In Vitro Studies on Intestinal, Longitudinal, and Circular Muscle Preparations
Over the past three decades, Pertwee and his coinvestigators have been the leading contributors to the understanding of the in vitro effects of A9-THC, its synthetic analogs, and anandamide on intestinal motility functions (Pertwee, 2001). Their studies evolved from the observation that A9-THC inhibits the contractile response produced by electrically evoked contractions of isolated whole ileum or isolated myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. In these preparations, the contractile response is thought to be due to the electrically induced release of acetylcholine from enteric cholinergic nerves. Cannabinoids of diverse structure and activity attenuate the induced contractions with a potency order (CP55940 > WIN55212-2 > nabilone > A9-THC > cannabinol > anandamide) that is similar to their affinity rank order for the cannabinoid CBi receptor (Breivogel and Childers, 2000; Breivogel et al., 2001). Moreover, the inhibitory effect of cannabinoids can be competitively and specifically antagonized by the selective CBi receptor antagonist/inverse agonist SR141716A (Pertwee et al., 1996; Coutts and Pertwee, 1997; Croci et al., 1998; Pertwee, 2001; Pinto, Izzo, et al., 2002). Anandamide also inhibits electrically evoked acetylcholine release (Mang et al., 2001) and electrically induced contractions of both MPLM (Pertwee et al., 1995; Mang et al., 2001) and circular muscle preparations of the guinea-pig ileum (Izzo et al., 1998; Pertwee et al., 1995). However, the role of CBi receptors on the inhibitory effect of anandamide on MPLM is questioned because a large concentration of SR141716A (1 uA/) was required to reverse the induced inhibition (Mang et al., 2001) though the CBi antagonist was more effective in the circular muscle preparation (Izzo et al., 1998). These, as well as similar findings in other test systems (see Mang et al., 2001) suggest: (1) the existence of subtypes of CBi receptors to which SR141716A binds with a lower affinity and (2) that anandamide acts on both CBi and non-CBi receptors to inhibit the discussed electrically evoked effects on GI motility. In addition, anandamide is also an endovanilloid and stimulates both basal acetylcholine release and basal MPLM tone in the guinea-pig ileum via the activation of vanilloid VR1 receptor (Mang et al., 2001). Moreover, other VR1 agonists such as capsaicin and pipeline cause an initial contraction of guinea-pig isolated ileum followed by blockade of contraction of circular and longitudinal muscles of this tissue evoked either by electrical field stimulation (ESF) or mesenteric nerve stimulation (Takai et al., 1990; Jin et al., 1990). Pertwee (2001) has summarized published evidence supporting the notion that cannabinoids act selectively and prejunctionally via CB, receptors to inhibit electrically evoked contractions of guinea-pig MPLM. The following are the characteristics of cannabinoid actions in this tissue:
1. Inhibition of evoked fast excitatory synaptic potentials of myenteric neurons by CB, agonists.
2. Presence of CB, receptor markers in the myenteric and submucosal plexuses.
3. Cannabinoid-induced CB,-receptor-mediated inhibition of acetylcholine release.
4. Blockade of the evoked contractions by tetrodotoxin.
5. Only CB, receptor antagonists counter the inhibitory effects of cannabinoids.
6. Cannabinoid agonists do not reduce the contractile response of such preparations to exogenously added acetylcholine, carbachol, substance P, or histamine, each of which directly acts on the smooth muscle. However, cannabinoids prevent the ability of 5-HT or GABA to produce contractions of these muscle preparations as the latter agents act presynaptically to increase acetylcholine release.
7. The more stable analog of anandamide, methanandamide, does not affect indomethacin-induced phasic contractions of guinea-pig ileum that are generated at the muscle level as they are left unchanged by tetrodotoxin (Heinemann et al., 1999).
8. CEi, receptor stimulation does not directly suppress smooth muscle activity (Izzo et al., 1998; Coutts and Pertwee, 1997).
Although 2-AG was the first endocannabinoid to be discovered in the intestine, virtually nothing has been published on its effects in the GI system. A recent study shows that 2-AG in the presence of a nonselective COX inhibitor (indomethacin) and an NO synthase inhibitor (NG-nitro-L-arginine) causes concentration-dependent and tetrodotoxin-sensitive contractions of longitudinal muscle strips of isolated distal colon of the guinea pig (Kojima et al., 2002). Thus, the observed contractions appear to be due to the stimulation of enteric nerves rather than the activation of release of prostanoids or NO. The induced contractions were markedly attenuated by atropine and partially blocked by either a ganglionic blocker (hexamethonium) or a lipoxygenase inhibitor (nordihy-droguaiaretic acid) pretreatment but were not affected by cannabinoid CB (SR141716A)-, CB2 (SR144528)- or vanilloid VR1 (capsazepine)-receptor antagonists. In this study, anandamide also produced similar but less robust neurogenic contractions, whereas the synthetic cannabinoid WIN55212-2 had no effect. In addition, both 2-AG and anandamide failed to contract circular smooth muscle preparations of the guinea-pig distal colon. These results suggest that 2-AG and anandamide induce contractions of the longitudinal muscle preparations of the guinea-pig colon mainly via stimulation of myenteric cholinergic neurons and that neither cannabinoid CB[/CB2 or vanilloid VR1 receptors contribute to the contractile response, but the effect may be mediated by one or more lipoxygenase metabolites of 2-AG. For example, leukotrienes LTC4, LTD4, and LTE4 (but not LTB4) cause concentration-dependent contractions of guinea-pig ileum (Gardiner et al., 1990). Downstream COX metabolites of endocannabinoids such as prostaglandins (PGs) PGE,, PGE2, 8-iso-PGE2, and PGE2a also cause contractions of isolated guinea-pig ileum (Grobovic and Radmanovic, 1987; Ishizawa and Miyaraki, 1975; Sametz et al., 2000).
From the earlier discussion, it is apparent that GI motility, in general, and peristalsis, in particular, are mainly controlled by the ENS. Peristalsis is a coordinated motor activity that allows the intestine to propel its content anal-ward. Peristalsis consists of a preparatory phase in which the longitudinal muscle contracts in response to effluent infusion and a subsequent emptying phase in which coordinated contractions of circular muscle expel the effluent from the mouth to the anal end.
Recent studies by the leading investigator in this field (Izzo, Mascolo, et al., 2000) have demonstrated that synthetic cannabinoids (CP55940 and WIN55212-2) inhibit peristalsis induced in segments of guinea pig isolated ileum by continuous luminal fluid infusion. Indeed, during the preparatory phase of peristalsis, these CB CB2 cannabinoid agonists decreased both the longitudinal smooth muscle reflex contraction and resistance of the intestinal wall to the infused liquid (compliance) but increased threshold pressure and the volume required to elicit peristalsis in a selective and SR141716A-sensitive manner. In addition, the cited agents decreased maximal ejection pressure during the emptying phase of peristalsis, which was also countered by SR141716A pretreatment. The inhibitory effect of cannabinoids seems to be extended to inhibition of electrically evoked peristaltic activity in isolated distal colon of mice, because WIN55212-2 reduced intraluminal pressure, longitudinal displacement of intestinal segment (shortening), and ejected fluid volume during peristaltic contractions in a dose-dependent and SR141716A-sensitive manner (Mancinelli et al., 2001). Heinemann and co-workers (1999) have recently shown that methanandamide also inhibits distension-induced propulsive motility of luminally perfused guinea-pig isolated ileum by increasing peristaltic pressure threshold in a SR141716A-sensitive and a dose-dependent fashion. To explain this effect, the authors have proposed that methanandamide stimulates CB! receptors to: (1) activate inhibitory enteric motor neurons that oppose distension-induced peristalsis by releasing both NO- and apamine-sensitive neurotransmitters, as L-NAME (an NO synthase inhibitor) and apamine (an inhibitor of fast inhibitory junction potentials mediated by transmitters of inhibitory motor neurons in the guinea-pig small intestine) attenuate the induced peristaltic activity and (2) inhibit excitatory enteric motor neurons that mediate ascending enteric reflex (AER) contraction of guinea-pig intestinal circular muscle in response to distension. Heinemann et al. (1999) further suggest that activation of CB, receptors in the guinea-pig ileum suppresses propulsive peristalsis and AER contractions by inhibiting both cholinergic and noncholinergic transmission, because these inhibitory effects of methanandamide were not only preserved but enhanced under conditions in which peristalsis is maintained via endogenous TKs (i.e., the blockade of cholinergic transmission with atropine or hexamethonium and the production of peristalsis by naloxone via release of endogenous TKs (Holzer et al., 1998). Supporting evidence for the latter notion comes from the ability of synthetic cannabinoid WIN55212-2 to inhibit cholinergic and nonadrenergic noncholinergic contractions evoked by electrical stimulation of circular muscle of the guinea-pig ileum in a SR141716A-sensitive manner (Izzo et al., 1998). Following an initial contraction, the vanilloid VR1 agonist capsaicin can depress intestinal peristalsis via the release of NO (Bartho and Holzer et al., 1995), an effect is also shared by anandamide (Stefano et al., 1997). Although the effects of anandamide on in vitro intestinal peristalsis remain unknown, it does stimulate basal release of acetylcholine and longitudinal muscle tone in the guinea-pig ileum preparation via VR1 receptor stimulation (see earlier). Capsaicin-induced neurogenic contraction of the smooth muscle of guinea-pig intestine also involves release of acetylcholine and TKs from enteric neurons (Bartho and Holzer, 1995). In addition, although both anandamide and pipeline (a vanilloid VR1 agonist) inhibit GI transit in vivo (Izzo, Capasso, et al., 2001), the VR1 receptor is not involved in ananda-mide-induced inhibition, as the effect was sensitive to SR141716A but not to pretreatment with either capsazepine (a VR1 receptor antagonist) or capsaicin (another VR1 agonist).
The effect of 2-AG on peristaltic activity has not yet been reported. However, downstream metabolites of both endocannabinoids can differentially affect propulsive peristalsis in fluid-perfused segments of the guinea-pig small intestine (Shahbazian et al., 2002). These findings indicate that PGs PGE! and PGE2 decrease peristaltic performance, whereas leukotriene LTD4 and PG PGD2 increase peristalsis pressure threshold.
The basic propulsive movement of the GIT is peristalsis in which a contractile ring appears around the gut and then moves forward. The usual stimulus for peristalsis is distension of the gut wall, which stimulates the ENS to initiate peristaltic movement thus allowing the food to be propelled analward.
The discussed in vitro studies have well established that CB C "H2 agonists inhibit electrically evoked contractions of ileum, guinea-pig MPLM contractions as well as peristalsis in isolated intestine through the activation of prejunctional cannabinoid CE^ receptors via inhibition of release of acetylcholine and other excitatory neurotransmitters from the enteric, cholinergic, nonadrenergic, and noncholinergic nerves, respectively. In vivo studies investigating the effects of drugs on GIT motility employ techniques which measure the passage of a nonadsorbable transit marker or glass beads in the GIT of several species, and these agents are administered either orally, intraduodenally, or in distal colon.
In line with the in vitro findings, in vivo studies show that structurally diverse exogenous cannabinoids [A8-THC, A9-THC, nabilone, cannabinol, WIN55212-2, CP55940, HU210, and arachidonoyl-2'-chloroethylamide (ACEA, a selective CE^ agonist)] attenuate gut motility, intestinal transit, and intestinal secretions in several species (Casu et al., 2003; Pertwee, 2001; Pinto, Capasso, et al., 2002; Pinto, Izzo, et al., 2002; Izzo, Capasso, et al., 2001; Landi et al., 2002). The inhibitory in vivo GIT effects of these cannabinoids were also SR141716A sensitive, implying an important role for the cannabinoid CBi receptor. The endocannabinoid anandamide causes similar inhibitory actions on intestinal motility, as it was shown to dose-dependently and selectively reduce the passage of both charcoal marker in the upper GIT (Calignano et al., 1997; Izzo, Capasso, et al., 2001) and glass beads in distal colon (Pinto, Izzo, et al., 2002) of mice in an SR141716A-sensitive manner. Moreover, the selective CB! receptor agonist and an analog of anandamide, ACEA, more potently inhibited the colonic propulsion. As expected, a selective inhibitor of anandamide cellular reuptake, VDM11, with little affinity for either CBi or CB2 receptors, was shown to significantly inhibit colonic propulsion in a manner sensitive to SR141716A. However, another inhibitor of anandamide reuptake, AM404, which is structurally similar and equipotent to VDM11 on the anandamide transporter, was found to be inactive on small intestine motility in mice (Calignano et al., 1997). This lack of effect on intestinal motility is puzzling and is probably either due to a tissue specificity of the tonic action of endocannabinoids or because AM404 also stimulates vanilloid VR1 receptors, activation of which might have masked the inhibitory action of AM404 on intestinal motility (Pinto, Izzo et al., 2002). The latter notion appears less likely because the same group of investigators, in a previous study, has shown that exogenous administration of the endocannabinoid/endovanilloid agonist anandamide reduces intestinal motility in mice via cannabinoid CBi sites and not vanilloid VR1 sites (Izzo, Capasso, et al., 2001). However, the in vivo effects of VR1 receptor agonists seem to be complex as pipeline inhibited but capsaicin failed to alter GIT motility. The effect of pipeline is thought to involve capsaicin-sensitive neurons but not vanilloid VR1 receptors, because the inhibitory effect of pipeline (Izzo, Capasso, et al., 2001) was strongly inhibited in capsaicin-pretreated mice. In addition, the effects of capsaicin differ in different sections of GIT (Shibata et al., 2002).
The basal anandamide level in the mouse colon is about 10 times greater than its concentration in the small intestine (Pinto, Izzo, et al., 2002), which implies that the colon requires more anandamide to control its motility and other functions. In line with this notion, the antitransit activity of A9-THC appears to be somewhat selective for the small intestine relative to the colon (Shook and Burks, 1989). Noladin ether (2-arachidonoylglycerol ether) is a putative endocannabinoid which also possesses antitransit properties because it reduces defecation in mice (Hanus et al., 2001). Although 2-AG is present both in small intestine and colon (Pinto, Izzo, et al., 2002), the in vivo effects of 2-AG on intestinal motility have not yet been investigated. However, downstream metabolites of endocannabinoids (e.g., PGs) have region- and muscle-layer-specific in vivo effects in different areas of the GIT. For example, PGE2 in the proximal colon inhibits myoelectric and mechanical activity at low doses but at higher doses causes marked excitation, whereas in the distal colon only excitation is observed (Burakoff and Percy, 1992).
In Vitro Isolated Gastric Tissue and In Vivo Gastric Emptying Studies
Emptying of the stomach is controlled to a moderate degree by stomach factors (such as degree of filling and excitatory effect of gastrin) but mainly by feedback signals from the duodenum including enterogastric nervous system feedback reflexes and hormonal feedback. Stomach emptying is promoted by the intense peristaltic contractions of the stomach antrum. In general, vagal stimulation increases the force and frequency of contractions, whereas sympathetic stimulation decreases both of these parameters. The endocannabinoid anandamide has recently been shown to attenuate cholinergically mediated contractions of isolated rat gastric smooth muscle preparation produced by EFS (Storr et al., 2002). Anandamide also attenuated nonadrenergic, noncholinergic-mediated relaxant neural responses (in the presence of atropine and guanethidine) produced by EFS in this preparation. Because the depressant effects of anandamide were countered by the CB1 antagonist AM630, it is suggested that CB1 receptors play an important role in the attenuation of excitatory cholinergic and inhibitory NANC neurotransmission in the rat isolated gastric fundus preparation. The aminoalkylindole cannabinoid WIN55212-2 also produces similar depressant effects in this fundus preparation. However, AM630 failed to counter WIN55212-2's inhibitory effect, suggesting further cannabinoid receptors may be involved. The effect of cannabinoid agonists seems to be prejunctional and not postjunctional because: (1) they did not alter NO- and VIP-induced relaxation of the stomach preparation and (2) CB1 receptor markers are found in prejunctional nerve terminals rather than the stomach tissue (see preceding text). To date, the effect of endocannabinoids on in vivo models of gastric emptying has not been reported. However, in line with the above in vitro findings, several in vivo studies (Izzo, Mascolo, Pinto, et al., 1999; Izzo, Mascolo, Capasso, et al., 1999; Landi et al., 2002; Shook and Burks, 1989) have shown that structurally diverse exogenous cannabinoids can delay gastric emptying in several species with an ED50 order of potency (CP55940 > WIN55212-2 >)9-THC > cannabinol) that is similar to: (1) their potency rank order in attenuating electrically evoked contractions of isolated ileum preparations and (2) their affinity rank order for cannabinoid CB1 receptor (see earlier). In addition, the cited studies show that the CB1 receptor antagonist SR141716A but not the CB2 antagonist SR144528, in a dose-dependent manner, counters the inhibitory effects of these CB1/CB2 receptor agonists on gastric emptying in several species. In addition to delaying gastric emptying, such cannabinoids (e.g., A9-THC) reduce intragastric pressure, pyloric contractility, and greater curvature contractile activity of the stomach in an SR141716A-sensitive manner (Krowicki et al., 1999). Besides preventing such parameters of gastric contractility, some cannabinoids (e.g., WIN55212-2) have been reported to inhibit transient lower esophageal sphincter relaxation (TLESR) and gastroesophageal reflux (major aberrant factors in gastroesophageal disease) in a dose-dependent and SR141716A-sensitive manner (Lehmann et al., 2002).
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