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.     

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.     

In vitro functional evidence of neuronal cannabinoid CB1 receptors in human ileum

We investigated the effect of the cannabinoid agonist (+)WIN-55212-2 on human ileum longitudinal smooth muscle preparations, either electrically stimulated or contracted by carbachol. Electrical field stimulation mostly activated cholinergic neurons, since atropine and tetrodotoxin (TTX), alone or coincubated, reduced twitch responses to a similar degree (85%). (+)WIN-55212-2 concentration-dependently inhibited twitch responses (IC50 73 nM), but had no additive effect with atropine or TTX. The cannabinoid CB1 receptor antagonist SR 141716 (pA2 8.2), but not the CB2 receptor antagonist, SR 144528, competitively antagonized twitch inhibition by (+)WIN-55212-2. Atropine but not (+)WIN-55212-2 or TTX prevented carbachol-induced tonic contraction. These results provide functional evidence of the existence of prejunctional cannabinoid CB1-receptors in the human ileum longitudinal smooth muscle. Agonist activation of these receptors prevents responses to electrical field stimulation, presumably by inhibiting acetylcholine release. SR 141716 is a potent and competitive antagonist of cannabinoid CB1 receptors naturally expressed in the human gut.     

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.     

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.     

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.     

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.     

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.     

Binding of the non-classical cannabinoid CP 55,940, and the diarylpyrazole AM251 to rodent brain cannabinoid receptors

The binding of [123I]AM251 (a radioiodinated analog of the cannabinoid CB1 receptor antagonist SR141716A) was compared to that of [3H]CP 55,940 in mouse and rat brain preparations. Scatchard analysis of the binding of [123I]AM251 and [3H]CP 55,940 to membranes prepared from mouse cerebellum, striatum and hippocampus yielded similar Bmax values (15-41 pmol/g wet wt tissue). Kd values were lower for [123I]AM251 (0.23-0.62 nM) than for [3H]CP 55,940 (1.3-4 nM). CP 55,940 and SR141716A increased dissociation of [123I]AM251 from binding sites in mouse cerebellar homogenates to a similar extent. The structurally dissimilar cannabinoid receptor ligands THC, methanandamide, WIN 55, 212-2, CP 55,940 and SR141716A were each able to fully compete with binding of both [123I]AM251 and [3H]CP 55,940 in mouse cerebellum. In vitro autoradiography demonstrated that the distribution of binding sites for [123I]AM251 in rat brain was very similar to published distributions of binding sites for [3H]CP 55,940. Together, these observations suggest that AM251 binds to the same site (the cannabinoid CB1 receptor) in rodent brains as CP 55,940. However, the binding site domains which interact with AM251 and CP 55,940 may not be identical, since IC50 values for cannabinoid receptor ligands depended on whether [123I]AM251 or [3H]CP 55,940 was used as radioligand.     

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.     

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.     

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.     

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.     

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.     

Differential requirements of sodium for coupling of cannabinoid receptors to adenylyl cyclase in rat brain membranes

Sodium is generally required for optimal inhibition of adenylyl cyclase by Gi/o-coupled receptors. Cannabinoids bind to specific receptors that act like other members of the Gi/o-coupled receptor superfamily to inhibit adenylyl cyclase. However, assay of cannabinoid inhibition of adenylyl cyclase in rat cerebellar membranes revealed that concentrations of NaCl ranging from 0 to 150 mM had no effect on agonist inhibition. This lack of effect of sodium was not unique to cannabinoid receptors, because the same results were observed using baclofen as an agonist for GABAB receptors in cerebellar membranes. The lack of sodium dependence was region-specific, because assay of cannabinoid and opioid inhibition of adenylyl cyclase in striatum revealed an expected sodium dependence, with 50 mM NaCl providing maximal inhibition levels by both sets of agonists. This difference in sodium requirements between these two regions was maintained at the G protein level, because agonist-stimulated low Km GTPase activity was maximal at 50 mM NaCl in striatal membranes, but was maximal in the absence of NaCl in cerebellar membranes. Assay of [3H]WIN 55212-2 binding in cerebellar membranes revealed that the binding of this labeled agonist was sensitive to sodium and guanine nucleotides like other Gi/o-coupled receptors, because both NaCl and the nonhydrolyzable GTP analogue Gpp(NH)p significantly inhibited binding. These results suggest that differences in receptor-G protein coupling exist for cannabinoid receptors between these two brain regions.     

Absence of tolerance to the aversive stimulus properties of ethanol following oral ethanol self-administration

Aluminum has been reported to inhibit long-term potentiation (LTP) following in vivo administration and decrease glutamate release following in vitro exposure. Because glutamate release is critical for synaptic transmission and the development and maintenance of LTP in the hippocampus, we examined the effects of aluminum chloride (AlCl3) on depolarization-induced glutamate release and LTP in rat hippocampal slices. The effects of AlCl3 on [14C]glutamate release were examined by incubation of slices in depolarizing (56 mM)K+ buffer solution in the absence or presence of 2 mM CaCl2. After 15 min depolarization, AlCl3 (100-1000 microM) did not significantly affect Ca(2+)-dependent [14C]glutamate release from slices, whereas a known Ca2+ channel blocker (100 microM CdCl2) decreased Ca(2+)-dependent [14C]glutamate release by approximately 50%. In contrast to a previous report, acute exposure to AlCl3 was without effect on depolarization-dependent glutamate release. LTP of the population spike (PS) in CA1 of hippocampus was induced by the delivery of stimulus trains to the stratum radiatum. LTP of the PS was observed in both control slices and slices bathed in solution containing 100 microM AlCl3. Neither the magnitude nor longevity (measured up to 1 h posttrain) of LTP distinguished control from aluminum-exposed slices. The lack of sensitivity in rat to the encephalopathic changes induced by aluminum, or methodological differences in exposure conditions may account for the lack of effect of aluminum on in vitro LTP in rat hippocampus.     

Appetite suppression and weight loss after the cannabinoid antagonist SR 141716

The effect of the cannabinoid CB1 receptor antagonist, SR 141716, on food intake and body weight was assessed in adult, non-obese Wistar rats. The daily administration of SR 141716 (2.5 and 10 mg/kg; i.p.) reduced dose-dependently both food intake and body weight. Tolerance to the anorectic effect developed within 5 days; in contrast, body weight in SR 141716-treated rats remained markedly below that of vehicle-treated rats throughout the entire treatment period (14 days). The results suggest that brain cannabinoid receptors are involved in the regulation of appetite and body weight.     

Role of the subthalamic nucleus in cannabinoid actions in the substantia nigra of the rat

The effect of cannabinoids on the excitatory input to the substantia nigra reticulata (SNr) from the subthalamic nucleus was explored. For this purpose a knife cut was performed rostral to the subthalamic nucleus to isolate the subthalamic nucleus and the SNr from the striatum, a major source of cannabinoid receptors to the SNr. The data showed that the cannabinoid agonist WIN55,212-2 blocked the increase in the firing rate of SNr neurons induced by stimulation of the subthalamic nucleus with bicuculline. Furthermore, the cannabinoid antagonist SR141716A antagonized the effect of the cannabinoid agonist. This study showed that cannabinoids regulate not only the striatonigral pathway, as previously reported, but also the subthalamonigral pathway. The opposite influences of these two inputs to the SNr, inhibitory and excitatory respectively, suggest that endogenous cannabinoids play a major role in the physiological regulation of the SNr.     

Inhibition of intestinal motility by anandamide, an endogenous cannabinoid

The endogenous cannabinoid ligand anandamide (arachidonylethanolamide) inhibited the intestinal passage of a charcoal meal when administered s.c. in mice at doses ranging from 0.1 to 50 mg/kg. This effect was prevented by the cannabinoid CB1 receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide x HCl] (1 mg/kg s.c.), but it was not affected by the anandamide transport inhibitor, N-(4-hydroxyphenyl) arachidonylethanolamide (AM404) (50 mg/kg, s.c.). The results indicate that anandamide modulates intestinal motility in mice by activating cannabinoid CB1 receptors. They also suggest that anandamide transport, which was previously shown to participate in terminating neural and vascular responses to anandamide, does not contribute to anandamide inactivation in intestinal tissue.     

SR 144528, the first potent and selective antagonist of the CB2 cannabinoid receptor

Based on both binding and functional data, this study introduces SR 144528 as the first, highly potent, selective and orally active antagonist for the CB2 receptor. This compound which displays subnanomolar affinity (Ki = 0.6 nM) for both the rat spleen and cloned human CB2 receptors has a 700-fold lower affinity (Ki = 400 nM) for both the rat brain and cloned human CB1 receptors. Furthermore it shows no affinity for any of the more than 70 receptors, ion channels or enzymes investigated (IC50 > 10 microM). In vitro, SR 144528 antagonizes the inhibitory effects of the cannabinoid receptor agonist CP 55,940 on forskolin-stimulated adenylyl cyclase activity in cell lines permanently expressing the h CB2 receptor (EC50 = 10 nM) but not in cells expressing the h CB1 (no effect at 10 microM). Furthermore, SR 144528 is able to selectively block the mitogen-activated protein kinase activity induced by CP 55,940 in cell lines expressing h CB2 (IC50 = 39 nM) whereas in cells expressing h CB1 an IC50 value of more than 1 microM is found. In addition, SR 144528 is shown to antagonize the stimulating effects of CP 55,940 on human tonsillar B-cell activation evoked by cross-linking of surface Igs (IC50 = 20 nM). In vivo, after oral administration SR 144528 totally displaced the ex vivo [3H]-CP 55,940 binding to mouse spleen membranes (ED50 = 0.35 mg/kg) with a long duration of action. In contrast, after the oral route it does not interact with the cannabinoid receptor expressed in the mouse brain (CB1). It is expected that SR 144528 will provide a powerful tool to investigate the in vivo functions of the cannabinoid system in the immune response.