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Relationship between anti-diarrheal activity and binding to calmodulin
European Journal of Pharmacology, 78 (1982) 375-377
375
Elsevier Biomedical Press
Short communication RELATIONSHIP BETWEEN A N T I - D I A R R H E A L A C T I V I T Y A N D B I N D I N G T O C A L M O D U L I N JAMES H. ZAVECZ *, THOMAS E. JACKSON, GERALD L. LIMP and TOBIAS O. YELLIN ** Biomedical Research Department, ICI Americas Inc., Wilmington, DE 19897, U.S.A.
Received I1 November 1981, accepted 18 January 1982
J.H. ZAVECZ, T.E. JACKSON, G.L. LIMP and T.O. YELLIN, Relationship between anti-diarrheal activity and binding to calmodulin, European J. Pharmacol. 78 (1982) 375-377. Several neuroleptics known to bind to calmodulin were tested for anti-diarrheal activity and were compared with the opiate anti-diarrheals loperamide and diphenoxylate. All inhibited the intestinal fluid secretion induced by 16,16-dimethylprostaglandin E 2 and castor oil-induced diarrhea in rats as a function of dose, the order of potency being loperamide ~ diphenoxylate> chlorpromazine > promethazine> amitriptyline. The opiates loperamide and diphenoxylate were found to compete with [ 3H]trifluoperazine binding to calmodulin in the presence of calcium. These opiates were approximately 3 times more potent inhibitors of [3H]trifluoperazine binding than chlorpromazine. A positive correlation between calmodulin binding and anti-diarrheal activity was demonstrated. Calmodulin
Diarrhea
Loperamide
Diphenoxylate
Chlorpromazine
I. Introduction
2. Materials and methods
Chlorpromazine has proven to be effective in preventing intestinal fluid secretion induced by cholera toxin, E. coli enterotoxin, prostaglandin El, and dibutyryl cyclic A M P in experimental animals (Holmgren et al., 1978). It has also proven to be beneficial in reducing fluid loss in cholera patients (Rabbani et al., 1979). Since the binding of chlorpromazine to calmodulin has been proposed as a mechanism by which it m a y exert some of its pharmacologic actions (Levin and Weiss, 1979), we investigated the effect of several agents k n o w n to bind to calmodulin for intestinal antisecretory arid anti-diarrheal action. In addition we investigated whether the highly effective opiate anti-diarrheal drugs loperamide and diphenoxylate interact with calmodulin.
Intestinal fluid secretion which leads to diarrhea was induced by 16,16-dimethyl prostaglandin E 2 (diMe P G E 2 ) and measured by the enteropooling method of Robert et al. (1976). Drugs were administered by gavage to fasted female Wistar rats (170-210 g) 1 h prior to giving 5 0 / ~ g / k g diMe P G E 2 by gavage. Thirty rain later the entire small intestine was removed and the volume contained therein measured. For determining efficacy against castor oilinduced diarrhea, drugs were administered 1 h prior to 1 ml oil also by gavage. The animals were observed for the appearance of diarrhea over the next 6 h. Displacement of Ca 2+-dependent (0.1 raM) [3H]trifluoperazine binding from bovine brain calmodulin by loperamide, diphenoxylate and chlorpromazine was performed in a single blind assay as previously described (Levin and Weiss, 1979) in the laboratory by Dr. Benjamin Weiss. Chlorpromazine was purchased from Sigma Chemical Co., St. Louis, MO. Loperamide, diphenoxylate, amitriptyline, and promethazine were gifts from Ortho Pharmaceuticals Corp., Raritan,
* Present address: Dept. of Pharmacology, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, VA 23501, U.S.A. To whom all correspondence should be addressed. ** Present address: Beckman Instruments, Inc., Bioproducts Operations, 1117 California Avenue, Palo Alto, CA 94304, U.S.A. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
376
N J; Searle and Co., San Juan, Puerto Rico; Merck, Sharp and D o h m e , Rahway, N J; and Wyeth Laboratories, Philadelphia, PA, respectively. D i M e P G E 2 was kindly supplied by Dr. Peter Marsham, I C I Ltd., Macclesfield, England.
3. Results
Chlorpromazine, promethazine and amitriptyline inhibited diMe P G E 2-induced fluid accumulation in the small intestine of the rat in a dosedependent manner, and were between 10 and 70 times less potent than the opiate anti-diarrheals loperamide and diphenoxylate (table 1). The effectiveness of these drugs against diarrhea induced by castor oil was of the same rank order as their effect on fluid secretion, i.e., loperamide diphenoxylate > chlorpromazine > promethazine > amitriptyline. Loperamide and diphenoxylate appeared to have greater affinity for calmodulin, since their ICs0 concentrations for displacing [3H]trifluoperazine were approximately 3 times lower than chlorpromazine (table 1). A plot of the IDs0 (the dose which p r o d u c e d 50% inhibition of the maxim u m secretory response to diMe P G E 2 ) against
TABLE 1 I n h i b i t o r y c o n c e n t r a t i o n s of various agents on i n t e s t i n a l fluid a c c u m u l a t i o n and C a 2 + - d e p e n d e n t [3 H ] t r i f l u o p e r a z i n e b i n d i n g to c a l m o d u l i n .
Drug
IDs0 (/~mol/kg) * (n=5-10)
ICs0 (~M) ** (n=3)
Loperamide Diphenoxylate Chlorpromazine Promethazine A m i trip tyline
1.2 2.0 18 70 140
12 10 30 60 *** 100 * * *
* Dose which inhibits maximal diMe PGEi-induced fluid accumulation by 50%. Values were obtained from doseresponse curves. ** Concentration of drug required 50% of [3H]trifluoperazine bound to calmodulin in the presence of calcium (0.1 mM). Values were obtained from dose-response curves. *** From Levin and Weiss (1979).
140 ~O E
120 100
a Z
o_ k-
ao
~
6o
0 u < a
40 CPZ
20 0
DP 10
20
30
I 40
I 50
I 60
t 70
I SO
I 90
I 100
CALMODULIN I C 5 0 ( # M )
Fig. 1. Relationship between the IDs0 (/~mol/kg) for the inhibition of 16,16-diMe PGE2-induced fluid accumulation and the IDso concentration (/~M) required to inhibit Ca2+-dependent [3H]trifluoperazine binding to calmodulin. The IDs0 and the ICs0 values were obtained from the dose-response curve for each drug and are the mean of 5-10 experiments for liquid accumulation and the mean of 3 experiments for binding to calmodulin. The ICs0 values for calmodulin binding of promethazine and amitriptyline are from Levin and Weiss (1979). The line was drawn by least squares regression analysis. DIP: diphenoxylate; LOP: loperamide; CPZ: chlorpromazine: PRO: promethazine; AMI: amitriptyline.
the IC50 for displacing [3H]trifluoperazine from calmodulin (fig. 1) demonstrates that a positive correlation (r = 0.995, P < 0.0025) exists between the capacity of the drugs to displace [3H]trifluoperazine from calmodulin and their ability to inhibit secretion of fluid into the rat small intestine.
4. D i s c u s s i o n Speculation that chlorpromazine and other neuroleptics m a y act as antidiarrheal agents by interfering with intestinal calmodulin was stimulated by the close parallel between the efficacy of neuroleptics and their affinity for calmodulin (Levin and Weiss, 1979) and the fact that trifluoperazine and several other related drugs which bind to calmodulin inhibit the C l - secretion associated with the secretory diarrhea induced by a n u m b e r of agents such as cholera toxin, prostaglandins and vasoactive intestinal polypeptide (Ilundain and Naftalin, 1979; Sandhu et al., 1979; Smith and
377 Field, 1980). Since it is highly probable that calcium is involved in the regulation of intestinal adsorption and secretion (Berridge, 1979), a cause effect relationship between Ca 2+-dependent binding of these drugs to calmodulin and their antisecretory properties seemed possible. The results presented here demonstrate that chlorpromazine, promethazine and amitriptyline, all of which bind to calmodulin, have some effectiveness against diarrhea, and that the opiate anti-diarrheal agents loperamide and diphenoxylate displace Ca2+-dependent [3H]trifluoperazine binding from calmodulin. The affinity of these drugs for calmodulin binding sites correlated positively with their ability to inhibit intestinal fluid secretion (fig. 1), which is known to lead to diarrhea (Robert et al., 1976). These data suggest a relationship between diarrhea and calmodulin, since the anti-diarrheal effect of loperamide, diphenoxylate, and certain neuroleptics parallel their affinity for calmodulin. C a u t i o n m u s t be observed, however, since morphine, an effective opiate anti-diarrheal agent, does not appear to bind to calmodulin (Levin and Weiss, 1979).
Acknowledgment Our thanks to Dr. Benjamin Weiss for performing the calmodulin binding assays.
References Berridge, M.J., 1979, Relationship between calcium and the cyclic nucleotides in ion secretion, in: Mechanisms of Intestinal Secretion, Kroc Foundation Series, Vol. 12, ed. H.J. Binder (Alan R. Liss, Inc., New York) p. 65. Holmgren, J., S. Lange and I. L6nnroth, 1978, Reversal of cyclic AMP-mediated intestinal secretion in mice by chlorpromazine, Gastroenterology 75, 1103. lllundain, A. and R.J. Naftalin, 1979, Role of Ca2+-dependent regulator protein in intestinal secretion, Nature 279, 446. Levin, R.M. and B. Weiss, 1979, Selective binding of antipsychotics and other psychoactive agents to the calciumdependent activator of cyclic nucleotide phosphodiesterase, J. Pharmacol. Exp. Ther. 208, 454. Rabbani, G.I., W.B. Grennough, J. Holmgren and I. L6nnroth, 1979, Chlorpromazine reduces fluid-loss in cholera, Lancet 1,410. Robert, A., J.E. Nezamis, C. Lancaster, A.J. Hanchar and M.S. Klepper, 1976, Enterpooling assay: a test for diarrhea produced by prostaglandins, Prostaglandins 11,809. Sandhu, B., J.H. Tripp, D.C.A. Candy and J.T. Harries, 1979, Loperamide inhibits cholera-toxin-induced small intestinal secretion, Lancet 2, 689. Smith, P.L. and M. Field, 1980, In vitro antisecretory effects of trifluoperazine and other neuroleptics in rabbit and human small intestine, Gastroenterology 78, 1545.
375
Elsevier Biomedical Press
Short communication RELATIONSHIP BETWEEN A N T I - D I A R R H E A L A C T I V I T Y A N D B I N D I N G T O C A L M O D U L I N JAMES H. ZAVECZ *, THOMAS E. JACKSON, GERALD L. LIMP and TOBIAS O. YELLIN ** Biomedical Research Department, ICI Americas Inc., Wilmington, DE 19897, U.S.A.
Received I1 November 1981, accepted 18 January 1982
J.H. ZAVECZ, T.E. JACKSON, G.L. LIMP and T.O. YELLIN, Relationship between anti-diarrheal activity and binding to calmodulin, European J. Pharmacol. 78 (1982) 375-377. Several neuroleptics known to bind to calmodulin were tested for anti-diarrheal activity and were compared with the opiate anti-diarrheals loperamide and diphenoxylate. All inhibited the intestinal fluid secretion induced by 16,16-dimethylprostaglandin E 2 and castor oil-induced diarrhea in rats as a function of dose, the order of potency being loperamide ~ diphenoxylate> chlorpromazine > promethazine> amitriptyline. The opiates loperamide and diphenoxylate were found to compete with [ 3H]trifluoperazine binding to calmodulin in the presence of calcium. These opiates were approximately 3 times more potent inhibitors of [3H]trifluoperazine binding than chlorpromazine. A positive correlation between calmodulin binding and anti-diarrheal activity was demonstrated. Calmodulin
Diarrhea
Loperamide
Diphenoxylate
Chlorpromazine
I. Introduction
2. Materials and methods
Chlorpromazine has proven to be effective in preventing intestinal fluid secretion induced by cholera toxin, E. coli enterotoxin, prostaglandin El, and dibutyryl cyclic A M P in experimental animals (Holmgren et al., 1978). It has also proven to be beneficial in reducing fluid loss in cholera patients (Rabbani et al., 1979). Since the binding of chlorpromazine to calmodulin has been proposed as a mechanism by which it m a y exert some of its pharmacologic actions (Levin and Weiss, 1979), we investigated the effect of several agents k n o w n to bind to calmodulin for intestinal antisecretory arid anti-diarrheal action. In addition we investigated whether the highly effective opiate anti-diarrheal drugs loperamide and diphenoxylate interact with calmodulin.
Intestinal fluid secretion which leads to diarrhea was induced by 16,16-dimethyl prostaglandin E 2 (diMe P G E 2 ) and measured by the enteropooling method of Robert et al. (1976). Drugs were administered by gavage to fasted female Wistar rats (170-210 g) 1 h prior to giving 5 0 / ~ g / k g diMe P G E 2 by gavage. Thirty rain later the entire small intestine was removed and the volume contained therein measured. For determining efficacy against castor oilinduced diarrhea, drugs were administered 1 h prior to 1 ml oil also by gavage. The animals were observed for the appearance of diarrhea over the next 6 h. Displacement of Ca 2+-dependent (0.1 raM) [3H]trifluoperazine binding from bovine brain calmodulin by loperamide, diphenoxylate and chlorpromazine was performed in a single blind assay as previously described (Levin and Weiss, 1979) in the laboratory by Dr. Benjamin Weiss. Chlorpromazine was purchased from Sigma Chemical Co., St. Louis, MO. Loperamide, diphenoxylate, amitriptyline, and promethazine were gifts from Ortho Pharmaceuticals Corp., Raritan,
* Present address: Dept. of Pharmacology, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, VA 23501, U.S.A. To whom all correspondence should be addressed. ** Present address: Beckman Instruments, Inc., Bioproducts Operations, 1117 California Avenue, Palo Alto, CA 94304, U.S.A. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
376
N J; Searle and Co., San Juan, Puerto Rico; Merck, Sharp and D o h m e , Rahway, N J; and Wyeth Laboratories, Philadelphia, PA, respectively. D i M e P G E 2 was kindly supplied by Dr. Peter Marsham, I C I Ltd., Macclesfield, England.
3. Results
Chlorpromazine, promethazine and amitriptyline inhibited diMe P G E 2-induced fluid accumulation in the small intestine of the rat in a dosedependent manner, and were between 10 and 70 times less potent than the opiate anti-diarrheals loperamide and diphenoxylate (table 1). The effectiveness of these drugs against diarrhea induced by castor oil was of the same rank order as their effect on fluid secretion, i.e., loperamide diphenoxylate > chlorpromazine > promethazine > amitriptyline. Loperamide and diphenoxylate appeared to have greater affinity for calmodulin, since their ICs0 concentrations for displacing [3H]trifluoperazine were approximately 3 times lower than chlorpromazine (table 1). A plot of the IDs0 (the dose which p r o d u c e d 50% inhibition of the maxim u m secretory response to diMe P G E 2 ) against
TABLE 1 I n h i b i t o r y c o n c e n t r a t i o n s of various agents on i n t e s t i n a l fluid a c c u m u l a t i o n and C a 2 + - d e p e n d e n t [3 H ] t r i f l u o p e r a z i n e b i n d i n g to c a l m o d u l i n .
Drug
IDs0 (/~mol/kg) * (n=5-10)
ICs0 (~M) ** (n=3)
Loperamide Diphenoxylate Chlorpromazine Promethazine A m i trip tyline
1.2 2.0 18 70 140
12 10 30 60 *** 100 * * *
* Dose which inhibits maximal diMe PGEi-induced fluid accumulation by 50%. Values were obtained from doseresponse curves. ** Concentration of drug required 50% of [3H]trifluoperazine bound to calmodulin in the presence of calcium (0.1 mM). Values were obtained from dose-response curves. *** From Levin and Weiss (1979).
140 ~O E
120 100
a Z
o_ k-
ao
~
6o
0 u < a
40 CPZ
20 0
DP 10
20
30
I 40
I 50
I 60
t 70
I SO
I 90
I 100
CALMODULIN I C 5 0 ( # M )
Fig. 1. Relationship between the IDs0 (/~mol/kg) for the inhibition of 16,16-diMe PGE2-induced fluid accumulation and the IDso concentration (/~M) required to inhibit Ca2+-dependent [3H]trifluoperazine binding to calmodulin. The IDs0 and the ICs0 values were obtained from the dose-response curve for each drug and are the mean of 5-10 experiments for liquid accumulation and the mean of 3 experiments for binding to calmodulin. The ICs0 values for calmodulin binding of promethazine and amitriptyline are from Levin and Weiss (1979). The line was drawn by least squares regression analysis. DIP: diphenoxylate; LOP: loperamide; CPZ: chlorpromazine: PRO: promethazine; AMI: amitriptyline.
the IC50 for displacing [3H]trifluoperazine from calmodulin (fig. 1) demonstrates that a positive correlation (r = 0.995, P < 0.0025) exists between the capacity of the drugs to displace [3H]trifluoperazine from calmodulin and their ability to inhibit secretion of fluid into the rat small intestine.
4. D i s c u s s i o n Speculation that chlorpromazine and other neuroleptics m a y act as antidiarrheal agents by interfering with intestinal calmodulin was stimulated by the close parallel between the efficacy of neuroleptics and their affinity for calmodulin (Levin and Weiss, 1979) and the fact that trifluoperazine and several other related drugs which bind to calmodulin inhibit the C l - secretion associated with the secretory diarrhea induced by a n u m b e r of agents such as cholera toxin, prostaglandins and vasoactive intestinal polypeptide (Ilundain and Naftalin, 1979; Sandhu et al., 1979; Smith and
377 Field, 1980). Since it is highly probable that calcium is involved in the regulation of intestinal adsorption and secretion (Berridge, 1979), a cause effect relationship between Ca 2+-dependent binding of these drugs to calmodulin and their antisecretory properties seemed possible. The results presented here demonstrate that chlorpromazine, promethazine and amitriptyline, all of which bind to calmodulin, have some effectiveness against diarrhea, and that the opiate anti-diarrheal agents loperamide and diphenoxylate displace Ca2+-dependent [3H]trifluoperazine binding from calmodulin. The affinity of these drugs for calmodulin binding sites correlated positively with their ability to inhibit intestinal fluid secretion (fig. 1), which is known to lead to diarrhea (Robert et al., 1976). These data suggest a relationship between diarrhea and calmodulin, since the anti-diarrheal effect of loperamide, diphenoxylate, and certain neuroleptics parallel their affinity for calmodulin. C a u t i o n m u s t be observed, however, since morphine, an effective opiate anti-diarrheal agent, does not appear to bind to calmodulin (Levin and Weiss, 1979).
Acknowledgment Our thanks to Dr. Benjamin Weiss for performing the calmodulin binding assays.
References Berridge, M.J., 1979, Relationship between calcium and the cyclic nucleotides in ion secretion, in: Mechanisms of Intestinal Secretion, Kroc Foundation Series, Vol. 12, ed. H.J. Binder (Alan R. Liss, Inc., New York) p. 65. Holmgren, J., S. Lange and I. L6nnroth, 1978, Reversal of cyclic AMP-mediated intestinal secretion in mice by chlorpromazine, Gastroenterology 75, 1103. lllundain, A. and R.J. Naftalin, 1979, Role of Ca2+-dependent regulator protein in intestinal secretion, Nature 279, 446. Levin, R.M. and B. Weiss, 1979, Selective binding of antipsychotics and other psychoactive agents to the calciumdependent activator of cyclic nucleotide phosphodiesterase, J. Pharmacol. Exp. Ther. 208, 454. Rabbani, G.I., W.B. Grennough, J. Holmgren and I. L6nnroth, 1979, Chlorpromazine reduces fluid-loss in cholera, Lancet 1,410. Robert, A., J.E. Nezamis, C. Lancaster, A.J. Hanchar and M.S. Klepper, 1976, Enterpooling assay: a test for diarrhea produced by prostaglandins, Prostaglandins 11,809. Sandhu, B., J.H. Tripp, D.C.A. Candy and J.T. Harries, 1979, Loperamide inhibits cholera-toxin-induced small intestinal secretion, Lancet 2, 689. Smith, P.L. and M. Field, 1980, In vitro antisecretory effects of trifluoperazine and other neuroleptics in rabbit and human small intestine, Gastroenterology 78, 1545.