-VLIET BJ, WARDEH G, et al.: Muscarine receptor-mediated modulation of (3H)dopamine and (14C)acetylchbline release from rat neostriatal slices: selective antagonism by gallamine but not pirenzepine. Eur J Pharmacol1986; 128: 291-294. 15. SINGH M, SIMSEK H: Ethanol and the pancreas. Current status. Gastroenterology 1990; 98: 1051-1062. 16. TISCORNJA OM, DREILING DA: Physiopathogenetic hypothesis of alcoholic pancreatitis: Supranorrnal ecbolic stimulation of the "pancreon" units secondary to the loss of the negative component of pancreas innervation. Pancreas 1987; 2: 604-612. 17. WILSON JS, PiROLA RC: Pathogenesis of alcoholic pancreatitis. Aust N Z J Med 1983; 13: 307-310.
Exp Toxic Pathol 45 (1993/94) 8
505
Departments of Surgery, Pharmacology and Pathology, The University of Turku, Turku, Finland
Alcohol, pancreatic muscarinic receptors and acute pancreatitis JUHA M. GRONROOS, TIMO KArLA, and ANTII J. HIETARANTA With 1 figure Received: February 9,1993; Accepted: March 2, 1993
Address for correspondence: JUHA M. GRONROOS, M. D., Department of Surgery, The University Central Hospital of Turku, Kiinamyllynkatu 4-8, 20520 Turku, Finland .
Key words: Muscarinic receptors; Receptors, muscarinic; Pancreatitis, acute; Alcohol, pancreatitis; Cholinergic stimulation; Acetylcholine.
Summary Recently, a new theory about the pathogenesis of acute alcoholic pancreatitis was proposed. The aim of the present work was to further study the basis of this cholinergic theory about the pathogenesis of acute alcoholic pancreatitis. The results indicated that already a short-term alcohol consumption induces in some rats a dramatic decrease in the number of pancreatic muscarinic receptors. This decrease may predispose to acute alcoholic pancreatitis by increasing the cholinergic tone, since excessive cholinergic tone invariably leads to acute pancreatitis both in experimental animals and in man. Thus, the pathogenetic mechanism triggering acute alcoholic pancreatitis might be similar to the mechanism triggering acute pancreatitis caused by the scorpion sting, intoxication with an antiacetylcholine-esterase-containing insecticides or after excessive cholinergic stimulation.
riably to acute pancreatitis both in animals (1, 3, 6, 11) and in man (2, 5). Since the effects of short-term alcohol intake on pancreatic muscarinic receptors are unknown, the question still remains, how the theory accounts for those attacks of acute alcoholic pancreatitis after only a few hours or days of alcohol consumption without preceding chronic alcohol exposure. Although this group consisting mostly of young patients is clearly a minority among those suffering from acute alcoholic pancreatitis, we all meet these patients every now and then in our clinical practice. Accordingly, the aim of the present investigation was to characterize the effects of short-term alcohol intake on pancreatic muscarinic receptors in our rat model of alcoholism.
Introduction
Material and methods
The pathogenesis of acute alcoholic pancreatitis is still poorly understood. Research into the pathogenesis of acute alcoholic pancreatitis has progressed from a "big duct" to a "small duct" approach and finally, in recent times, to studies of the effects of alcohol on pancreatic acinar cells themselves (15-17) .
Twenty male Wistar rats were kept on a standard laboratory diet (Ewos R3, Ewos Ab, SOdertalje, Sweden) under controlled conditions (22 ± 1°C, 60 % humidity, lights on from 7.00 to J9.()(). At the age of two months the animals were randomly divided into two groups. Ten rats were offered alcohol only as 15 % (w/V) solution of ethanol in water for four days. Ten control animals were identical to the test animals in all respects but were offered water instead of alcohol. The animals were housed in individual cages and had free access to food. The daily fluid consumption per rat (x ± SD) was 18.5 ± 4.8 ml (range 11.3-23.8 ml) in alcoholic group and 36.6 ± 6.0 ml (range 27.5-46.2 ml) in control group. After the four days test period the animals were decapitated and the pancreas was removed, trimmed free of fat and weighed. The number and affinity of pancreatic muscarinic receptors were measured by using N-3H-methylscopolamine (NMS , 73.8 Ci/mmol), New England Nuclear) as a radioligand. Preparation of pancreatic plasma membranes, the time course
Recently, a new theory about the pathogenesis of acute alcoholic pancreatitis was proposed (7, 8). This theory is based on the studies indicating that chronic alcohol consumption decreases by 60 % the number of rat pancreatic muscarinic receptors (9) and, by feedback mechanism, increases manifoldly the ratio of pancreatic acetylcholine concentration/muscarinic receptor density (4, 7, 8, 9, 12). These changes may playa crucial part in the pathogenesis of acute alcoholic pancreatitis, since excessive cholinergic stimulation of pancreatic muscarinic receptors leads inva-
Exp Toxic Pathol 4S (1993/94) 8
503
fmol/m~
I)rO'. n
80
•
70
&0
•
50
•
• •
•
It
• • ••
10
•
I
•
A
c
Fig. 1. The maximal binding capacity to NMS of rat pancreatic muscarinic receptors/mg protein after four days of alcohol/water intake. A = alcoholic rats (n = 10); C = control rats (n
= 10).
studies of NMS binding, saturation analysis and protein determinations were performed as described earlier (9). Scatchard analyses were performed for saturation binding studies and LIGAND-program (Elsevier-BIO-SOFT, Cambridge, UK) was utilized in non-linear regression analysis. Mann-Whitney U-test was used in statistical analysis.
Results The effect of four days of alcohol intake on the number of rat pancreatic muscarinic receptors is shown in fig. 1. Although the difference between the two groups was statistically suggestive only (P = 0,09), it is of interest to notice that there was a most conspicuous decrease in the number of pancreatic muscarinic receptors in three alcoholic animals out of ten. In the alcoholic group, no correlation was found between the decrease in the number of muscarinic receptors and the amount of alcohol consumed. There were no differences in the receptor affinities for NMS between the alcohol-exposed and control animals, indicating that the function of the remaining muscarinic receptors in the alcoholic animals is normal.
Discussion The present study indicated that already a short-term alcohol consumption induces in some rats a dramatic decrease in the number of pancreatic muscarinic receptors. The lowest number of pancreatic muscarinic receptors in control animals was lO-fold higher than the lowest number 504
Exp Toxic Pathol 45 (1993/94) 8
in alcoholic animals. As we know that the decrease in the number of muscarinic receptors increases, by feedback mechanism, the cholinergic tone (10, 13, 14) and that excessive cholinergic tone (lO-fold higher than the tone causing maximum secretory response) affecting pancreatic acinar cells leads invariably to acute pancreatitis both in laboratory animals (1, 3, 6, 11) and in man (2, 5), it is fascinating to propose that those having the most dramatic decrease in the number of pancreatic muscarinic receptors after alcohol consumption are predisposed to an attack of acute alcoholic pancreatitis. In conclusion, the results of our present and former studies indicate that short-term alcohol consumption in some rats and long-term alcohol consumption in all rats decreases the number of pancreatic muscarinic receptors, which may predispose those with downregulated muscarinic receptors to acute alcoholic pancreatitis by increasing the cholinergic tone bombarding pancreatic acinar cells. The results are in accordance with the clinical finding that those with acute alcoholic pancreatitis after a short-term alcohol consumption without preceding chronic alcohol exposure are clearly a minority among patients suffering from acute alcoholic pancreatitis. Our results suggest that the pathogenetic mechanism triggering acute alcoholic pancreatitis would be similar to the mechanism triggering acute pancreatitis caused by the scorpion sting (2, 6), intoxication with an antiacetylcholinesterase-containing insecticides (5), or after excessive cholinergic stimulation (1, 3, 11). In future, it is essential to study the effects of alcohol consumption on human pancreatic muscarinic receptors and cholinergic tone and, thus, clarify their possible role in the pathogenesis of acute alcoholic pancreatitis. Acknowledgements: This study was supported by the Finnish Foundation for Alcohol studies.
References 1. ADLER G, GERHARDS G, SCHICK J, et al.: Effects of in vivo
2. 3.
4.
5.
6.
7.
cholinergic stimulation of rat exocrine pancreas. Am J Physiol 1983; 244: G624-G629. BARTHOLOMEW C: Acute scorpion pancreatitis in Trinidad. Br Med J 1970; 1: 666-668. BILCHIK AJ, ZUCKER KA, ADRIAN TE et al.: Amelioration of cholinergic-induced pancreatitis with a selective cholecystokinin receptor antagonist. Arch Surg 1990; 125: 1546-1549. CELENER P, DE LA PORTE P, TISCORNIA OM, et al.: Histochemical study of cholinergic activities in exocrine pancreas of dogs: modifications related to chronic alcoholism. Biomed (Express) 1977; 27: 161-165. DRESSEL TD, GOODALE RL, ARNESON MA, et al.: Pancreatitis as a complication of anticholinesterase insecticide intoxication. Ann Surg 1979; 189: 199-204. GALLAGHER S, HARIHARAN S, WILLIAMS JA: Mechanism of scorpion toxin-induced enzyme secretion in rat pancreas. Gastroenterology 1981; 80: 970-973. GRONROOS JM: Pathogenesis of acute alcoholic pancreatitis. Lancet 1990; 335: 1046.
8. GRONROOS JM, AHO HJ, NEVALAlNEN TJ: Cholinergic hypothesis of alcoholic pancreatitis. Dig Dis 1992; 10: 38-45. 9. GRONROOS JM, KAlLA T, AHO HJ, et al.: Decrease in the number of muscarinic receptors in rat pancreas after chronic alcohol intake. Pharmacol Toxicol1989; 64: 356-359. 10. KILBINGER H, NAFZIGER M: Two types of neuronal muscarine receptors modulating acetylcholine release from guinea-pig myenteric plexus. Naunyn Schmiedeberg's Arch PharmacoI1985;328:304-309. 11. LAMPEL M, KERN HF: Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue. Virchows Arch A 1977; 373: 97-117. 12. PEREc CJ, CELENER D, TISCORNJA OM, et al.: Effects of chronic ethanol administration on the autonomic innervation of salivary glands, pancreas and heart. Am J Gastroenterol1979; 72: 46-59.
13. SCHMIDT DN: Does ethanol exert its effect on the canine pancreas by mediation of cholinergic nerves? Scand J Gastroenterol1986; 126: 69-74. 14. SCHOEFFELMEER AN, VAN-VLIET BJ, WARDEH G, et al.: Muscarine receptor-mediated modulation of (3H)dopamine and (14C)acetylchbline release from rat neostriatal slices: selective antagonism by gallamine but not pirenzepine. Eur J Pharmacol1986; 128: 291-294. 15. SINGH M, SIMSEK H: Ethanol and the pancreas. Current status. Gastroenterology 1990; 98: 1051-1062. 16. TISCORNJA OM, DREILING DA: Physiopathogenetic hypothesis of alcoholic pancreatitis: Supranorrnal ecbolic stimulation of the "pancreon" units secondary to the loss of the negative component of pancreas innervation. Pancreas 1987; 2: 604-612. 17. WILSON JS, PiROLA RC: Pathogenesis of alcoholic pancreatitis. Aust N Z J Med 1983; 13: 307-310.
Exp Toxic Pathol 45 (1993/94) 8
505