Comparison of cannabinoid binding sites in guinea-pig forebrain and small intestine

We have investigated the nature of cannabinoid receptors in guinea-pig small intestine by establishing whether this tissue contains cannabinoid receptors with similar binding properties to those of brain CB1 receptors. The cannabinoids used were the CB1-selective antagonist SR141716A, the CB2-selective antagonist SR144528, the novel cannabinoid receptor ligand, 6'-azidohex-2'-yne-delta8-tetrahydrocannabinol (O-1184), and the agonists CP55940, which binds equally well to CB1 and CB2 receptors, and WIN55212-2, which shows marginal CB2 selectivity. [3H]-CP55940 (1 nM) underwent extensive specific binding both to forebrain membranes (76.3%) and to membranes obtained by sucrose density gradient fractionation of homogenates of myenteric plexus-longitudinal muscle of guinea-pig small intestine (65.2%). Its binding capacity (Bmax) was higher in forebrain (4281 fmol mg(-1)) than in intestinal membranes (2092 fmol mg(-1)). However, the corresponding KD values were not significantly different from each other (2.29 and 1.75 nM respectively). Nor did the Ki values for its displacement by CP55940, WIN55212-2, O-1184, SR141716A and SR144528 from forebrain membranes (0.87, 4.15, 2.85, 5.32 and 371.9 respectively) differ significantly from the corresponding Ki values determined in experiments with intestinal membranes (0.99, 5.03, 3.16, 4.95 and 361.5 nM respectively). The Bmax values of [3H]-CP55940 and [3H]-SR141716A in forebrain membranes did not differ significantly from each other (4281 and 5658 fmol mg(-1)) but were both greater than the Bmax of [3H]-WIN55212-2 (2032 fmol mg(-1)). O-1184 (10 or 100 nM) produced parallel dextral shifts in the log concentration-response curves of WIN55212-2 and CP55940 for inhibition of electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation, its KD values being 0.20 nM (against WIN55212-2) and 0.89 nM (against CP55940). We conclude that cannabinoid binding sites in guinea-pig small intestine closely resemble CB1 binding sites of guinea-pig brain and that 0-1184 behaves as a cannabinoid receptor antagonist in the guinea-pig myenteric plexus-longitudinal muscle preparation.     

Excitatory transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of cannabinoid CB1 receptors

1. The effect of cannabinoid drugs has been investigated on cholinergic and non-adrenergic non-cholinergic (NANC) contractile responses to the circular smooth muscle of guinea-pig ileum elicited by electrical field stimulation (EFS). 2. The cannabinoid receptor agonist WIN 55,212-2 (1-1000 nM) and the putative endogenous ligand anandamide (0.1-100 microM) both produced a concentration-dependent inhibition of the cholinergic (9-57% and 1-51% inhibition) and NANC (9 55% and 2-57% inhibition) contractile responses. WIN 55,212-2 and anandamide did not modify the contractions produced by exogenous acetylcholine or substance P. 3. Apamin (30 nM), a blocker of Ca2+-activated K+ channels, reduced the inhibitory effect of WIN 55,212-2 on cholinergic, but not NANC, contractile response. NG-nitro-L-arginine methyl ester (100 microM), an inhibitor of nitric oxide synthase, or naloxone (1 microM), an opioid receptors antagonist, did not modify the inhibitory effect of WIN 55,212-2 on both cholinergic and NANC contractions. 4. The inhibitory effects of WIN 55,212-2 and anandamide on both cholinergic and NANC contractile response was competitively antagonized by the cannabinoid CB1 receptor antagonist SR 141716A (10-1000 nM). 5. In absence of other drugs, SR 141716A (1-1000 nM) enhanced cholinergic (1-45% increase) and NANC (2-38% increase) contractile responses elicited by electrical stimulation, but did not modify the contractions produced by acetylcholine or substance P. 6. It is concluded that activation of prejunctional cannabinoid CB1 receptors produces inhibition of cholinergic and NANC excitatory responses in the guinea-pig circular muscle. The inhibition of cholinergic (but not NANC) transmission involves activation of apamin-sensitive K+ channels. In addition, an endogenous cannabinoid ligand could inhibit cholinergic and NANC transmission in the guinea-pig ileal circular muscle.     

Antagonism between the anti-inflammatory activity of the cannabinoid WIN 55212-2 and SR 141716A

The CB1/CB2 receptor agonist WIN 55212-2 (0.75 mg/kg, i.v.) caused a significant reduction in neurogenic plasma extravasation induced by electrical stimulation of the saphenous nerve in anesthetized rats; WIN 55212-2 at 2.5-10 mg/kg, s.c., also produced a significant reduction in the carrageenan-induced paw edema in conscious rats. The selective CB1 antagonist SR 141716A (0.075-0.75 mg/kg i.v.) antagonized the WIN 55212-2 effects in the plasma extravasation model and antagonized the WIN 55212-2 (2.5 mg/kg, s.c.)-induced decreases in rectal temperature and increases in tail-flick latencies. However, SR 141716A (10 mg/kg, p.o.) failed to antagonize the effects of Win 55212-2 (2.5 mg/kg, s.c.) in the carrageenan model, suggesting that cannabinoid receptors found in the periphery may be able to modulate inflammatory processes in rats.     

Systemic candidiasis with acute Epstein-Barr virus infection

The binding of a classical cannabinoid agonist, [3H]R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2 ,3-de]-1,4-benzoxazin-6-yl)(1-napthalenyl)methanone monomethanesulfonate ([3H] WIN55212-2), and a selective cannabinoid receptor (CB1) antagonist, N-(piperidin-1-yl)-5-(4-chlorophenyl)1-(2,4-dichlorophenyl)-4-meth yl-1H-pyrazole-3-carboxamide hydrochloride ([3H]SR141716A), to rat cannabinoid receptors was evaluated using rat cerebellar membranes. Guanine nucleotides inhibited [3H]WIN55212-2 binding by approximately 50% at 10 microM and enhanced [3H]SR141716A binding very slightly. In the same tissue, the binding of guanosine 5'-O-[gamma-[35S]thio]triphosphate ([35S]GTP-gamma-S) was characterized and the influence of cannabinomimetics evaluated on this binding. Cannabinoid receptor agonists enhanced [35S]GTP-gamma-S binding, whereas SR141716A was devoid of action by itself but antagonized the action of cannabinoid receptor agonists. The good correlation obtained between the half maximum efficient concentration (EC50) values in [35S]GTP-gamma-S binding and the IC50 values [3H]WIN55212-2 binding shows that [35S]GTP-gamma-S binding could be a good functional assay for brain cannabinoid receptors.     

Electrically evoked acetylcholine release from hippocampal slices is inhibited by the cannabinoid receptor agonist, WIN 55212-2, and is potentiated by the cannabinoid antagonist, SR 141716A

This study examined the effect of the cannabinoid receptor agonist, WIN 55212-2, on the electrically evoked release of [14C]acetylcholine (ACh) from superfused brain slices from the hippocampus, a region with a high density of cannabinoid receptors. A comparison was also made with [14C]ACh release from the nucleus accumbens, which has relatively fewer cannabinoid receptors. In the hippocampal slices, WIN 55212-2 produced a dose-dependent inhibition of [14C]ACh release, with an EC50 of 0.03 microM and a maximal inhibition of 81% at 1 microM. In the nucleus accumbens slices, WIN 55212-2 produced a weak inhibition of [14C]ACh release, which did not quite reach statistical significance. The inhibition of electrically evoked hippocampal [14C]ACh release by WIN 55212-2 could be prevented by the cannabinoid receptor antagonist, SR 141716A (EC50, 0.3-1.0 microM). In addition to antagonizing the effects of WIN 55212-2, SR 141716A alone produced a 2-fold potentiation of the electrically stimulated [14C]ACh release in this region (EC50, 0.1-0.3 microM). By contrast, in nucleus accumbens slices, no potentiation of the stimulated release of [14C]ACh release by SR 141716A was observed. Basal [14C]ACh release was unaffected by WIN 55212-2 or SR 141716A in either area. These results suggest that cannabinoid receptor activation can produce a strong inhibition of ACh release in the hippocampus. Furthermore, the potentiation of ACh release in the hippocampus by SR 141716A alone suggests either that this compound is an inverse agonist at cannabinoid receptors or it is antagonizing the actions of an endogenous ligand acting on these receptors.     

Classifying extraintestinal non-typhoid Salmonella infections

The cannabinoid receptors expressed in the mouse neuroblastoma X rat glioma NG108-15 cell and the rat pituitary tumor GH4C1 cell were determined by polymerase chain reaction, dideoxysequencing and pharmacologically. The CB1 but not the CB2 or CB1A cannabinoid receptor was found in both cell lines. The cDNA identified in GH4C1 cells corresponds to the rat CB1 receptor. Interestingly, NG108-15 cells express two distinct cDNAs, one corresponds to the rat and the other to the mouse CB1 receptor. The newly developed CB1 receptor selective antagonist SR141716A was found to reverse cannabinoid agonist (WIN55212-2 or CP55940)-induced adenylyl cyclase inhibition. These results provide more direct evidence that the CB1 receptor is mediating the pharmacological actions of cannabinoids in NG108-15 and GH4C1 cells.     

SR 141716A acts as an inverse agonist to increase neuronal voltage-dependent Ca2+ currents by reversal of tonic CB1 cannabinoid receptor activity

The CB1 cannabinoid receptor antagonist SR 141716A abolished the inhibition of Ca2+ currents by the agonist WIN 55,212-2. However, SR 141716A alone increased Ca2+ currents, with an EC50 of 32 nM, in neurons that had been microinjected with CB1 cRNA. For an antagonist to elicit an effect, some receptors must be tonically active. Evidence for tonically active CB1 receptors was seen as enhanced tonic inhibition of Ca2+ currents. Preincubation with anandamide failed to enhance the effect of SR 141716A, indicating that anandamide did not cause receptor activity. Under Ca2+-free conditions designed to block the Ca2+-dependent formation of anandamide and sn-2-arachidonylglycerol, SR 141716A again increased the Ca2+ current. The Ca2+ current was tonically inhibited in neurons expressing the mutant K192A receptor, which has no affinity for anandamide, demonstrating that this receptor is also tonically active. SR 141716A had no effect on the Ca2+ current in these neurons, but SR 141716A could still antagonize the effect of WIN 55, 212-2. Thus, the K192 site is critical for the inverse agonist activity of SR 141716A. SR 141716A appeared to become a neutral antagonist at the K192A mutant receptor. Native cannabinoid receptors were studied in male rat major pelvic ganglion neurons, where it was found that WIN 55,212-2 inhibited and SR 141716A increased Ca2+ currents. Taken together, our results demonstrate that a population of native and cloned CB1 cannabinoid receptors can exist in a tonically active state that can be reversed by SR 141716A, which acts as an inverse agonist.     

Characterization and distribution of binding sites for [3H]-SR 141716A, a selective brain (CB1) cannabinoid receptor antagonist, in rodent brain

SR 141716A belongs to a new class of compounds (diarylpyrazole) that inhibits brain cannabinoid receptors (CB1) in vitro and in vivo. The present study showed that [3H]-SR 141716A binds with high affinity (Kd=0.61 +/- 0.06 nM) to a homogenous population of binding sites (Bmax=0.72 +/- 0.05 pmol/mg of protein) in rate whole brain (minus cerebellum) synaptosomes. This specific binding was displaced by known cannabinoid receptor ligands with the following rank order of potency SR 141716A > CP 55,940 > WIN 55212-2 = delta9-THC > anandamide. Apart from anandamide, all these compounds were found to interact competitively with the binding sites labeled by [3H]-SR 141716A. On the other hand, agents lacking affinity for cannabinoid receptors were unable to displace [3H]-SR 141716A from its binding sites (IC50 > 10 microM). In addition, the binding of [3H]-SR 141716A was insensitive to guanyl nucleotides. Regional rat brain distribution of CB1 cannabinoid receptors detected by [3H]-SR 141716A saturation binding and autoradiographic studies, showed that this distribution was very similar to that found for [3H]-CP 55,940. In vivo, the [3H]-SR 141716A binding was displaced by SR 141716A with ED50 values of 0.39 +/- 0.07 and 1.43 +/- 0.29 mg/kg following intraperitoneal and oral administration, respectively. Finally, the [3H]-SR 141716A binding sites remained significantly occupied for at least 12 hr following oral administration of 3 mg/kg SR 141716A. Taken together, these results suggest that SR 141716A in its tritiated form is a useful research tool for labeling brain cannabinoid receptors (CB1) in vitro and in vivo.     

Effects of cannabinoids on preimplantation mouse embryo development and implantation are mediated by brain-type cannabinoid receptors

We examined the relative importance of G (Gi) protein-coupled brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors in preimplantation embryo development using agonists and antagonists specific to CB1-R and CB2-R. The results establish that endogenous cannabinoid ligands, anandamide and sn-2 arachidonoylglycerol, arrest embryo development in vitro, and this effect is reversed by CB1-R antagonists SR141716A or AM 251, but not by SR144528, a CB2-R antagonist. A CB2-R selective agonist AM 663 failed to affect embryo development. These results suggest that cannabinoid effects on embryo development are mediated by CB1-R. We also observed that delta9-tetrahydrocannabinol ([-]THC) infused in the presence of cytochrome P450 inhibitors interfered with blastocyst implantation. This adverse effect was reversed by coinfusion of SR141716A. The less active stereoisomer (+)THC plus the inhibitors failed to affect implantation. Analysis of tissue levels demonstrated that uterine accumulation of (-)THC occurred when it was infused in the presence of the P450 inhibitors. These results demonstrate that the uterus and perhaps the embryo have the cytochrome P450 enzymes to metabolize (-)THC and neutralize its adverse effects on implantation. Collectively, the present study demonstrates that cannabinoid effects on embryo development and implantation are mediated by embryonic and/or uterine CB1-R, but not CB2-R.     

Analgesic, respiratory and heart rate effects of cannabinoid and opioid agonists in rhesus monkeys: antagonist effects of SR 141716A

This study characterized the antinociceptive, respiratory and heart rate effects of the cannabinoid receptor agonists Delta-9-tetrahydrocannabinol (Delta-9-THC) and WIN 55212 ((R)-(+)-2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol-[1,2,3-de]-1, 4-benzoxazin-6-yl)(1-naphtalenyl)methanone monomethanesulfonate), N-arachidonyl ethanolamide (anandamide) and the mu and kappa opioid receptor agonists heroin and U69593, alone and in conjunction with a cannabinoid receptor antagonist, SR 141716A [N-(piperidin-1-1-yl)-5-(4-chlorophenyl)-1(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] and an opioid receptor antagonist, quadazocine, in rhesus monkeys (Macaca mulatta). Using 12 adult rhesus monkeys, latencies to remove the tail from a 50 degrees C water bath, respiration in 5% CO2 and heart rate were measured. When administered alone, SR 141716A (1.8, 5.6 mg/kg i.m.) did not alter nociception, respiration or heart rate. Delta-9-THC (0.1-10 mg/kg i.m.) and WIN 55212 (0.1-10 mg/kg i.m.) dose-dependently increased antinociception and dose-dependently decreased respiratory minute and tidal volumes and heart rate. These antinociceptive, respiratory and heart rate effects were reversed by SR 141716A but not by the opioid antagonist quadazocine (1 mg/kg i.m.). Anandamide (10 mg/kg i.m.) also produced antinociception. Heroin (0.01-10 mg/kg i.m.) and U69593 (0.01-3.2 mg/kg i.m.) also dose-dependently increased antinociception and decreased respiratory and heart rate measures; these effects were antagonized by quadazocine but not by SR 141716A. These results demonstrate selective and reversible antagonism of cannabinoid behavioral effects by SR 141716A in rhesus monkeys.     

Cannabinoid-induced hypotension and bradycardia in rats mediated by CB1-like cannabinoid receptors

Previous studies indicate that the CB1 cannabinoid receptor antagonist, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1H-pyrazole-3-carboxamide HCl (SR141716A), inhibits the anandamide- and delta9-tetrahydrocannabinol- (THC) induced hypotension and bradycardia in anesthetized rats with a potency similar to that observed for SR141716A antagonism of THC-induced neurobehavioral effects. To further test the role of CB1 receptors in the cardiovascular effects of cannabinoids, we examined two additional criteria for receptor-specific interactions: the rank order of potency of agonists and stereoselectivity. A series of cannabinoid analogs including the enantiomeric pair (-)-11-OH-delta9-THC dimethylheptyl (+)-11-OH-delta9-THC dimethylheptyl were evaluated for their effects on arterial blood pressure and heart rate in urethane anesthetized rats. Six analogs elicited pronounced and long lasting hypotension and bradycardia that were blocked by 3 mg/kg of SR141716A. The rank order of potency was (-)-11-OH-delta9-THC dimethylheptyl > or = (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol > (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol > THC > anandamide > or = (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol, which correlated well with CB1 receptor affinity or analgesic potency (r = 0.96-0.99). There was no hypotension or bradycardia after palmitoylethanolamine or (+)-11-OH-delta9-THC dimethylheptyl. An initial pressor response was also observed with THC and anandamide, which was not antagonized by SR141716A. We conclude that the similar rank orders of potency, stereoselectivity and sensitivity to blockade by SR141716A indicate the involvement of CB1-like receptors in the hypotensive and bradycardic actions of cannabinoids, whereas the mechanism of the pressor effect of THC and anandamide remains unclear.     

Repression of Drosophila photoreceptor cell fate through cooperative action of two transcriptional repressors Yan and Tramtrack

Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 [[1alpha,2beta(R)5alpha]-(-)-5-(1,1-dimethylheptyl+ ++)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol], and the specific antagonist SR 141716 [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [3H] dopamine-prelabelled striatal slices were also investigated. CP 55940 (1 microM) and anandamide (10 microM) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 microM). CP 55940 (1 microM) had no effect on either KCl- or neurotransmitter-stimulated 3H-inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [3H]dopamine-prelabelied striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.     

Fertility and contraception in Europe: the case of low fertility in southern Europe

We have investigated whether there are cannabinoid CB2 receptors that can mediate cannabinoid-induced inhibition of electrically evoked contractions in the mouse vas deferens or guinea-pig myenteric plexus-longitudinal muscle preparation. Our results showed that mouse vas deferens and guinea-pig whole gut contain cannabinoid CB1 and CB2-like mRNA whereas the myenteric plexus preparation seemed to contain only cannabinoid CB1 mRNA. JWH-015 (1-propyl-2-methyl-3-( -naphthoyl)indole) and JWH-051 (1-deoxy-11-hydroxy-delta8-tetrahydrocannabinol-dimethylheptyl+ ++), which have higher affinities for CB2 than CB1 cannabinoid binding sites, inhibited electrically evoked contractions of both tissues in a concentration related manner. This inhibition was attenuated by 31.62 nM of the cannabinoid CB1 receptor selective antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride] only in the myenteric plexus preparation. Vasa deferentia from delta9-tetrahydrocannabinol-pretreated mice (20 mg/kg i.p. once daily for two days) showed reduced sensitivity to JWH-015 and JWH-051. The results suggest that these compounds exert their inhibitory effects through cannabinoid CB1 receptors in the myenteric plexus preparation, but mainly through CB2-like cannabinoid receptors in the vas deferens.     

Cannabinoid CB1 receptor and endothelium-dependent hyperpolarization in guinea-pig carotid, rat mesenteric and porcine coronary arteries

1. The purpose of these experiments was to determine whether or not the endothelium-dependent hyperpolarizations of the vascular smooth muscle cells (observed in the presence of inhibitors of nitric oxide synthase and cyclo-oxygenase) can be attributed to the production of an endogenous cannabinoid. 2. Membrane potential was recorded in the guinea-pig carotid, rat mesenteric and porcine coronary arteries by intracellular microelectrodes. 3. In the rat mesenteric artery, the cannabinoid receptor antagonist, SR 141716 (1 microM), did not modify either the resting membrane potential of smooth muscle cells or the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (17.3 +/- 1.8 mV, n = 4 and 17.8 +/- 2.6 mV, n = 4, in control and presence of SR 141716, respectively). Anandamide (30 microM) induced a hyperpolarization of the smooth muscle cells (12.6 +/- 1.4 mV, n = 13 and 2.0 +/- 3.0 mV, n = 6 in vessels with and without endothelium, respectively) which could not be repeated in the same tissue, whereas acetylcholine was still able to hyperpolarize the preparation. The hyperpolarization induced by anandamide was not significantly influenced by SR 141716 (1 microM). HU-210 (30 microM), a synthetic CB1 receptor agonist, and palmitoylethanolamide (30 microM), a CB2 receptor agonist, did not influence the membrane potential of the vascular smooth muscle cells. 4. In the rat mesenteric artery, the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (19.0 +/- 1.7 mV, n = 6) was not altered by glibenclamide (1 microM; 17.7 +/- 2.3 mV, n = 3). However, the combination of charybdotoxin (0.1 microM) plus apamin (0.5 microM) abolished the acetylcholine-induced hyperpolarization and under these conditions, acetylcholine evoked a depolarization (7.7 +/- 2.7 mV, n = 3). The hyperpolarization induced by anandamide (30 microM) (12.6 +/- 1.4 mV, n = 13) was significantly inhibited by glibenclamide (4.0 +/- 0.4 mV, n = 4) but not significantly affected by the combination of charybdotoxin plus apamin (17.3 +/- 2.3 mV, n = 4). 5. In the guinea-pig carotid artery, acetylcholine (1 microM) evoked endothelium-dependent hyperpolarization (18.8 +/- 0.7 mV, n = 15). SR 141716 (10 nM to 10 microM), caused a direct, concentration-dependent hyperpolarization (up to 10 mV at 10 microM) and a significant inhibition of the acetylcholine-induced hyperpolarization. Anandamide (0.1 to 3 microM) did not influence the membrane potential. At a concentration of 30 microM, the cannabinoid agonist induced a non-reproducible hyperpolarization (5.6 +/- 1.3 mV, n = 10) with a slow onset. SR 141716 (1 microM) did not affect the hyperpolarization induced by 30 microM anandamide (5.3 +/- 1.5 mV, n = 3). 6. In the porcine coronary artery, anandamide up to 30 microM did not hyperpolarize or relax the smooth muscle cells. The endothelium-dependent hyperpolarization and relaxation induced by bradykinin were not influenced by SR 141716 (1 microM). 7. These results indicate that the endothelium-dependent hyperpolarizations, observed in the guinea-pig carotid, rat mesenteric and porcine coronary arteries, are not related to the activation of cannabinoid CB1 receptors.     

Control of pain initiation by endogenous cannabinoids

The potent analgesic effects of cannabis-like drugs and the presence of CB1-type cannabinoid receptors in pain-processing areas of the brain and spinal cord indicate that endogenous cannabinoids such as anandamide may contribute to the control of pain transmission within the central nervous system (CNS). Here we show that anandamide attenuates the pain behaviour produced by chemical damage to cutaneous tissue by interacting with CB1-like cannabinoid receptors located outside the CNS. Palmitylethanolamide (PEA), which is released together with anandamide from a common phospholipid precursor, exerts a similar effect by activating peripheral CB2-like receptors. When administered together, the two compounds act synergistically, reducing pain responses 100-fold more potently than does each compound alone. Gas-chromatography/mass-spectrometry measurements indicate that the levels of anandamide and PEA in the skin are enough to cause a tonic activation of local cannabinoid receptors. In agreement with this possibility, the CB1 antagonist SR141716A and the CB2 antagonist SR144528 prolong and enhance the pain behaviour produced by tissue damage. These results indicate that peripheral CB1-like and CB2-like receptors participate in the intrinsic control of pain initiation and that locally generated anandamide and PEA may mediate this effect.     

Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): identification of cannabinoid receptor subtype selective ligands

Two cannabinoid receptors have been identified to date; one is located predominantly in the central nervous system (CB1), whereas the other is located exclusively in the periphery (CB2). The purposes of this study were to explore further the binding requirements of the CB2 receptor and to search for compounds displaying distinct affinities for either cannabinoid receptor. The binding affinities of a series of cannabinoids tested previously at the CB1 receptor were determined at cloned human CB1 and CB2 receptors using a filtration assay. In addition, possible allosteric regulation of the CB2 receptor was examined. Sodium and a GTP analog elicited a concentration-dependent decrease in specific binding to the CB2 receptor. The affinity of cannabinol for CB2 receptors (Ki = 96.3 +/- 14 nM) was confirmed to be in approximately the same range as that of delta 9-THC (Ki = 36.4 +/- 10 nM). Affinities at cloned CB1 and CB2 receptors were compared with affinities determined in the brain. Although most of the chosen compounds did not discriminate between CB1 and CB2, several ligands were identified that showed selectivity. Affinity ratios demonstrated that two 2'-fluoro analogs of anandamide were over 23-fold selective for the CB1 receptor and confirmed the CB1 selectivity of SR141716A {N- (piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamidehydrochloride}. In addition, WIN-55, 212-2 {(R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl) methyl] pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl) methanone} and a closely related propyl indole analog were shown to be 6.75- and 27.5- fold selective, respectively, for the CB2 receptor. These ligands can now serve as a basis for the design of compounds with even greater selectivity.     

Activation of peripheral CB1 cannabinoid receptors in haemorrhagic shock

Anandamide, an endogenous cannabinoid ligand, binds to CB1 cannabinoid receptors in the brain and mimics the neurobehavioural actions of marijuana. Cannabinoids and anandamide also elicit hypotension mediated by peripheral CB1 receptors. Here we report that a selective CB1 receptor antagonist, SR141716A, elicits an increase in blood pressure in rats subjected to haemorrhagic shock, whereas similar treatment of normotensive rats or intracerebroventricular administration of the antagonist during shock do not affect blood pressure. Blood from haemorrhaged rats causes hypotension in normal rats, which can be prevented by SR141716A but not by inhibition of nitric oxide synthase in the recipient. Macrophages and platelets from haemorrhaged rats elicit CB1 receptor-mediated hypotension in normotensive recipients, and incorporate arachidonic acid or ethanolamine into a product that co-elutes with anandamide on reverse-phase high-performance liquid chromatography. Also, macrophages from control rats stimulated with ionomycin or bacterial phospholipase D produce anandamide, as identified by gas chromatography and mass spectrometry. These findings indicate that activation of peripheral CB1 cannabinoid receptors contributes to haemorrhagic hypotension, and anandamide produced by macrophages may be a mediator of this effect.     

Expression cloning and biochemical characterizations of recombinant cyclophilin proteins from Schistosoma mansoni

Cannabinoid receptor agonists inhibit electrically evoked isometric contractions of the myenteric plexus--longitudinal muscle preparation of the guinea-pig small intestine (MPLM), probably by reducing release of acetylcholine (ACh) through the activation of prejunctional CB1 receptors. As CB1 receptors are thought to be negatively coupled through Gi/o proteins to both N-type Ca2+ channels and adenylate cyclase, we have now further investigated the involvement of CB1 receptors by monitoring the effects of forskolin, 8-bromo-cAMP, 3-isobutyl-1-methylxanthine (IBMX), and extracellular Ca2+ on the ability of the cannabinoid agonist, (+)-WIN 55212 to inhibit electrically evoked contractions of the MPLM (0.1 Hz, 0.5 ms, and 110% maximal voltage). Some experiments were performed with normorphine instead of (+)-WIN 55212. At 10(-7) M, forskolin, 8-bromo-cAMP, and IBMX were found to reduce significantly the maximum inhibitory response to (+)-WIN 55212 by 49.4, 48.4, and 40.2%, respectively, without affecting control contractions or responses to exogenous ACh. Low external Ca2+ (0.64 mM) significantly increased the maximum response to (+)-WIN 55212 and shifted the curve slightly leftwards, whereas high external Ca2+ (5.08 mM) reduced the maximum response by 27.2%. The concentration-response curve to normorphine, which also reduces evoked contractions of this preparation as a result of a presynaptic inhibition of ACh release via opioid mu receptors, was affected similarly. These results support the hypothesis that cannabinoid-induced inhibition in the MPLM is mediated by CB1 receptors.     

Assessment of anandamide interaction with the cannabinoid brain receptor: SR 141716A antagonism studies in mice and autoradiographic analysis of receptor binding in rat brain

Anandamide is the newly discovered endogenous cannabinoid ligand that binds to brain cannabinoid receptors and shares most, but not all, of the pharmacological properties of delta 9-THC. Therefore, this study was undertaken to determine whether its interaction with the CB1 receptor in brain was identical to that of delta 9-THC. Anandamide depressed spontaneous activity and produced hypothermia, antinociception and immobility in mice after i.v. administration. However, none of these effects was blocked by pretreatment with the selective CB1 antagonist, SR 141716A. However, the metabolically stable analog 2-methyl-2'-fluoroethylanandamide produced reductions in motor activity and antinociception in mice, effects that were blocked by the antagonist. To determine whether anandamide's receptor binding mimicked that of other cannabinoids, an autoradiographic comparison of anandamide, SR 141716A and CP 55,940 competition for [3H]CP55,940 binding was conducted throughout rat brain. The receptor affinities for all three compounds did not change according to brain area. As expected, Bmax values differed dramatically among differ brain areas. However, the Bmax values for each brain area were similar regardless of the compound used for displacement. These data suggest that anandamide, SR 141716A and CP 55,940 compete for the same cannabinoid receptor throughout brain despite SR 141716A's failure to block anandamide's pharmacological effects. Although there is no question that anandamide binds to the cannabinoid receptor, failure of SR 141716A to block its pharmacological effects in mice poses a dilemma. The results presented herein raise the possibility that anandamide may not be producing all of its effects by a direct interaction with the CB1 receptor.     

Delta9-tetrahydrocannabinol activates [Ca2+]i increases partly sensitive to capacitative store refilling

Delta9-tetrahydrocannabinol induces [Ca2+]i increases in DDT1MF-2 smooth muscle cells. Both Ca2+ entry and release from intracellular Ca2+ stores were concentration dependently activated. The Ca2+ entry component contributed most to the increases in [Ca2+]i. Stimulation with delta9-tetrahydrocannabinol after functional downregulation of intracellular Ca2+ stores by longterm thapsigargin treatment, still induced a major Ca2+ entry and a minor Ca2+ release component. Thapsigargin sensitive influx and release were selectively inhibited by the cannabinoid CB1 receptor antagonist SR141716A. No effects on [Ca2+]i were obtained after stimulation with the CB2 receptor agonist palmitoylethanolamide. This study is the first demonstration of (1) Ca2+ release from thapsigargin sensitive intracellular stores and capacitative Ca2+ entry via CB1 receptor stimulation and of (2) an additional delta9-tetrahydrocannabinol induced thapsigargin insensitive component, mainly representing Ca2+ influx which is neither mediated by CB1 nor CB2 receptor stimulation.