Naunyn-Schmiedeberg's
Archivesof
Naunyn-Schmiedeberg's Arch. Pharmacol. 301,195-199 (1978)
Pharmacology 9 by Springer-Verlag1978
Effects of PGE2 and Colchicine on the Intestinal Fluid Volume E. BEUBLER, F. LEMBECK, and M. SCHWEDITSCH
Institut fiir experimentelle und klinische Pharmakologie der Universitfit Graz, Universitfitsplatz4, A-8010 Graz, Austria
Summary. a) Colchicine, prostaglandin E2 (PGE2) and carbachol increase the intestinal fluid volume in the rat. b) The effect of carbachol and prostaglandin E2 is augmented whereas that of colchicine is diminished in the pithed rat. c) The effect of colchicine is diminished in anaesthesia whereas that of PGEz remains unchanged. d) The effect of PGE2 and of colchicine are both inhibited by atropine. e) The effects of PGE2 and o f colchicine are both enhanced by phentolamine. f) Pretreatment with indomethacin decreased the effect of colchicine whereas that of PGEz remained unchanged. g) The PGEz-induced increase in intestinal fluid volume m a y partly be mediated by a peripheral mechanism involving acetylcholine receptors. h) The increase in intestinal fluid volume by colchicine can mainly be explained by a central action of the drug which is mediated by cholinergic neurones.
conditions (rheumatoid synovial tissue: Robinson et al., 1975; urate crystal arthritis: Glatt et al., 1976; isolated perfused ear: Seewann et al., 1977) it seemed likely that the colchicine induced diarrhoea is caused by stimulation of intestinal PG-synthesis. Ferguson (1952) obtained experimental evidence for the involvement of the central nervous system in the gastrointestinal effects of colchicine. We have therefore examined the possible influence of anaesthesia (pentobarbital, urethane), of despinalisation and of drugs affecting the autonomic nervous system (atropine, phentolamine, reserpine) on colchicine induced diarrhoea in order to elucidate the mechanism of action of colchicine. Since only little is known about the mode of action of PGs on water secretion into the gut experiments were also performed with PGE2 in order to obtain some information on the way in which PGs act on intestinal water transport.
Key words." Intestinal fluid volume PGE2 - Indomethacin.
METHODS
Colchicine -
Animals. Female Sprague Dawley rats (breeder: Mus-Rattus,
Brunnthal), (200 + 20 g) were used. They were deprived of food for 24 h prior to the experiments. INTRODUCTION Colchicine is used in the treatment of acute attacks of gout. Its therapeutical application is severely limited by characteristic side effects like diarrhoea and abdominal pain. The mechanism of these side effects is still unknown. Since prostaglandins (PGs) also produce diarrhoea and since colchicine stimulates the synthesis of prostaglandins under certain experimental Send offprint requests to E. Beubler at the above address Preliminary reports: E. Beubler and M. Schweditsch: NaunynSchmiedeberg's Arch. Pharmacol. 297, R56 (1977) and F. Atzlinger and F. Lembeck: Naunyn-Schmiedeberg's Arch. Pharmacol. 293, R34 (1976)
Procedures. The intestinal fluid volume (IFV) was measured by the "enteropooling assay" of Robert (1976). Twenty min after the i.p. injection of PG or carbachol or 75 min after colchicine the animals were killed by a blow on the head, the entire small intestine was removed and its content squeezed into a calibrated test tube. IFV was expressed in ml/100 g rat. The pithed rats were prepared according to the method of Shipley and Tilden (1959). After an interval of 20 rain colchicine, PG or carbachol was injected intraperitoneally. Substances. Atropine (Merck), carbachol (Fluka), colchicine (Merck), indomethacin (Merck, Sharp & Dohme), pentobarbital (Abbott-Lab.), phentolamine (Ciba Geigy SA), propranolol (I.C.I.), prostaglandin E2 (Upjohn Co.), reserpine (Ciba Geigy SA), urethane (Merck). Statistics. The experimental data were evaluated by an unpaired t-test.
0028-1298/78/0301/0195/$ 01.00
196
Naunyn-Schmiedeberg'sArch. Pharmacol. 301 (1978)
0.8-
08t
0.6 0.~ r2~
g o
E ~- 0.4
Z
E ~'0.4
/
0.2
~02
--O-- cotcbicine,3mg/kg 9 control Fig. 1.
untreated
--#X--- PGE2,202Jg/kg
Time-course of the increase of intestinal fluid volume (IFV)
after i.p. injection of colchicine (3 mg/kg) or PGE2 (20 gg/kg) in the rat (Y 4- s~, n = 6)
[]
pentobarb, urethane
despina[.
control M colchicine,10 mg/kg [ ] PGE2, 20,ug/kg
Fig. 3. Influence of pentobarbital (50 gg/kg), urethane (1.25 g/kg) and despinalising on the increase in IFV induced by colchicine (J0 mg/kg) and PGE2 (20 gg/kg) (~ 4- s~, n = 6-- 10)
the administration of PGEz the IFV was back to the basal value (Fig. 1). The influence of drugs on the increase in IFV caused by PGE2 or colchicine was in each case assessed at the time of the maximal effect, i. e. 20 min after PGEz and 75 rain after colchicine injection.
1.0-
0,8
g
o K 0.6
B. Effect of PGEz~ on IFV
"d "5
PGF2~ at a dose of 20 gg/kg did not affect the IFV. The increase in IFV caused by 150 gg/kg PGF2~ amounted to only about 25 % of that seen after injecting 20 lag/kg PGE2.
-~ 0,4
0.2
C. Lffect of Carbachol on IFV 0
untreated
[ ] control [ ]
despinal.
carbachot, 25,ug/kg
Carbachol (25 gg/kg i.p., 20 rain before death) increased the IFV by about 140% (p < 0.001). In the pithed rat the effect of carbachol was much larger (p < 0.001) than in the intact, conscious rat (Fig. 2).
Fig. 2. Effect of carbachol (25 gg/kg i. p.) on IFV in untreated and pithed rats (2 4- s~, n = 6)
D. Effect of Drug or Despinalisation on the Changes in IFV Caused by Colchicine or PGs RESULTS
A. Effect of Colchicine and Prostaglandin E2 on Intestinal Fluid Volume Both colchicine (3 mg/kg i.p.) and PGE2 (20 gg/kg i.p.) increased the IFV. The onset of action of colchicine was slow, the maximal effect was observed after 75 min (Fig. 1). During the following hours the effect declined slowly: Six hours after the administration the IFV was still increased. The maximal effect of PGE2 was reached already after 20 min. One hour after
a) Anaesthetic Drugs. Pentobarbital (50 gg/kg i.p.) and urethane (1.25 g/kg i.p.) anaesthesia did not influence the basal IFV (p > 0.1). Both drugs decreased significantly (p < 0.001) the effect of colchicine (10 rag/ kg), whereas the effect of PGE2 (20 gg/kg) on the IFV remained unchanged (Fig. 3). b) Despinalisation. Destruction of the brain and the spinal cord did not influence (p > 0.1) the basal IFV. In the pithed rat the effect of colchicine was significantly reduced (p < 0.001) whereas that of PGE2 was significantly (p < 0.001) increased (Fig. 3).
197
E. Beubler et al. IntestinalFluid Volume: Colchicineand PGE2
e) Phentolamine. Pretreatment with phentolamine (1 mg/kg i.p., 20 rain before the experiment did not increase the basal IFV (p > 0.05). The rise in IFV caused by colchicine (p < 0.05) as well as that caused by PGE2 (p < 0.025) were slightly enhanced (Fig. 5). g
f) Reserpine. Pretreatment with reserpine (2.5 mg/kg
o
and day s.c. starting 2 days prior to the experiment) increased basal IFV (p < 0.005). The effect of colchicine was slightly inhibited (p < 0.05), that of PGE2 slightly enhanced (p < 0.02). Additional pretreatment with atropine abolished the effect of reserpine on basal IFV and the increase of the effect of PGE2 (p < 0.001), but did not further reduce the effect of colchicine (Fig. 5).
5
untreated []
Fig.4.
control [ ]
atropine
despinal,
co[chicine,10mg/kg
[]
indometh,
PGE2,20JJg/kg
Influence of atropine (1 mg/kg) in the conscious and the
pithed rat (control see Fig.3) and of indomethacin(4 mg/kg and day, s. c., startingtwo daysprior to the experiment)on the increasein IFV inducedby colchicine(10 mg/kg) and PGEz (20 p.g/kg)(ff _+s~, n = 6) 08-
g
o K
~- o~
0
untreated []
Fig. 5.
control [ ]
phentol
propr,
colchieine,10 mg/kg
reserpine []
reserpine +atropine
PGE2,20~ug/kg
Influence of phentolamine (1 mg/kg), propranolol (1 rag/
kg), reserpine (2 x 2.5 mg/kg) and reserpine plus atropine (1 nag/ kg) on basal IFV and on the increase in IFV inducedby colchicine (10 mg/kg) and PGE2 (20 gg/kg) (~ 4- s~, n = 6-10)
c) Atropine. Atropine (1 mg/kg i.p.) did not change the basal IFV. When given 10 rain prior to the injection of PGE2 or colchicine it reduced the effect of both compounds significantly (p < 0.001). Atropine also reduced the augmented effect of PGE2 in the pithed rat (Fig. 4). d) Indomethaein. Inhibition of PG synthesis by pretreatment with indomethacin (4 mg/kg and day s.c., starting 2 days prior to the experiment) had no effect on basal IFV or on the increase in IFV caused by PGE2. However it did reduce the rise in IFV after colchicine by more than 50 ~ (p < 0.001 ; Fig. 4).
DISCUSSION
A. Mode of Action of Colchicine Colchicine provides relief in acute attacks of gout and is also used as a prophylactic agent against such attacks. However, gastrointestinal side effects like diarrhoea and abdominal pain make prolonged administration often impracticable. Colchicine has a half life time of 24 rain in human blood plasma (Wallace et al., 1970). Its distribution volume is much larger than the extracellular fluid volume which indicates a rapid entrance into cells. Colchicine is very slowly excreted. Only about 10 % of a given dose appear in urine and faeces within 48 h (Walaszek et al., 1960). The intestinal effects of colchicine were attributed to its antimitotic activity. Colchicine decreased the absorption of xylose in conscious rats (Levin, 1966), caused severe morphological and cytochemical damage in the duodenal epithelium of the mouse (Raymackers and Hugon, 1973) and subcellular necrosis within the rapidly proliferating cells in the rat duodenal mucosa (Dinsdale, 1975). Ferguson (1952) observed that vomiting and diarrhoea induced by colchicine in cats could be inhibited by atropine and also by barbiturate anaesthesia. Since colchicine is inactive on the isolated intestine, Ferguson suggested a neurogenic basis for its action on the gut. Our observations on the inhibition of the colchicine induced increase in IFV by anaesthesia and by atropine confirm the results of Ferguson. The fact that in the pithed rat the effect of colchicine on IFV is decreased provides additional evidence for the participation of the central nervous system in the intestinal action of the drug. As this action is also reduced by atropine, cholinergic neurones must be involved. The effect of colchicine was slightly enhanced by blocking ~-adrenoceptors with phentolamine. This
198 enhancement might be due to changed hemodynamics after phentolamine treatment, or a mechanism involving ~-adrenoceptors inhibits the action of colchicine in the untreated rat. The decrease of the effect of colchicine in reserpine treated rats with or without pretreatment with atropine remains unexplained. The mechanism which mediates the reserpine induced sedation may involve similar central nervous structures as anaesthetic drugs do. Some of the intestinal effects of colchicine could be explained by an increased formation of prostaglandins. Most nonsteroidal antiinflammatory drugs inhibit the synthesis of PGs (Vane, 1971; Smith and Willis, 1971 ; Fen'eira et al., 1971). In contrast, colchicine, in spite of its slight antiinflammatory actions (Fitzgerald et al., 1971) increases the formation of PGs in cultured rheumatoid synovial cells (Robinson et al., 1973, 1975) and in fibroblast cultures (Robinson et al., 1974). Increased concentrations of PGs were also found in the synovial fluid of inflamed joints of chickens during colchicine treatment (Glatt et al., 1976). A stimulation of PG synthesis by colchicine was observed in bull seminal vesicles (Collier et al., 1976) and in the perfused rabbit ear (Seewann et al., 1977). PGs are known to be synthesized in the gut under physiological conditions (Beubler and Juan, 1977) and in response to various stimuli (Collier, 1974; Ferreira et al., 1976). Administration of PGs causes accumulation of water and electrolytes in the lumen of the small intestine, abdominal pain and diarrhoea (Horton et al., 1968; Misiewicz et al., 1969; Pierce et al., 1971; Matuchansky and Bernier, 1973; Milton-Thompson et al., 1975; Beubler and Juan, 1977). Indomethacin, a drug which inhibits PG-synthesis (Ferreira et al., 1971; Smith and Willis, 1971; Vane, 1971) did not reduce the basal IFV in the present experiments. This is not necessarily in conflict with previous observations on the perfused jejunal loop (Beubler and Juan, 1977) in which indomethacin increased water absorption, since the perfusion method can detect much smaller variations in fluid volume than the "enteropooling assay". The observation that the colchicine induced increase in IFV is reduced in indomethacin pretreated rats indicates that colchicine diarrhoea may partly be caused by an increased PG-synthesis in the gut. B. Mode of Action of PGE2 Drugs with acetylcholine like actions as betanechol (Tidball, 1961) or carbachol increase IFV. The effect of carbachol is about 50 ~ larger in pithed rats than in intact, conscious rats. This may be due to the loss of sympathetic activity after despinalisation. The effect of PGEz was much larger than that of PGF2~, which
Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1978)
is in agreement with the results of Robert (1976) and Beubler and Juan (1977). In preliminary experiments we observed a larger effect of PGE2 in female than in male rats. Therefore only females were used in the present experiments. No explanation for this sex differences is so far available. The effect of PGE2 on IFV was reduced by pretreatment with atropine. Despinalisation increased the effect of PGEz on IFV and also that of carbachol. These observations suggest that a peripheral mechanism involving acetylcholine receptors participates in the intestinal action of PGEz. Pentobarbital or urethane anaesthesia did not influence PGE2 increase in IFV which indicates that PGs act on a peripheral site. After pretreatment with phentolamine and propranolol the basal IFV remained unchanged. Phentolamine and despinalisation increased the effect of PGE2 which could be explained by a loss of sympathetic activity which normally inhibits the effect of PGE2 on the IFV. An effect of catecholamines on intestinal ion absorption was reported by Field and McColl (1973) who found that catecholamines increase the absorption of sodium and of chloride. PGs inhibit the absorption of sodium from the intestine and increase chloride and water secretion (Pierce et al., 1971 ; Matuchansky and Bernier, 1973). Pretreatment with reserpine increased basal IFV and enhanced the effect of PGE2. The increase in basal IFV in the reserpine treated rat cannot be ascribed to the loss of catecholamines since phentolamine did not change basal IFV. The effect of reserpine could be inhibited by pretreatment with atropine which indicates the involvement of acetylcholine receptors. Choleratoxin is known to stimulate intestinal adenylate cyclase (Kimberg et al., 1971 ; Schafer et al., 1970). The resulting increase in cyclic 3',5'-AMP enhances intestinal blood flow, water absorption and secretion. These effects were also obtained by inhibition of phosphodiesterase with theophylline (Pierce et al., 1971; Beubler and Lembeck, 1976). PGE2 stimulates intestinal adenylate cyclase (Kimberg et al., 1971 ; /974). The rise in IFV after PGE2 c o u l d only be reduced but not fully inhibited by blocking acetylcholine receptors with atropine. The residual effect might be due to the stimulating action of PGE2 on intestinal adenylate cyclase. Acknowledgements. The authors wish to thank Dr. J. Pike for the generous gift of prostaglandius. The investigation was supported by grant No. 2574 from the Fonds zur F6rderung der wissenschaftlichen Forschung ~sterreichs and by the Osterreichische Krebsgesellschaft.
REFERENCES Beubler, E., Juan, H. : The function of prostaglandins in transmucosal water movement and blood flow in the rat jejunum. Naunyn-Schmiedeberg's Arch. Pharmacol. 299, 8 9 - 9 4 (t977)
E. Beubler et al. : Intestinal Fluid Volume: Colchicine and PGE2 Beubler, E., Lembeck, F.: Methylxanthines and intestinal drug absorption. Naunyn-Schmiedeberg's Arch. Pharmacol. 292, 7 3 - 7 7 (1976) Collier, H. O. J. : Prostaglandin synthetase inhibitors and the gut. In: Prostaglandin synthetase inhibitors. (Robinson, H. J., Vane, J. R., eds.), pp. 121-133. New York: Raven Press, 1974 Collier, H. O. J., McDonald-Gibson, W. J., Saeed, S. A. : Stimulation of prostaglandin biosynthesis by drugs: Effects in vitro of some drugs affecting gut function. Br. J. Pharmacol. 58, 193 - 199 (1976) Dinsdale, D. : Colchicine-induced lesions in the rat duodenum. Pathol. Eur. 10, 9 5 - 104 (1975) Field, M., McColl, I. : Ion transport in rabbit ileal mucosa. III. Effects of catecholamines. Am. J. Physiol. 225, 852-857 (1973) Ferguson, F. C. : Colchicine. I. General Pharmacology. J. Pharmacol. Exp. Ther. 106, 261-270 (1952) Ferreira, S. H., Herman, A. G., Vane, J. R. : Prostaglandin production by rabbit isolated jejunum and its relationship to the inherent tone of the preparation. Br. J. Pharmacol. 56, 4 6 9 - 477 (1976) Ferreira, S. H., Moncada, S., Vane, J. R.: Indomethacin and aspirin abolish prostaglandin release from the spleen. Nature New Biol. 231,237-239 (1971) Fitzgerald, T. J., Williams, B., Uyeki, E. M. : Effects of antimitotic and antiinflammatory agents on sodium urate-induced paw swelling in mice. Pharmacology, 6, 265-273 (1971) Glatt, M., Wagner, K., Brune, K. : The effects of antiinflammatory doses of colchicine on prostaglandin content in inflamed tissue. In: Advances in prostaglandin and thromboxane research. (Samuelsson, B., Paoletti, R., eds.), pp. 111-115. New York: Raven Press 1976 Horton, E. W., Main, I. H. M., Thompson, C. J., Wright, P. M.: Effect of orally administered prostaglandin E1 on gastric secretion and gastro-intestinal motility in man. Gut 9, 655-658 (1968) Kimberg, D. V., Field, M., Gershon, E., Henderson, A. : Effects of prostaglandins and cholera enterotoxin on intestinal mucosal cyclic AMP accumulation. J. Clin. Invest. 53, 941 - 9 4 9 (1974) Kimberg, D. V., Field, M., Johnson, J., Henderson, A., Gershon, E. : Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins. J. Clin. Invest. 50, 1218-1230 (1971) Levin, R. J. : Effect of eolchicine on intestinal function in the rat. Gut 7, 250-257 (1966) Matuchansky, C., Bernier, J. J. : Effect of prostaglandin E1 on glucose, water, and electrolyte absorption in the human jejunum. Gastroenterology 64, 1111 - 1118 (1973) Milton-Thompson, G. J., Cummings, J. H., Newman, A., Billings, J. A., Misiewicz, J. J. : Colonic and small intestinal response to intravenous prostaglandin Fa~ and Ea in man. Gut 16, 4 2 - 4 6 (1975)
199 Misiewicz, J. J., Waller, S. L., Kiley, N., Horton, E. W. : Effect of oral prostaglandin E1 on intestinal transit in man. Lancet I, 648-651 (1969) Pierce, N. F., Carpenter, C. C. J., Elliott, H. L., Greenough, W. B. : Effects of prostaglandins, theophylline, and cholera exotoxin upon transmucosal water and electrolyte movement in the canine jejunum. Gastroenterology 60, 2 2 - 32 (1971) Raymackers, E., Hugon, J. S.: Morphological and cytochemical modification induced by colchicine treatment in the duodenal epithelium of mouse. Histochemie 34, 65-76 (1973) Robert, A.: Antisecretory, antiulcer, cytoprotective and diarrheogenic properties of prostaglandins. In: Advances in prostaglandin and thromboxane research. (Samuelson, B., Paoletti, R., eds.), pp. 507-520. New York: Raven Press 1976 Robinson, D. R., Levine, L. : Prostaglandin concentrations in synovial fluid in rheumatic diseases: Action of indomethacin and aspirin. In: Prostaglandin synthease inhibitors. (Robinson, H. J., Vane, J. R., eds.), pp. 223- 228. New York: Raven Press 1974 Robinson, D. R., Smith, H., Levine, L. : Prostaglandin synthesis by human synovial cultures and its stimulation by colchicine. Arthritis Rheum. 16, 129-134 (1973) Robinson, D. W., Smith, H., McGnire, M. B., Levine, L. : Prostagtandin synthesis by rheumatoid synovium and its stimulation by colchicine. Prostaglandins 10, 67-85 (1975) Schafer, D. E., Lust, W. D., Sircar, B.: Elevated concentration of adenosin 3',5'-cyclic monophosphate in intestinal mucosa after treatment with colera toxin. Proc. Nat. Acad. Sci. U.S.A. 67, 851-856 (1970) Seewann, S., Wutte, U., Juan, H.: Aconitine, capsaicin, colchicine and SC 19220: effect on pain receptors and PG-synthesis in the rabbit ear. Naunyn-Schmiedeberg's Arch. Pharmacol. 297, R58 (1977) Shipley, R. E., Tilden, J. N.: A pithed rat preparation suitable for assaying pressor substances. Proc. Soc. Exp. Biol. Med. 64, 453 (1959) Smith, J. B., Willis, A. L. : Aspirin selectively inhibits prostaglandin production in human platelets. Nature New Biol. 231, 235-237 (1971) Tidball, C. S. : Active chloride transport during intestinal secretion. Am. J. Physiol. 200, 309-312 (1961) Vane, J. R. : Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature New Biol. 231,232-235 (1971) Walaszek, E. J., Kocsis, J. J., Leroy, G. V., Geiling, E. M. K. : Studies on the excretion of radioactive colchicine. Arch. Intern. Phannacodyn. 125, 371 - 382 (1960) Wallace, S.L., Omokoku, B., Ertel, N. H. : Colchicine plasma levels. Implications as to pharmacology and mechanism of action. Am. J. Med. 48, 443-448 (1970) Received July 15/Accepted October 25, 1977
Archivesof
Naunyn-Schmiedeberg's Arch. Pharmacol. 301,195-199 (1978)
Pharmacology 9 by Springer-Verlag1978
Effects of PGE2 and Colchicine on the Intestinal Fluid Volume E. BEUBLER, F. LEMBECK, and M. SCHWEDITSCH
Institut fiir experimentelle und klinische Pharmakologie der Universitfit Graz, Universitfitsplatz4, A-8010 Graz, Austria
Summary. a) Colchicine, prostaglandin E2 (PGE2) and carbachol increase the intestinal fluid volume in the rat. b) The effect of carbachol and prostaglandin E2 is augmented whereas that of colchicine is diminished in the pithed rat. c) The effect of colchicine is diminished in anaesthesia whereas that of PGEz remains unchanged. d) The effect of PGE2 and of colchicine are both inhibited by atropine. e) The effects of PGE2 and o f colchicine are both enhanced by phentolamine. f) Pretreatment with indomethacin decreased the effect of colchicine whereas that of PGEz remained unchanged. g) The PGEz-induced increase in intestinal fluid volume m a y partly be mediated by a peripheral mechanism involving acetylcholine receptors. h) The increase in intestinal fluid volume by colchicine can mainly be explained by a central action of the drug which is mediated by cholinergic neurones.
conditions (rheumatoid synovial tissue: Robinson et al., 1975; urate crystal arthritis: Glatt et al., 1976; isolated perfused ear: Seewann et al., 1977) it seemed likely that the colchicine induced diarrhoea is caused by stimulation of intestinal PG-synthesis. Ferguson (1952) obtained experimental evidence for the involvement of the central nervous system in the gastrointestinal effects of colchicine. We have therefore examined the possible influence of anaesthesia (pentobarbital, urethane), of despinalisation and of drugs affecting the autonomic nervous system (atropine, phentolamine, reserpine) on colchicine induced diarrhoea in order to elucidate the mechanism of action of colchicine. Since only little is known about the mode of action of PGs on water secretion into the gut experiments were also performed with PGE2 in order to obtain some information on the way in which PGs act on intestinal water transport.
Key words." Intestinal fluid volume PGE2 - Indomethacin.
METHODS
Colchicine -
Animals. Female Sprague Dawley rats (breeder: Mus-Rattus,
Brunnthal), (200 + 20 g) were used. They were deprived of food for 24 h prior to the experiments. INTRODUCTION Colchicine is used in the treatment of acute attacks of gout. Its therapeutical application is severely limited by characteristic side effects like diarrhoea and abdominal pain. The mechanism of these side effects is still unknown. Since prostaglandins (PGs) also produce diarrhoea and since colchicine stimulates the synthesis of prostaglandins under certain experimental Send offprint requests to E. Beubler at the above address Preliminary reports: E. Beubler and M. Schweditsch: NaunynSchmiedeberg's Arch. Pharmacol. 297, R56 (1977) and F. Atzlinger and F. Lembeck: Naunyn-Schmiedeberg's Arch. Pharmacol. 293, R34 (1976)
Procedures. The intestinal fluid volume (IFV) was measured by the "enteropooling assay" of Robert (1976). Twenty min after the i.p. injection of PG or carbachol or 75 min after colchicine the animals were killed by a blow on the head, the entire small intestine was removed and its content squeezed into a calibrated test tube. IFV was expressed in ml/100 g rat. The pithed rats were prepared according to the method of Shipley and Tilden (1959). After an interval of 20 rain colchicine, PG or carbachol was injected intraperitoneally. Substances. Atropine (Merck), carbachol (Fluka), colchicine (Merck), indomethacin (Merck, Sharp & Dohme), pentobarbital (Abbott-Lab.), phentolamine (Ciba Geigy SA), propranolol (I.C.I.), prostaglandin E2 (Upjohn Co.), reserpine (Ciba Geigy SA), urethane (Merck). Statistics. The experimental data were evaluated by an unpaired t-test.
0028-1298/78/0301/0195/$ 01.00
196
Naunyn-Schmiedeberg'sArch. Pharmacol. 301 (1978)
0.8-
08t
0.6 0.~ r2~
g o
E ~- 0.4
Z
E ~'0.4
/
0.2
~02
--O-- cotcbicine,3mg/kg 9 control Fig. 1.
untreated
--#X--- PGE2,202Jg/kg
Time-course of the increase of intestinal fluid volume (IFV)
after i.p. injection of colchicine (3 mg/kg) or PGE2 (20 gg/kg) in the rat (Y 4- s~, n = 6)
[]
pentobarb, urethane
despina[.
control M colchicine,10 mg/kg [ ] PGE2, 20,ug/kg
Fig. 3. Influence of pentobarbital (50 gg/kg), urethane (1.25 g/kg) and despinalising on the increase in IFV induced by colchicine (J0 mg/kg) and PGE2 (20 gg/kg) (~ 4- s~, n = 6-- 10)
the administration of PGEz the IFV was back to the basal value (Fig. 1). The influence of drugs on the increase in IFV caused by PGE2 or colchicine was in each case assessed at the time of the maximal effect, i. e. 20 min after PGEz and 75 rain after colchicine injection.
1.0-
0,8
g
o K 0.6
B. Effect of PGEz~ on IFV
"d "5
PGF2~ at a dose of 20 gg/kg did not affect the IFV. The increase in IFV caused by 150 gg/kg PGF2~ amounted to only about 25 % of that seen after injecting 20 lag/kg PGE2.
-~ 0,4
0.2
C. Lffect of Carbachol on IFV 0
untreated
[ ] control [ ]
despinal.
carbachot, 25,ug/kg
Carbachol (25 gg/kg i.p., 20 rain before death) increased the IFV by about 140% (p < 0.001). In the pithed rat the effect of carbachol was much larger (p < 0.001) than in the intact, conscious rat (Fig. 2).
Fig. 2. Effect of carbachol (25 gg/kg i. p.) on IFV in untreated and pithed rats (2 4- s~, n = 6)
D. Effect of Drug or Despinalisation on the Changes in IFV Caused by Colchicine or PGs RESULTS
A. Effect of Colchicine and Prostaglandin E2 on Intestinal Fluid Volume Both colchicine (3 mg/kg i.p.) and PGE2 (20 gg/kg i.p.) increased the IFV. The onset of action of colchicine was slow, the maximal effect was observed after 75 min (Fig. 1). During the following hours the effect declined slowly: Six hours after the administration the IFV was still increased. The maximal effect of PGE2 was reached already after 20 min. One hour after
a) Anaesthetic Drugs. Pentobarbital (50 gg/kg i.p.) and urethane (1.25 g/kg i.p.) anaesthesia did not influence the basal IFV (p > 0.1). Both drugs decreased significantly (p < 0.001) the effect of colchicine (10 rag/ kg), whereas the effect of PGE2 (20 gg/kg) on the IFV remained unchanged (Fig. 3). b) Despinalisation. Destruction of the brain and the spinal cord did not influence (p > 0.1) the basal IFV. In the pithed rat the effect of colchicine was significantly reduced (p < 0.001) whereas that of PGE2 was significantly (p < 0.001) increased (Fig. 3).
197
E. Beubler et al. IntestinalFluid Volume: Colchicineand PGE2
e) Phentolamine. Pretreatment with phentolamine (1 mg/kg i.p., 20 rain before the experiment did not increase the basal IFV (p > 0.05). The rise in IFV caused by colchicine (p < 0.05) as well as that caused by PGE2 (p < 0.025) were slightly enhanced (Fig. 5). g
f) Reserpine. Pretreatment with reserpine (2.5 mg/kg
o
and day s.c. starting 2 days prior to the experiment) increased basal IFV (p < 0.005). The effect of colchicine was slightly inhibited (p < 0.05), that of PGE2 slightly enhanced (p < 0.02). Additional pretreatment with atropine abolished the effect of reserpine on basal IFV and the increase of the effect of PGE2 (p < 0.001), but did not further reduce the effect of colchicine (Fig. 5).
5
untreated []
Fig.4.
control [ ]
atropine
despinal,
co[chicine,10mg/kg
[]
indometh,
PGE2,20JJg/kg
Influence of atropine (1 mg/kg) in the conscious and the
pithed rat (control see Fig.3) and of indomethacin(4 mg/kg and day, s. c., startingtwo daysprior to the experiment)on the increasein IFV inducedby colchicine(10 mg/kg) and PGEz (20 p.g/kg)(ff _+s~, n = 6) 08-
g
o K
~- o~
0
untreated []
Fig. 5.
control [ ]
phentol
propr,
colchieine,10 mg/kg
reserpine []
reserpine +atropine
PGE2,20~ug/kg
Influence of phentolamine (1 mg/kg), propranolol (1 rag/
kg), reserpine (2 x 2.5 mg/kg) and reserpine plus atropine (1 nag/ kg) on basal IFV and on the increase in IFV inducedby colchicine (10 mg/kg) and PGE2 (20 gg/kg) (~ 4- s~, n = 6-10)
c) Atropine. Atropine (1 mg/kg i.p.) did not change the basal IFV. When given 10 rain prior to the injection of PGE2 or colchicine it reduced the effect of both compounds significantly (p < 0.001). Atropine also reduced the augmented effect of PGE2 in the pithed rat (Fig. 4). d) Indomethaein. Inhibition of PG synthesis by pretreatment with indomethacin (4 mg/kg and day s.c., starting 2 days prior to the experiment) had no effect on basal IFV or on the increase in IFV caused by PGE2. However it did reduce the rise in IFV after colchicine by more than 50 ~ (p < 0.001 ; Fig. 4).
DISCUSSION
A. Mode of Action of Colchicine Colchicine provides relief in acute attacks of gout and is also used as a prophylactic agent against such attacks. However, gastrointestinal side effects like diarrhoea and abdominal pain make prolonged administration often impracticable. Colchicine has a half life time of 24 rain in human blood plasma (Wallace et al., 1970). Its distribution volume is much larger than the extracellular fluid volume which indicates a rapid entrance into cells. Colchicine is very slowly excreted. Only about 10 % of a given dose appear in urine and faeces within 48 h (Walaszek et al., 1960). The intestinal effects of colchicine were attributed to its antimitotic activity. Colchicine decreased the absorption of xylose in conscious rats (Levin, 1966), caused severe morphological and cytochemical damage in the duodenal epithelium of the mouse (Raymackers and Hugon, 1973) and subcellular necrosis within the rapidly proliferating cells in the rat duodenal mucosa (Dinsdale, 1975). Ferguson (1952) observed that vomiting and diarrhoea induced by colchicine in cats could be inhibited by atropine and also by barbiturate anaesthesia. Since colchicine is inactive on the isolated intestine, Ferguson suggested a neurogenic basis for its action on the gut. Our observations on the inhibition of the colchicine induced increase in IFV by anaesthesia and by atropine confirm the results of Ferguson. The fact that in the pithed rat the effect of colchicine on IFV is decreased provides additional evidence for the participation of the central nervous system in the intestinal action of the drug. As this action is also reduced by atropine, cholinergic neurones must be involved. The effect of colchicine was slightly enhanced by blocking ~-adrenoceptors with phentolamine. This
198 enhancement might be due to changed hemodynamics after phentolamine treatment, or a mechanism involving ~-adrenoceptors inhibits the action of colchicine in the untreated rat. The decrease of the effect of colchicine in reserpine treated rats with or without pretreatment with atropine remains unexplained. The mechanism which mediates the reserpine induced sedation may involve similar central nervous structures as anaesthetic drugs do. Some of the intestinal effects of colchicine could be explained by an increased formation of prostaglandins. Most nonsteroidal antiinflammatory drugs inhibit the synthesis of PGs (Vane, 1971; Smith and Willis, 1971 ; Fen'eira et al., 1971). In contrast, colchicine, in spite of its slight antiinflammatory actions (Fitzgerald et al., 1971) increases the formation of PGs in cultured rheumatoid synovial cells (Robinson et al., 1973, 1975) and in fibroblast cultures (Robinson et al., 1974). Increased concentrations of PGs were also found in the synovial fluid of inflamed joints of chickens during colchicine treatment (Glatt et al., 1976). A stimulation of PG synthesis by colchicine was observed in bull seminal vesicles (Collier et al., 1976) and in the perfused rabbit ear (Seewann et al., 1977). PGs are known to be synthesized in the gut under physiological conditions (Beubler and Juan, 1977) and in response to various stimuli (Collier, 1974; Ferreira et al., 1976). Administration of PGs causes accumulation of water and electrolytes in the lumen of the small intestine, abdominal pain and diarrhoea (Horton et al., 1968; Misiewicz et al., 1969; Pierce et al., 1971; Matuchansky and Bernier, 1973; Milton-Thompson et al., 1975; Beubler and Juan, 1977). Indomethacin, a drug which inhibits PG-synthesis (Ferreira et al., 1971; Smith and Willis, 1971; Vane, 1971) did not reduce the basal IFV in the present experiments. This is not necessarily in conflict with previous observations on the perfused jejunal loop (Beubler and Juan, 1977) in which indomethacin increased water absorption, since the perfusion method can detect much smaller variations in fluid volume than the "enteropooling assay". The observation that the colchicine induced increase in IFV is reduced in indomethacin pretreated rats indicates that colchicine diarrhoea may partly be caused by an increased PG-synthesis in the gut. B. Mode of Action of PGE2 Drugs with acetylcholine like actions as betanechol (Tidball, 1961) or carbachol increase IFV. The effect of carbachol is about 50 ~ larger in pithed rats than in intact, conscious rats. This may be due to the loss of sympathetic activity after despinalisation. The effect of PGEz was much larger than that of PGF2~, which
Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1978)
is in agreement with the results of Robert (1976) and Beubler and Juan (1977). In preliminary experiments we observed a larger effect of PGE2 in female than in male rats. Therefore only females were used in the present experiments. No explanation for this sex differences is so far available. The effect of PGE2 on IFV was reduced by pretreatment with atropine. Despinalisation increased the effect of PGEz on IFV and also that of carbachol. These observations suggest that a peripheral mechanism involving acetylcholine receptors participates in the intestinal action of PGEz. Pentobarbital or urethane anaesthesia did not influence PGE2 increase in IFV which indicates that PGs act on a peripheral site. After pretreatment with phentolamine and propranolol the basal IFV remained unchanged. Phentolamine and despinalisation increased the effect of PGE2 which could be explained by a loss of sympathetic activity which normally inhibits the effect of PGE2 on the IFV. An effect of catecholamines on intestinal ion absorption was reported by Field and McColl (1973) who found that catecholamines increase the absorption of sodium and of chloride. PGs inhibit the absorption of sodium from the intestine and increase chloride and water secretion (Pierce et al., 1971 ; Matuchansky and Bernier, 1973). Pretreatment with reserpine increased basal IFV and enhanced the effect of PGE2. The increase in basal IFV in the reserpine treated rat cannot be ascribed to the loss of catecholamines since phentolamine did not change basal IFV. The effect of reserpine could be inhibited by pretreatment with atropine which indicates the involvement of acetylcholine receptors. Choleratoxin is known to stimulate intestinal adenylate cyclase (Kimberg et al., 1971 ; Schafer et al., 1970). The resulting increase in cyclic 3',5'-AMP enhances intestinal blood flow, water absorption and secretion. These effects were also obtained by inhibition of phosphodiesterase with theophylline (Pierce et al., 1971; Beubler and Lembeck, 1976). PGE2 stimulates intestinal adenylate cyclase (Kimberg et al., 1971 ; /974). The rise in IFV after PGE2 c o u l d only be reduced but not fully inhibited by blocking acetylcholine receptors with atropine. The residual effect might be due to the stimulating action of PGE2 on intestinal adenylate cyclase. Acknowledgements. The authors wish to thank Dr. J. Pike for the generous gift of prostaglandius. The investigation was supported by grant No. 2574 from the Fonds zur F6rderung der wissenschaftlichen Forschung ~sterreichs and by the Osterreichische Krebsgesellschaft.
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