Angela A. Coutts

Further evidence for the presence of cannabinoid CB1 receptors in guinea-pig small intestine.

1 CP 50,556, CP 55,940, nabilone, CP 56,667, Δ9‐tetrahydrocannabinol (THC) and cannabinol each inhibited electrically‐evoked contractions of the myenteric plexus‐longitudinal muscle preparation of guinea‐pig small intestine in a concentration‐related manner. The IC50 values of these cannabinoids, respectively 3.45, 3.46, 30.61, 162.94, 214.63, and 3913.5 nM, correlate well with previously obtained potency values for displacement of [3H]‐CP 55,940 from cannabinoid binding sites. 2 Electrically‐evoked contractions of the myenteric plexus‐longitudinal muscle preparation were also inhibited by AM 630 (6‐iodo‐pravadoline) and by WIN 55,212‐2 (IC50 = 1923.0 and 5.54 nM, respectively). The present finding that AM 630 is an agonist, contrasts with a previous observation that it behaves as a cannabinoid receptor antagonist in the mouse isolated vas deferens. 3 SR141716A produced dose‐related parallel rightward shifts in the log concentration‐response curves of CP 55,940, WIN 55,212‐2, THC and AM 630 for inhibition of electrically‐evoked contractions of the myenteric plexus‐longitudinal muscle preparation. SR141716A (1 μm) did not reverse the inhibitory effects of normorphine and clonidine on electrically‐evoked contractions or potentiate the contractile response to acetylcholine. 4 Doses of naloxone and yohimbine that reversed the inhibitory effects of normorphine or clonidine on electrically‐evoked contractions of the myenteric plexus‐longitudinal muscle preparation did not affect the inhibitory response to WIN 55,212‐2. 5 Electrically‐evoked release of acetylcholine from strips of myenteric plexus‐longitudinal muscle was decreased by 200 nM CP 55,940 and this inhibitory effect was almost completely reversed by 1 μm SR141716A. Acetylcholine‐induced contractions were not affected by 200 nM CP 55,940. 6 These results support the hypothesis that guinea‐pig small intestine contains prejunctional cannabinoid CB1 receptors through which cannabinoids act to inhibit electrically‐evoked contractions by reducing release of the contractile transmitter, acetylcholine. 7 THC was found to be more susceptible to antagonism by SR141716A than CP 55,940 or AM 630, raising the possibility that guinea‐pig small intestine contains more than one type of cannabinoid receptor. 8 At concentrations of 10 nM and above, SR141716A produced small but significant increases in the amplitude of electrically‐evoked contractions of the myenteric plexus‐longitudinal muscle preparation suggesting that this tissue may release an endogenous cannabinoid receptor agonist or that some cannabinoid receptors in this tissue are precoupled and that SR141716A can reduce the number of receptors in this state.

Actions of cannabinoid receptor ligands on rat cultured sensory neurones: implications for antinociception

Cannabinoids modulate nociceptive processing in models of acute, inflammatory and neuropathic pain. We have investigated the location and function of cannabinoid receptors on cultured neonatal dorsal root ganglion (DRG) neurones and F-11 cells, a dorsal root ganglionxneuroblastoma hybridoma which displays several of the features of authentic DRG neurones. CB(1) receptor immunolabelling was observed on the cell bodies and as fine puncta on processes of both cultured DRG neurones and F-11 cells. Additionally, fluorescence-activated cell sorting (FACS) analysis provided evidence that both CB(1) and CB(2) receptors are expressed on populations of cells within the cultured DRG and F-11 cells. The cannabinoid receptor agonist (+)-WIN55212 (10 and 100 nM) inhibited the mean voltage-activated Ca(2+) current in DRG neurones by 21% and 30%, respectively. The isomer, (-)-WIN55212 (10 and 100 nM) produced significantly less inhibition of 6% and 10% respectively. The CB(1) selective receptor antagonist SR141716A (100 nM) enhanced the peak high voltage-activated Ca(2+) current by 24% and simultaneous application of SR141716A (100 nM) and (+)-WIN55212 (100 nM) resulted in a significant attenuation of the inhibition obtained with (+)-WIN55212 alone. These data give functional evidence for the hypothesis that the analgesic actions of cannabinoids may be mediated by presynaptic inhibition of transmitter release in sensory neurones.

Inhibition by cannabinoid receptor agonists of acetylcholine release from the guinea‐pig myenteric plexus

1 The dose‐related inhibition of the twitch responses of the myenteric plexus‐longitudinal muscle preparation of the guinea‐pig small intestine by cannabinoid (CB) agonists, (+)‐WIN 55212 and CP 55940 during stimulation at 0.1 Hz with supramaximal voltage was confirmed. These agonists inhibited acetylcholine (ACh) release in the presence of physostigmine (7.7 μM) thus indicating a prejunctional site of action. 2 Inhibition of twitch responses and ACh release by CB agonists was reversed by the CB1‐selective cannabinoid receptor antagonist, SR141716A. Dose‐response curves to (+)‐WIN 55212 and CP 55940 were shifted to the right, with no reduction of maximal response, by pretreatment with SR141716A (31.6–1000 nM), but not its vehicle, Tween 80 (1 μM). However, at very high concentrations (25–400 μM), Tween 80 itself caused a dose‐related inhibition of the twitch response which was significantly reduced in the presence of SR141716A (1 μM). The opioid receptor antagonist, naloxone (1 μM) had no significant effect on the inhibition by CP 55940 of the twitch response. 3 (+)‐WIN 55212, CP 55940 and Tween 80 (50 μM) had no effect on responses to exogenous ACh, confirming that their actions were prejunctional. SR141716A (1 μM) did not increase the sensitivity of the longitudinal muscle to either ACh or histamine, but inhibited the responses to high doses of ACh. 4 The (−)‐enantiomer of WIN 55212, was approximately 300 times less active than the (+) enantiomer in inhibiting the twitch response, had no CB1 antagonist activity against the active isomer and did not inhibit the release of ACh in the presence of physostigmine. 5 The dissociation constant (KD) values for SR 141716A against the inhibitory effect of (+)‐WIN 55212 and CP 55940 on the twitch response were 12.07 nM (95% confidence intervals 8.55 and 20.83) and 6.44 nM (95% confidence intervals 4.70 and 10.24), respectively. In experiments in which the release of ACh was inhibited by (+)‐WIN 55212, the KD values were 9.21 nM and 10.53 nM at SR141716A concentrations of 31.6 nM and 100 nM, respectively. The KD values for the antagonism by naloxone of the inhibition of the twitch responses and the inhibition of ACh release by normorphine in this preparation were found to be 2.38±0.69 nM and 2.00±0.9 nM, respectively. 6 During maximal inhibition of ACh release by (+)‐WIN 55212, the addition of normorphine (400 nM) caused a further significant decrease in ACh output. 7 SR141716A alone produced a significant increase in ACh release in both the absence and presence of exogenous cannabinoid drugs, hence we conclude that it has a presynaptic site of action. We also conclude that SR141716A acts either by antagonizing the effect of an endogenous CB1 receptor agonist or by having an inverse agonist effect at these receptors.

Localisation of cannabinoid CB1 receptor immunoreactivity in the guinea pig and rat myenteric plexus

Activation of cannabinoid CB1 receptors inhibits gastrointestinal motility, propulsion, and transit, whereas selective antagonism of these receptors has the opposite effects, suggesting the presence of endocannabinoid tone. Supporting evidence for presynaptic CB1 receptors on myenteric neurons has been found in vitro. In this study, selective CB1 receptor antibodies and neuronal markers were used to identify and characterise myenteric neurons expressing cannabinoid receptors. Whole mounts of rat and guinea pig myenteric preparations were dually labelled with antibodies against the CB1 receptor and choline acetyltransferase, neurofilament proteins, calbindin, calretinin, synapsin I, microtubule‐associated protein‐2, calcitonin gene‐related peptide, or substance P. The pattern of CB1 receptor labelling and the neurochemical classification of CB1 receptor‐positive cells were markedly influenced by the species and fixation procedure. Virtually all choline acetyltransferase‐immunoreactive myenteric neurons expressed CB1 receptors in ganglia from both species. Subpopulations of neurons identified with calbindin, calretinin, and microtubule‐associated protein‐2 did not express CB1 receptors. A few calcitonin gene‐related peptide‐ and substance P‐positive somata coexpressed CB1 receptor immunoreactivity but showed little colocalisation on individual fibres. There was a close association between CB1 receptor immunoreactivity and fibres labelled for synaptic protein, suggesting a role in the modulation of transmitter release. Functional responses to cannabinoids in the presence of hexamethonium suggest further that CB1 receptors occur on excitatory motoneurons. In conclusion, CB1 receptors are expressed on a variety of cholinergic sensory, interneuronal, and motor neurons in myenteric ganglia. J. Comp. Neurol. 448:410–422, 2002.

Functional expression of cell surface cannabinoid CB1 receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons

At present, little is known about the mechanisms by which cannabinoids exert their effects on the central nervous system. In this study, fluorescence imaging and electrophysiological techniques were used to investigate the functional relationship between cell surface cannabinoid type 1 (CB(1)) receptors and GABAergic synaptic transmission in cultured hippocampal neurons. CB(1) receptors were labelled on living neurons using a polyclonal antibody directed against the N-terminal 77 amino acid residues of the rat cloned CB(1) receptor. Highly punctate CB(1) receptor labelling was observed on fine axons and at axonal growth cones, with little somatic labelling. The majority of these sites were associated with synaptic terminals, identified either with immunohistochemical markers or by using the styryl dye FM1-43 to label synaptic vesicles that had undergone active turnover. Dual labelling of neurons for CB(1) receptors with either the inhibitory neurotransmitter GABA or its synthesising enzyme glutamate decarboxylase, demonstrated a strong correspondence. The immunocytochemical data was supported by functional studies using whole-cell patch-clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs). The cannabinoid agonist WIN55,212-2 (100nM) markedly inhibited (by 77+/-6.3%) the frequency of pharmacologically-isolated GABAergic mIPSCs. The effects of WIN55,212-2 were blocked in the presence of the selective CB(1) receptor antagonist SR141716A (100nM).In conclusion, the present data show that cell surface CB(1) receptors are expressed at presynaptic GABAergic terminals, where their activation inhibits GABA release. Their presence on growth cones could indicate a role in the targeting of inhibitory connections during development.

Anti-inflammatory property of the cannabinoid receptor-2-selective agonist JWH-133 in a rodent model of autoimmune uveoretinitis

Previous studies have shown that cannabinoids have anti‐inflammatory and immune‐modulating effects, but the precise mechanisms of action remain to be elucidated. In this study, we investigated the effect of JWH 133, a selective agonist for cannabinoid receptor 2, the main receptor expressed on immune cells, in a model of autoimmune disease, experimental autoimmune uveoretinitis (EAU). JWH 133 suppressed EAU in a dose‐dependent manner (0.015–15 mg/kg), and the suppressive effect could be achieved in the disease‐induction stage and the effector stage. Leukocytes from mice, which had been treated with JWH 133, had diminished responses to retinal peptide and mitogen Con A stimulation in vitro. In vivo JWH 133 treatment also abrogated leukocyte cytokine/chemokine production. Further in vitro studies indicated that JWH 133 down‐regulated the TLR4 via Myd88 signal transduction, which may be responsible for its moderate, suppressive effect on antigen presentation. In vivo JWH 133 treatment (1 mg/kg) also suppressed leukocyte trafficking (rolling and infiltration) in inflamed retina as a result of an effect on reducing adhesion molecules CD162 (P‐selectin glycoprotein ligand 1) and CD11a (LFA‐1) expression on T cells. In conclusion, the cannabinoid agonist JWH 133 has a high in vivo, anti‐inflammatory property and may exert its effect via inhibiting the activation and function of autoreactive T cells and preventing leukocyte trafficking into the inflamed tissue.

Cannabinoids and the digestive tract.

In the digestive tract there is evidence for the presence of high levels of endocannabinoids (anandamide and 2-arachidonoylglycerol) and enzymes involved in the synthesis and metabolism of endocannabinoids. Immunohistochemical studies have shown the presence of CB1 receptors on myenteric and submucosal nerve plexuses along the alimentary tract. Pharmacological studies have shown that activation of CB1 receptors produces relaxation of the lower oesophageal sphincter, inhibition of gastric motility and acid secretion, as well as intestinal motility and secretion. In general, CB1-induced inhibition of intestinal motility and secretion is due to reduced acetylcholine release from enteric nerves. Conversely, endocannabinoids stimulate intestinal primary sensory neurons via the vanilloid VR1 receptor, resulting in enteritis and enhanced motility. The endogenous cannabinoid system has been found to be involved in the physiological control of colonic motility and in some pathophysiological states, including paralytic ileus, intestinal inflammation and cholera toxin-induced diarrhoea. Cannabinoids also possess antiemetic effects mediated by activation of central and peripheral CB1 receptors. Pharmacological modulation of the endogenous cannabinoid system could provide a new therapeutic target for the treatment of a number of gastrointestinal diseases, including nausea and vomiting, gastric ulcers, secretory diarrhoea, paralytic ileus, inflammatory bowel disease, colon cancer and gastro-oesophageal reflux conditions.

Comparison of novel cannabinoid partial agonists and SR141716A in the guinea-pig small intestine

The controversial nature of the CB1 receptor antagonist, SR141716A, in the guinea‐pig small intestine was investigated by comparing it with four analogues of Δ8‐tetrahydrocannabinol (Δ8‐THC): O‐1184, O‐1238, O‐584 and O‐1315. These compounds (10–1000 nM) inhibited the electrically‐evoked contractions with a rank order of potency of O‐1238>O‐1184>O‐584>O‐1315. Log concentration‐response curves for O‐1238, O‐1184 and O‐1315 were significantly shifted to the right by SR141716A and the maxima were significantly less than that of the CB1 agonist, WIN55212‐2, an indication of partial agonism. Partial saturation of the triple bond in O‐1184 to a cis double bond (O‐1238) increased its potency as an agonist (pEC50 from 6.42 to 7.63) and as an antagonist of WIN55212‐2, (pKB, from 8.36 to 9.49). Substitution of the terminal azide group by an ethyl group (O‐584) or removal of the phenolic hydroxyl group (O‐1315) had no significant effect on the agonist or antagonist potency. None of these analogues increased the twitch response in a manner resembling that of SR141716A. O‐1184 (10 and 100 nM) shifted the log concentration‐response curve of WIN55212‐2 for inhibition of the twitch responses to the right with pKB values of 8.29 and 8.38, respectively. We conclude that these Δ8‐THC analogues behave as partial agonists rather than silent antagonists at CB1 binding sites in this tissue. There was no evidence of antagonism of endocannabinoids thus supporting the hypothesis that, in this tissue, SR141716A is an inverse agonist of constitutively active CB1 receptors.

Cellular distribution of vanilloid VR1 receptor immunoreactivity in the guinea-pig myenteric plexus.

Recent investigations suggest that vanilloid receptor-1 (VR1) immunoreactivity occurs in the intestine. We have determined and quantified this immunoreactivity in the myenteric plexus with respect to cholinergic and neurofilament protein-positive neurones. Guinea-pig and rat preparations were dual-labelled with specific antibodies raised in rabbit or goat against vanilloid receptor-1 and against other neurochemical markers. In the rat ileum, both vanilloid receptor antibodies were co-distributed, whereas in the guinea-pig ileum and colon, tertiary fibres were also detected with the goat antibody. In the guinea-pig, all vanilloid receptor-1-immunoreactive cell bodies were choline acetyltransferase-immunopositive (100%) and showed some immunoreactivity to neurofilament proteins (NFP-200 kDa (79%) or triplet (10.8%)) or calretinin. Immunoreactive fibres in the secondary plexus co-localised with calcitonin gene-related peptide (CGRP) and with substance P, calretinin and synapsin I in the tertiary plexus. Subpopulations of cholinergic neurones including sensory, interneuronal and secretory neurones express vanilloid receptor-1. Co-localisation with substance P and calretinin in fibres suggests that vanilloid receptor-1 may be expressed by excitatory motor neurones. The association of vanilloid receptors with calcitonin gene-related peptide and synaptic protein in fibres implies a role for vanilloid receptors in neurotransmitter/neuropeptide release. Although it is likely that at least some of the vanilloid receptor-bearing fibres originate in immunopositive myenteric soma, the origin of all these fibres cannot be identified in the present study.

Vanilloid receptor type 1-immunoreactivity is expressed by intrinsic afferent neurones in the guinea-pig myenteric plexus

The enteric sensory nervous system consists of extrinsic and intrinsic components. The cellular distribution of vanilloid receptor type 1 (VR1) and its relation to the intrinsic sensory neurones were studied in myenteric plexus-longitudinal muscle preparations of rat ileum and guinea-pig ileum and colon. VR1-immunoreactivity was localized on fine fibres and expressed by ganglionic cells. In the guinea-pig myenteric plexus, a proportion of VR1-immunoreactive cells co-localized with calbindin, a marker for intrinsic afferent neurones. Reverse transcription-polymerase chain reaction with rat VR1-specific primers detected VR1 mRNA in rat but not in guinea-pig preparations. We conclude that VR1 is expressed on fibres and by myenteric neurones. In the guinea-pig, VR1 is expressed by intrinsic afferent neurones but its mRNA may differ from the rat sequence in the region of the primers.