Truth Seeker
New Member
Abstract
We have evaluated the effect of cannabinoid drugs, administered intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.) on upper gastrointestinal transit in control and in croton oil-treated mice.
The cannabinoid agonists, WIN 55,212-2 (2—239nmolmouse−1) and cannabinol (24—4027nmolmouse−1), decreased while the CB1 antagonist SR141716A (2—539nmolmouse−1) increased transit in control mice. WIN 55,212-2, cannabinol and SR141716A had lower ED50 values when administered i.c.v., than when administered i.p. The CB2 antagonist SR144528 (52nmol mouse−1, i.p.) was without effect.
During croton oil (0.01mlmouse−1, p.o.)-induced diarrhoea, the ED50 values of i.p.-injected WIN 55,212-2 and cannabinol (but not SR141716A) were significantly decreased (compared to control mice). However, the ED50 values of WIN 55,212-2 were similar after i.p. or i.c.v. administration.
The inhibitory effects of WIN 55,212-2 and cannabinol were counteracted by SR141716A (16nmolmouse−1, i.p.) but not by SR144528 (52nmolmouse−1, i.p.) both in control and croton-oil treated mice.
Ganglionic blockade with hexamethonium (69nmolmouse−1, i.p.) did not modify the inhibitory effect of i.p.-injected cannabinoid agonists either in control or in croton-oil treated mice.
The lower ED50 values of cannabinoid drugs after i.c.v. administration suggest a central (CB1) site of action. However, a peripheral site of action is suggested by the lack of effect of hexamethonium. In addition, croton oil-induced diarrhoea enhances the effect of cannabinoid agonists by a peripheral mechanism.
Introduction
Preparations of Cannabis sativa have been used medicinally for over 4000 years for the treatment of a variety of disorders, including migraine, muscle spasm, seizures, glaucoma, pain, nausea and diarrhoea (Felder & Glass, 1998). In 1964 Δ9-tetrahydrocannabinol (Δ9-THC) was isolated, which was later shown to be responsible for many of the pharmacological actions of Cannabis preparations (Mechoulam et al., 1998). With regard to the gastrointestinal tract, Dewey et al. (1972) were the first to report that Δ9-THC reduced the rate of passage of a charcoal meal in the mouse small intestine and these findings were confirmed by others (Chesher et al., 1973; Jackson et al., 1976; Shook & Burks, 1989).
Understanding of the mechanism by which Δ9-THC exerts its pharmacological actions has seen considerable progress in the last ten years following the discovery of two distinct cannabinoid receptors, named CB1, (expressed mainly by central and peripheral neurons) and CB2 (that occur mainly in immune cells) (Matsuda et al., 1990; Munro et al., 1993; Pertwee, 1998). The discovery of these receptors has led to the demonstration that there are endogenous agonists for these receptors, namely anandamide and 2-arachidonylglycerol (Devane et al., 1992; Stella et al., 1997), the latter found in the intestine of the dog (Mechoullam et al., 1995).
The myenteric plexus of the guinea-pig intestine contains CB1-, but not CB2-like cannabinoid receptor mRNA (Griffin et al., 1997). Activation of prejunctional CB1 receptors produces inhibition of excitatory transmission (Pertwee et al., 1996; Izzo et al., 1998) in the isolated guinea-pig ileum and these inhibitory effects are associated with a decrease in acetylcholine release from enteric nerves (Coutts & Pertwee, 1997). However, a preliminary report indicates that cannabinoid agonists potentiate electrically-induced contractions in the porcine ileum and this effect is mediated by CB2 receptors (Albasan et al., 1999).
The involvement of CB1 receptors in intestinal motility has been confirmed also in vivo. Indeed, the endogenous cannabinoid agonist anandamide (Calignano et al., 1997) and the synthetic cannabinoid agonist WIN 55,212-2 (Colombo et al., 1998; Izzo et al., 1999a) inhibited, whilst the CB1 receptor antagonist, SR141716A increased gastrointestinal transit in mice. However, in these studies, cannabinoid drugs were administered intraperitoneally or subcutaneously and therefore it was not clear if cannabinoids were acting at central or peripheral cannabinoid receptors. In addition, there are no data in the literature concerning the effects of cannabinoid drugs in the control of upper gastrointestinal motility during pathophysiological states.
The present study, therefore, has two objectives: (i) to compare the effect of cannabinoid drugs on intestinal motility after intracerebroventricular and intraperitoneal administration and (ii) to evaluate the effect of cannabinoid agonists on intestinal motility during experimental diarrhoea. In order to achieve this experimental condition, we have used croton oil, a well-known cathartic agent (Pol et al., 1996). The cannabinoid drugs used were: the natural agonist cannabinol (Petitet et al., 1998) and the synthetic agonist WIN 55,212-2 (Compton et al., 1992), the CB1 receptor antagonist SR141716A (Rinaldi-Carmona et al., 1995) and the CB2 receptor antagonist SR144528 (Rinaldi-Carmona et al., 1998).
Methods
Animals
Male ICR mice (Harlan Italy, Corezzana, MI) (24—26g) were used after 1week of acclimation (temperature 23±2°C; humidity 60%). Food was withheld 3h before experiments but there was free access to drinking water.
Upper gastrointestinal transit
Gastrointestinal transit was measured in control mice or 3h after treatment with croton oil (0.01mlmouse−1). At this time, 0.1ml of a black marker (10% charcoal suspension in 5% gum arabic) was administered orally to assess upper gastrointestinal transit as previously described (Pol et al., 1996; Izzo et al., 1999a). After 20min the mice were killed by asphyxiation with CO2 and the gastrointestinal tract removed. The distance travelled by the marker was measured and expressed as a percentage of the total length of the small intestine from pylorus to caecum (Izzo et al., 1999a).
The cannabinoid agonists WIN 55,212-2 (2—239nmol mouse−1), cannabinol (24—4027nmolmouse−1), the CB1 receptor antagonist SR141716A (2—539nmolmouse−1), the CB2 receptor antagonist SR144528 (52nmolmouse−1) or vehicle (DMSO, 4—8μlmouse−1) were given intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.) 20min before charcoal administration. In some experiments SR141716A (16nmolmouse−1=0.3mgkg−1), SR144528 (52nmolmouse −1=1mgkg−1) or hexamethonium (69nmolmouse −1=1mgkg−1) were given (i.p.) 10min before the cannabinoid agonists. The doses of hexamethonium and SR144528 were selected on the basis of previous published work (Schirgi-Degen & Beubler, 1995; Rinaldi-Carmona et al., 1998)
Intracerebroventricular injections
Intracerebroventricular injections were performed as described by Haley & McCormick (1957)). Mice were briefly anaesthetized with enflurane and the drugs were delivered in a volume of 4μl, using a Hamilton microlitre syringe fitted with 26-gauge needle.
Drugs
Drugs used were: WIN 55,212-2 mesylate (Tocris Cookson, Bristol, U.K.), hexamethonium bromide and cannabinol (SIGMA, Milan, Italy). SR141716A [(N-piperidin-l-yl)-5-(4-chlorophenyl)-1-2,4-dichlorophenyl)-4-methyl-lH-pyrazole-3-carboxamide hydrochloride and SR144528 (N-[-1S-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide-3-carboxamide) were a gift from Dr Madaleine Mossé and Dr Francis Barth (SANOFI-Recherche, Montpellier, France). Cannabinoid drugs were dissolved in DMSO, while hexamethonium was dissolved in saline.
Statistics
Data are mean±s.e.mean. To determine statistical significance, Student's t-test for unpaired data or one-way analysis of variance followed by Tukey—Kramer multiple comparisons test was used. A P-value less than 0.05 was considered significant. ED50 (dose which produced a 50% variation of gastrointestinal transit) and Emax (maximal effect) values were calculated using the computer program of Tallarida & Murray (1986).
Results
Effect of cannabinoid drugs on upper gastrointestinal transit in control mice
The effect of i.p.- or i.c.v.- injected WIN 55,212-2 (2—239nmolmouse−1) and cannabinol (24—4027nmolmouse−1) on percentage inhibition of upper gastrointestinal transit are presented in Figure 1. Both WIN 55,212-2 and cannabinol produce a dose-dependent inhibition of gastrointestinal transit. However, the ED50 values after i.p. or i.c.v. administration were statistically different. The ED50 and Emax values of cannabinoid drugs are shown in Table 1.
The CB1 receptor antagonist SR141716A (16nmol mouse−1, i.p.), but not the CB2 receptor antagonist SR144528 (52nmolmouse−1, i.p.) counteracted the inhibitory effect of WIN 55,212-2 (5nmolmouse−1, i.c.v. or 50nmolmouse−1, i.p.) and cannabinol (201nmolmouse−1, i.c.v. or 2010nmolmouse−1, i.p.) after both i.c.v. (Figure 2) and i.p. (Figure 3) routes of administration. Hexamethonium (69nmolmouse−1, i.p.) abolished the effect of both WIN 55,212-2 and cannabinol after i.c.v. (Figure 2) but not after i.p. (Figure 3) administration.
SR 14176A (i.p. or i.c.v.), per se, dose-dependently increased upper gastrointestinal transit (Figure 4a). However, the ED50 value after i.c.v. administration was significantly (P<0.01) lower than the ED50 after i.p. administration (Table 1). At a dose of 16nmolmouse−1, SR141716A (i.c.v.) significantly (P<0.05) increased intestinal motility (Figure 4a) and this effect was significantly (P<0.05) counteracted by hexamethonium (69nmolmouse−1 i.p.) (per cent increase of SR141716A: 44±3; per cent increase of SR141716A in the presence of hexamethonium; 1±3, n=10).
The CB2 receptor antagonist SR144528 (52nmol mouse−1, i.p.), given alone, did not significantly modify gastrointestinal transit (control 47±4%; SR144528 48±2%, n=10, P>0.2). Hexamethonium (69nmolmouse−1 i.p.) did not significantly modify gastrointestinal transit (17±8% increase, n=12). DMSO (4μlmouse−1 i.c.v. or 4—8μlmouse−1 i.p.) had no effect on the response under study (data not shown).
Effect of cannabinoid drugs on upper gastrointestinal transit during croton oil-induced diarrhoea
Oral administration of croton oil produced diarrhoea which was associated with a significant increase in gastrointestinal transit (per cent transit: control 46±2; croton oil, 56±2, P<0.01, n=24). Both WIN 55,212-2 (2—239nmolmouse−1, i.p.) and cannabinol (24—4027nmolmouse−1, i.p.) produced a dose-related inhibition of transit (Figure 5) and both agonists had a lower ED50 value compared to the corresponding i.p. treatment in control mice (Table 1). In croton oil-treated animals, WIN 55,212-2 (i.p.) and cannabinol (i.p.) had a significant inhibitory effect with threshold doses of 5nmolmouse−1 and 80nmolmouse−1 doses respectively whilst in control mice, significant inhibitory effects were achieved at doses of 14nmol mouse−1 (WIN 55,212-2) and 2010nmolmouse−1 (cannabinol) respectively (Figure 5).
Administered i.c.v. WIN 55,212-2 (2—239nmolmouse−1) also decreased intestinal motility, but the ED50 value (74±10nmolmouse−1) was not statistically different from the ED50 value (68±5nmolmouse−1) after i.p. administration (Table 1).
The inhibitory effect of i.p.-injected WIN 55,212-2 (14nmolmouse−1) or cannabinol (805nmolmouse−1) was reduced by the CB1 receptor antagonist SR141716A (16nmolmouse−1, i.p.) but not by the CB2 receptor antagonist SR144528 (52nmolmouse−1, i.p.) or by the ganglion blocker hexamethonium (69nmolmouse−1, i.p.) (Figure 6).
Figure 4b shows the potentiating effect of SR141716A (2—539nmolmouse, i.p.) in mice treated with croton oil. The ED50 value (418±32nmolmouse−1) was not statistically different from the corresponding ED50 value in control animals (375±31nmolmouse−1). By contrast, SR144528 (52nmol mouse−1, i.p.) or hexamethonium (69nmolmouse−1, i.p.) did not modify gastrointestinal transit (per cent transit: croton oil: 58±6, croton oil+SR144528 61±5, croton oil+hexamethonium 68±4, n=6, P>0.2).
Discussion
The role of cannabinoid receptors in control mice
It is now well known that cannabinoid agonists can reduce intestinal motility through activation of CB1 receptors. Indeed activation of CB1 receptors can mediate, (i) inhibition of electrically-evoked contractions in the isolated guinea-pig (Pertwee et al., 1996; Izzo et al., 1998) and human ileum (Croci et al., 1998), (ii) inhibition of fast and slow synaptic transmission in guinea-pig myenteric nerves (Lopez-Redondo et al., 1997), (iii) inhibition of electrically-evoked acetylcholine release from myenteric nerves (Coutts & Pertwee, 1997) and (iv) reduction of peristalsis efficiency in the isolated guinea-pig ileum (Heinemann et al., 1999; Izzo et al., 2000). These findings are in keeping with the presence of CB1, but not CB2-like receptor messenger RNA in the myenteric plexus of the guinea-pig small intestine (Griffin et al., 1997). Consistent with these in vitro findings, it has been shown that cannabinoid agonists reduced intestinal motility in mice (Calignano et al., 1997; Colombo et al., 1998; Izzo et al., 1999a) and rats (Izzo et al., 1999c) and this effect was counteracted by SR141716A, a specific CB1 antagonist. However, whether the effect of cannabinoid drugs in vivo is mediated via a central or a peripheral site of action was not demonstrated in these studies. Indeed the CB1 receptor is located within both the central nervous system (Matsuda et al., 1990) and within the enteric nervous system (Griffin et al., 1997).
In the present study we have shown that the synthetic cannabinoid agonist WIN 55,212-2 and the natural cannabinoid agonist cannabinol produced a dose-related inhibition of upper gastrointestinal transit when administered i.p. or i.c.v. The inhibitory effect of cannabinoid agonists was abolished by SR141716A, a specific CB1 antagonist, but not by SR144528, a CB2 receptor antagonist, indicating an involvement of CB1 but not CB2 receptors.
The ED50 values of WIN 55,212-2 and cannabinol after i.c.v. administration were significantly lower than the corresponding ED50 values after i.p. administration. The low doses that were needed to inhibit transit after i.c.v. injection implies that cannabinoid agonists may inhibit intestinal motility through activation of central CB1 receptors. However, the effect of i.p.-injected cannabinoid agonists was not modified by the ganglion blocker hexamethonium. These results probably indicate that the effect of i.p.-injected cannabinoid agonists is mediated by peripheral CB1 cannabinoid receptors.
Although some reports indicate that the CB1 receptor antagonist SR141716A does not affect intestinal motility in the isolated human ileum (Croci et al., 1998) and gastric emptying in the rat (Izzo et al., 1999b), other studies indicate that intestinal motility could be tonically inhibited by the endogenous cannabinoid system. Indeed SR141716A increased electrically-induced contractions in the isolated guinea-pig ileum (Pertwee et al., 1996; Izzo et al., 1998) and intestinal motility and defaecation in the mouse (Colombo et al., 1998; Izzo et al., 1999a). The observation that SR141716A, per se, increased intestinal motility does not necessary imply that endogenous cannabinoids are involved in the control of intestinal motility in view of the inverse agonist properties of SR141716A at human recombinant CB1 (Landsman et al., 1997) and both CB1 and CB2 receptors (MacLennan et al., 1998).
In the present study, we have shown that SR141716A (i.c.v. or i.p.) produced a dose-dependent increase in upper gastrointestinal transit. The ED50 value after i.c.v. administration was significantly lower than the ED50 value after i.p. administration, suggesting a central site of action of SR141716A. The most likely explanation of these results is that the endogenous cannabinoid system, within the central nervous system, can inhibit intestinal motility through activation of CB1 receptors. In a recent study, we have shown that SR141716A (i.p.)-induced changes in intestinal motility are not modified by the ganglionic blocker hexamethonium (Izzo et al., 1999a), suggesting a peripheral site of action of i.p.-injected SR141716A.
Effect of cannabinoid drugs during croton oil-induced diarrhoea
Croton oil is a well known irritant that has been widely used to produce experimental inflammation in different tissues, especially skin and mucosa, and induces diarrhoea associated with intestinal inflammation in the mouse small intestine (Pol et al., 1996). According to Pol et al., (1996), we have shown that croton oil increases upper gastrointestinal transit 3h after oral administration. The cannabinoid agonists WIN 55,212-2 and cannabinol blocked the increase in intestinal motility induced by croton oil; in addition, the ED50 values of i.p.-injected WIN 55,212-2 and cannabinol were significantly decreased (compared to control mice). However, during croton oil-induced diarrhoea the ED50 value of WIN 55,212-2 was similar after i.p. or i.c.v. treatment and ganglionic blockade with hexamethonium did not alter the inhibitory effect of i.p.-injected cannabinoids.
Taken together, these results indicate that the enhanced effect of cannabinoid agonists are mediated by peripheral receptors. By contrast, using the castor oil test, we have recently shown that cannabinoid agonists possess either weak or no antidiarrhoeal activity in the rat (Izzo et al., 1999c). The use of a different cathartic (castor oil vs croton oil), different species (rat vs mouse) and different region of the gut (whole gut vs upper gastrointestinal tract) could explain this discrepancy. Consistent with this hypothesis, Shook & Burks (1989) showed that Δ9-THC produced a greater inhibition of small intestinal transit than large bowel transit.
In line with the result obtained in control mice and those reported in the isolated guinea-pig ileum (Pertwee et al., 1996; Izzo et al., 1998), the antitransit response of cannabinoid agonists involves CB1, but not CB2 receptors, as the inhibitory effect of both WIN 55,212-2 and cannabinol were reduced by SR141716A, but not SR144528. Administration of SR141716A (i.p.), per se, increased intestinal motility in control mice and those given croton oil with a similar ED50 value, thus indicating that during the experimental diarrhoea the endogenous cannabinoid system is activated as in control animals. By contrast, SR144524, a specific CB2 receptor antagonist, at doses previously shown to bind the CB2 receptor in the rat spleen (Rinaldi-Carmona et al., 1998), failed to modify the inhibitory effect of both WIN 55,212-2 and cannabinol and did not modify, per se, intestinal motility during the diarrhoea induced by croton oil. Thus, a role for CB2 receptors in modulating intestinal motility during experimental diarrhoea seems unlikely.
Conclusions
Our results suggest that both central and peripheral CB1 receptors can modulate upper gastrointestinal motility. However, the effect of systemic (i.p.) cannabinoid drugs is probably mediated by peripheral receptors. Diarrhoea induced by the irritant croton oil enhances the inhibitory effect of cannabinoid agonists by a peripheral mechanism, while CB2 receptors are not involved in the control of intestinal motility, either in physiological or in pathophysiological states. Thus, selective non-psychotropic CB1 agonists could represent novel drugs to treat motility disorders associated with inflammatory diarrhoea.
Source, Graphs and Figures: Central and peripheral cannabinoid modulation of gastrointestinal transit in physiological states or during the diarrhoea induced by croton oil
We have evaluated the effect of cannabinoid drugs, administered intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.) on upper gastrointestinal transit in control and in croton oil-treated mice.
The cannabinoid agonists, WIN 55,212-2 (2—239nmolmouse−1) and cannabinol (24—4027nmolmouse−1), decreased while the CB1 antagonist SR141716A (2—539nmolmouse−1) increased transit in control mice. WIN 55,212-2, cannabinol and SR141716A had lower ED50 values when administered i.c.v., than when administered i.p. The CB2 antagonist SR144528 (52nmol mouse−1, i.p.) was without effect.
During croton oil (0.01mlmouse−1, p.o.)-induced diarrhoea, the ED50 values of i.p.-injected WIN 55,212-2 and cannabinol (but not SR141716A) were significantly decreased (compared to control mice). However, the ED50 values of WIN 55,212-2 were similar after i.p. or i.c.v. administration.
The inhibitory effects of WIN 55,212-2 and cannabinol were counteracted by SR141716A (16nmolmouse−1, i.p.) but not by SR144528 (52nmolmouse−1, i.p.) both in control and croton-oil treated mice.
Ganglionic blockade with hexamethonium (69nmolmouse−1, i.p.) did not modify the inhibitory effect of i.p.-injected cannabinoid agonists either in control or in croton-oil treated mice.
The lower ED50 values of cannabinoid drugs after i.c.v. administration suggest a central (CB1) site of action. However, a peripheral site of action is suggested by the lack of effect of hexamethonium. In addition, croton oil-induced diarrhoea enhances the effect of cannabinoid agonists by a peripheral mechanism.
Introduction
Preparations of Cannabis sativa have been used medicinally for over 4000 years for the treatment of a variety of disorders, including migraine, muscle spasm, seizures, glaucoma, pain, nausea and diarrhoea (Felder & Glass, 1998). In 1964 Δ9-tetrahydrocannabinol (Δ9-THC) was isolated, which was later shown to be responsible for many of the pharmacological actions of Cannabis preparations (Mechoulam et al., 1998). With regard to the gastrointestinal tract, Dewey et al. (1972) were the first to report that Δ9-THC reduced the rate of passage of a charcoal meal in the mouse small intestine and these findings were confirmed by others (Chesher et al., 1973; Jackson et al., 1976; Shook & Burks, 1989).
Understanding of the mechanism by which Δ9-THC exerts its pharmacological actions has seen considerable progress in the last ten years following the discovery of two distinct cannabinoid receptors, named CB1, (expressed mainly by central and peripheral neurons) and CB2 (that occur mainly in immune cells) (Matsuda et al., 1990; Munro et al., 1993; Pertwee, 1998). The discovery of these receptors has led to the demonstration that there are endogenous agonists for these receptors, namely anandamide and 2-arachidonylglycerol (Devane et al., 1992; Stella et al., 1997), the latter found in the intestine of the dog (Mechoullam et al., 1995).
The myenteric plexus of the guinea-pig intestine contains CB1-, but not CB2-like cannabinoid receptor mRNA (Griffin et al., 1997). Activation of prejunctional CB1 receptors produces inhibition of excitatory transmission (Pertwee et al., 1996; Izzo et al., 1998) in the isolated guinea-pig ileum and these inhibitory effects are associated with a decrease in acetylcholine release from enteric nerves (Coutts & Pertwee, 1997). However, a preliminary report indicates that cannabinoid agonists potentiate electrically-induced contractions in the porcine ileum and this effect is mediated by CB2 receptors (Albasan et al., 1999).
The involvement of CB1 receptors in intestinal motility has been confirmed also in vivo. Indeed, the endogenous cannabinoid agonist anandamide (Calignano et al., 1997) and the synthetic cannabinoid agonist WIN 55,212-2 (Colombo et al., 1998; Izzo et al., 1999a) inhibited, whilst the CB1 receptor antagonist, SR141716A increased gastrointestinal transit in mice. However, in these studies, cannabinoid drugs were administered intraperitoneally or subcutaneously and therefore it was not clear if cannabinoids were acting at central or peripheral cannabinoid receptors. In addition, there are no data in the literature concerning the effects of cannabinoid drugs in the control of upper gastrointestinal motility during pathophysiological states.
The present study, therefore, has two objectives: (i) to compare the effect of cannabinoid drugs on intestinal motility after intracerebroventricular and intraperitoneal administration and (ii) to evaluate the effect of cannabinoid agonists on intestinal motility during experimental diarrhoea. In order to achieve this experimental condition, we have used croton oil, a well-known cathartic agent (Pol et al., 1996). The cannabinoid drugs used were: the natural agonist cannabinol (Petitet et al., 1998) and the synthetic agonist WIN 55,212-2 (Compton et al., 1992), the CB1 receptor antagonist SR141716A (Rinaldi-Carmona et al., 1995) and the CB2 receptor antagonist SR144528 (Rinaldi-Carmona et al., 1998).
Methods
Animals
Male ICR mice (Harlan Italy, Corezzana, MI) (24—26g) were used after 1week of acclimation (temperature 23±2°C; humidity 60%). Food was withheld 3h before experiments but there was free access to drinking water.
Upper gastrointestinal transit
Gastrointestinal transit was measured in control mice or 3h after treatment with croton oil (0.01mlmouse−1). At this time, 0.1ml of a black marker (10% charcoal suspension in 5% gum arabic) was administered orally to assess upper gastrointestinal transit as previously described (Pol et al., 1996; Izzo et al., 1999a). After 20min the mice were killed by asphyxiation with CO2 and the gastrointestinal tract removed. The distance travelled by the marker was measured and expressed as a percentage of the total length of the small intestine from pylorus to caecum (Izzo et al., 1999a).
The cannabinoid agonists WIN 55,212-2 (2—239nmol mouse−1), cannabinol (24—4027nmolmouse−1), the CB1 receptor antagonist SR141716A (2—539nmolmouse−1), the CB2 receptor antagonist SR144528 (52nmolmouse−1) or vehicle (DMSO, 4—8μlmouse−1) were given intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.) 20min before charcoal administration. In some experiments SR141716A (16nmolmouse−1=0.3mgkg−1), SR144528 (52nmolmouse −1=1mgkg−1) or hexamethonium (69nmolmouse −1=1mgkg−1) were given (i.p.) 10min before the cannabinoid agonists. The doses of hexamethonium and SR144528 were selected on the basis of previous published work (Schirgi-Degen & Beubler, 1995; Rinaldi-Carmona et al., 1998)
Intracerebroventricular injections
Intracerebroventricular injections were performed as described by Haley & McCormick (1957)). Mice were briefly anaesthetized with enflurane and the drugs were delivered in a volume of 4μl, using a Hamilton microlitre syringe fitted with 26-gauge needle.
Drugs
Drugs used were: WIN 55,212-2 mesylate (Tocris Cookson, Bristol, U.K.), hexamethonium bromide and cannabinol (SIGMA, Milan, Italy). SR141716A [(N-piperidin-l-yl)-5-(4-chlorophenyl)-1-2,4-dichlorophenyl)-4-methyl-lH-pyrazole-3-carboxamide hydrochloride and SR144528 (N-[-1S-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide-3-carboxamide) were a gift from Dr Madaleine Mossé and Dr Francis Barth (SANOFI-Recherche, Montpellier, France). Cannabinoid drugs were dissolved in DMSO, while hexamethonium was dissolved in saline.
Statistics
Data are mean±s.e.mean. To determine statistical significance, Student's t-test for unpaired data or one-way analysis of variance followed by Tukey—Kramer multiple comparisons test was used. A P-value less than 0.05 was considered significant. ED50 (dose which produced a 50% variation of gastrointestinal transit) and Emax (maximal effect) values were calculated using the computer program of Tallarida & Murray (1986).
Results
Effect of cannabinoid drugs on upper gastrointestinal transit in control mice
The effect of i.p.- or i.c.v.- injected WIN 55,212-2 (2—239nmolmouse−1) and cannabinol (24—4027nmolmouse−1) on percentage inhibition of upper gastrointestinal transit are presented in Figure 1. Both WIN 55,212-2 and cannabinol produce a dose-dependent inhibition of gastrointestinal transit. However, the ED50 values after i.p. or i.c.v. administration were statistically different. The ED50 and Emax values of cannabinoid drugs are shown in Table 1.
The CB1 receptor antagonist SR141716A (16nmol mouse−1, i.p.), but not the CB2 receptor antagonist SR144528 (52nmolmouse−1, i.p.) counteracted the inhibitory effect of WIN 55,212-2 (5nmolmouse−1, i.c.v. or 50nmolmouse−1, i.p.) and cannabinol (201nmolmouse−1, i.c.v. or 2010nmolmouse−1, i.p.) after both i.c.v. (Figure 2) and i.p. (Figure 3) routes of administration. Hexamethonium (69nmolmouse−1, i.p.) abolished the effect of both WIN 55,212-2 and cannabinol after i.c.v. (Figure 2) but not after i.p. (Figure 3) administration.
SR 14176A (i.p. or i.c.v.), per se, dose-dependently increased upper gastrointestinal transit (Figure 4a). However, the ED50 value after i.c.v. administration was significantly (P<0.01) lower than the ED50 after i.p. administration (Table 1). At a dose of 16nmolmouse−1, SR141716A (i.c.v.) significantly (P<0.05) increased intestinal motility (Figure 4a) and this effect was significantly (P<0.05) counteracted by hexamethonium (69nmolmouse−1 i.p.) (per cent increase of SR141716A: 44±3; per cent increase of SR141716A in the presence of hexamethonium; 1±3, n=10).
The CB2 receptor antagonist SR144528 (52nmol mouse−1, i.p.), given alone, did not significantly modify gastrointestinal transit (control 47±4%; SR144528 48±2%, n=10, P>0.2). Hexamethonium (69nmolmouse−1 i.p.) did not significantly modify gastrointestinal transit (17±8% increase, n=12). DMSO (4μlmouse−1 i.c.v. or 4—8μlmouse−1 i.p.) had no effect on the response under study (data not shown).
Effect of cannabinoid drugs on upper gastrointestinal transit during croton oil-induced diarrhoea
Oral administration of croton oil produced diarrhoea which was associated with a significant increase in gastrointestinal transit (per cent transit: control 46±2; croton oil, 56±2, P<0.01, n=24). Both WIN 55,212-2 (2—239nmolmouse−1, i.p.) and cannabinol (24—4027nmolmouse−1, i.p.) produced a dose-related inhibition of transit (Figure 5) and both agonists had a lower ED50 value compared to the corresponding i.p. treatment in control mice (Table 1). In croton oil-treated animals, WIN 55,212-2 (i.p.) and cannabinol (i.p.) had a significant inhibitory effect with threshold doses of 5nmolmouse−1 and 80nmolmouse−1 doses respectively whilst in control mice, significant inhibitory effects were achieved at doses of 14nmol mouse−1 (WIN 55,212-2) and 2010nmolmouse−1 (cannabinol) respectively (Figure 5).
Administered i.c.v. WIN 55,212-2 (2—239nmolmouse−1) also decreased intestinal motility, but the ED50 value (74±10nmolmouse−1) was not statistically different from the ED50 value (68±5nmolmouse−1) after i.p. administration (Table 1).
The inhibitory effect of i.p.-injected WIN 55,212-2 (14nmolmouse−1) or cannabinol (805nmolmouse−1) was reduced by the CB1 receptor antagonist SR141716A (16nmolmouse−1, i.p.) but not by the CB2 receptor antagonist SR144528 (52nmolmouse−1, i.p.) or by the ganglion blocker hexamethonium (69nmolmouse−1, i.p.) (Figure 6).
Figure 4b shows the potentiating effect of SR141716A (2—539nmolmouse, i.p.) in mice treated with croton oil. The ED50 value (418±32nmolmouse−1) was not statistically different from the corresponding ED50 value in control animals (375±31nmolmouse−1). By contrast, SR144528 (52nmol mouse−1, i.p.) or hexamethonium (69nmolmouse−1, i.p.) did not modify gastrointestinal transit (per cent transit: croton oil: 58±6, croton oil+SR144528 61±5, croton oil+hexamethonium 68±4, n=6, P>0.2).
Discussion
The role of cannabinoid receptors in control mice
It is now well known that cannabinoid agonists can reduce intestinal motility through activation of CB1 receptors. Indeed activation of CB1 receptors can mediate, (i) inhibition of electrically-evoked contractions in the isolated guinea-pig (Pertwee et al., 1996; Izzo et al., 1998) and human ileum (Croci et al., 1998), (ii) inhibition of fast and slow synaptic transmission in guinea-pig myenteric nerves (Lopez-Redondo et al., 1997), (iii) inhibition of electrically-evoked acetylcholine release from myenteric nerves (Coutts & Pertwee, 1997) and (iv) reduction of peristalsis efficiency in the isolated guinea-pig ileum (Heinemann et al., 1999; Izzo et al., 2000). These findings are in keeping with the presence of CB1, but not CB2-like receptor messenger RNA in the myenteric plexus of the guinea-pig small intestine (Griffin et al., 1997). Consistent with these in vitro findings, it has been shown that cannabinoid agonists reduced intestinal motility in mice (Calignano et al., 1997; Colombo et al., 1998; Izzo et al., 1999a) and rats (Izzo et al., 1999c) and this effect was counteracted by SR141716A, a specific CB1 antagonist. However, whether the effect of cannabinoid drugs in vivo is mediated via a central or a peripheral site of action was not demonstrated in these studies. Indeed the CB1 receptor is located within both the central nervous system (Matsuda et al., 1990) and within the enteric nervous system (Griffin et al., 1997).
In the present study we have shown that the synthetic cannabinoid agonist WIN 55,212-2 and the natural cannabinoid agonist cannabinol produced a dose-related inhibition of upper gastrointestinal transit when administered i.p. or i.c.v. The inhibitory effect of cannabinoid agonists was abolished by SR141716A, a specific CB1 antagonist, but not by SR144528, a CB2 receptor antagonist, indicating an involvement of CB1 but not CB2 receptors.
The ED50 values of WIN 55,212-2 and cannabinol after i.c.v. administration were significantly lower than the corresponding ED50 values after i.p. administration. The low doses that were needed to inhibit transit after i.c.v. injection implies that cannabinoid agonists may inhibit intestinal motility through activation of central CB1 receptors. However, the effect of i.p.-injected cannabinoid agonists was not modified by the ganglion blocker hexamethonium. These results probably indicate that the effect of i.p.-injected cannabinoid agonists is mediated by peripheral CB1 cannabinoid receptors.
Although some reports indicate that the CB1 receptor antagonist SR141716A does not affect intestinal motility in the isolated human ileum (Croci et al., 1998) and gastric emptying in the rat (Izzo et al., 1999b), other studies indicate that intestinal motility could be tonically inhibited by the endogenous cannabinoid system. Indeed SR141716A increased electrically-induced contractions in the isolated guinea-pig ileum (Pertwee et al., 1996; Izzo et al., 1998) and intestinal motility and defaecation in the mouse (Colombo et al., 1998; Izzo et al., 1999a). The observation that SR141716A, per se, increased intestinal motility does not necessary imply that endogenous cannabinoids are involved in the control of intestinal motility in view of the inverse agonist properties of SR141716A at human recombinant CB1 (Landsman et al., 1997) and both CB1 and CB2 receptors (MacLennan et al., 1998).
In the present study, we have shown that SR141716A (i.c.v. or i.p.) produced a dose-dependent increase in upper gastrointestinal transit. The ED50 value after i.c.v. administration was significantly lower than the ED50 value after i.p. administration, suggesting a central site of action of SR141716A. The most likely explanation of these results is that the endogenous cannabinoid system, within the central nervous system, can inhibit intestinal motility through activation of CB1 receptors. In a recent study, we have shown that SR141716A (i.p.)-induced changes in intestinal motility are not modified by the ganglionic blocker hexamethonium (Izzo et al., 1999a), suggesting a peripheral site of action of i.p.-injected SR141716A.
Effect of cannabinoid drugs during croton oil-induced diarrhoea
Croton oil is a well known irritant that has been widely used to produce experimental inflammation in different tissues, especially skin and mucosa, and induces diarrhoea associated with intestinal inflammation in the mouse small intestine (Pol et al., 1996). According to Pol et al., (1996), we have shown that croton oil increases upper gastrointestinal transit 3h after oral administration. The cannabinoid agonists WIN 55,212-2 and cannabinol blocked the increase in intestinal motility induced by croton oil; in addition, the ED50 values of i.p.-injected WIN 55,212-2 and cannabinol were significantly decreased (compared to control mice). However, during croton oil-induced diarrhoea the ED50 value of WIN 55,212-2 was similar after i.p. or i.c.v. treatment and ganglionic blockade with hexamethonium did not alter the inhibitory effect of i.p.-injected cannabinoids.
Taken together, these results indicate that the enhanced effect of cannabinoid agonists are mediated by peripheral receptors. By contrast, using the castor oil test, we have recently shown that cannabinoid agonists possess either weak or no antidiarrhoeal activity in the rat (Izzo et al., 1999c). The use of a different cathartic (castor oil vs croton oil), different species (rat vs mouse) and different region of the gut (whole gut vs upper gastrointestinal tract) could explain this discrepancy. Consistent with this hypothesis, Shook & Burks (1989) showed that Δ9-THC produced a greater inhibition of small intestinal transit than large bowel transit.
In line with the result obtained in control mice and those reported in the isolated guinea-pig ileum (Pertwee et al., 1996; Izzo et al., 1998), the antitransit response of cannabinoid agonists involves CB1, but not CB2 receptors, as the inhibitory effect of both WIN 55,212-2 and cannabinol were reduced by SR141716A, but not SR144528. Administration of SR141716A (i.p.), per se, increased intestinal motility in control mice and those given croton oil with a similar ED50 value, thus indicating that during the experimental diarrhoea the endogenous cannabinoid system is activated as in control animals. By contrast, SR144524, a specific CB2 receptor antagonist, at doses previously shown to bind the CB2 receptor in the rat spleen (Rinaldi-Carmona et al., 1998), failed to modify the inhibitory effect of both WIN 55,212-2 and cannabinol and did not modify, per se, intestinal motility during the diarrhoea induced by croton oil. Thus, a role for CB2 receptors in modulating intestinal motility during experimental diarrhoea seems unlikely.
Conclusions
Our results suggest that both central and peripheral CB1 receptors can modulate upper gastrointestinal motility. However, the effect of systemic (i.p.) cannabinoid drugs is probably mediated by peripheral receptors. Diarrhoea induced by the irritant croton oil enhances the inhibitory effect of cannabinoid agonists by a peripheral mechanism, while CB2 receptors are not involved in the control of intestinal motility, either in physiological or in pathophysiological states. Thus, selective non-psychotropic CB1 agonists could represent novel drugs to treat motility disorders associated with inflammatory diarrhoea.
Source, Graphs and Figures: Central and peripheral cannabinoid modulation of gastrointestinal transit in physiological states or during the diarrhoea induced by croton oil
