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.     

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.     

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.     

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.     

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.     

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.     

Effect of sodium rhein on electrically-evoked and agonist-induced contractions of the guinea-pig isolated ileal circular muscle

1. This study examined the effects of sodium rhein (0.03-30 microM) on the contractions of the isolated circular muscle of guinea-pig ileum induced by acetylcholine (100 nM), substance P (3 nM) and electrical stimulation (10 Hz for 0.3 s, 100 mA, 0.5 ms pulse duration). The effect of sodium rhein was also evaluated on the ascending excitatory reflex using a partitioned bath (oral and anal compartments). Ascending excitatory enteric nerve pathways were activated by electrical field stimulation (10 Hz for 2 s, 20 mA, 0.5 pulse duration) in the anal compartment and the resulting contraction of the guinea-pig intestinal circular muscle in the oral compartment was recorded. 2. Sodium rhein (0.3, 3 and 30 microM) significantly potentiated (52+/-11% at 30 microM) acetylcholine-induced contractions. In the presence of tetrodotoxin (0.6 microM) or omega-conotoxin GVIA (10 nM) sodium rhein (3 and 30 microM) did not enhance, but significantly reduced (49+/-10% and 44+/-8%, respectively, at 30 microM) acetylcholine-induced contractions. 3. Sodium rhein (0.3, 3 and 30 microM) significantly increased (65+/-11% at 30 microM) substance P-induced contractions. In the presence of tetrodotoxin (0.6 microM), omega-conotoxin GVIA (10 nM) or atropine (0.1 microM), sodium rhein (3 and 30 microM) significantly reduced (50+/-10%, 55+/-8% and 46+/-10%, respectively, at 30 microM) substance P-induced contractions. 4. NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) abolished the potentiating effect of sodium rhein on acetylcholine and substance P-induced contractions. At the highest concentration (30 microM), sodium rhein, in presence of L-NAME, reduced the acetylcholine (30+/-6%)- or substance P (36+/-6%)-induced contractions. 5. Sodium rhein (30 microM) significantly potentiated (29+/-9%) the electrically-evoked contractions. L-NAME (100 microM), but not phentolamine, enhanced the effect of sodium rhein. Sodium rhein (30 microM) significantly increased (32+/-9%) the ascending excitatory reflex when applied in the oral, but not in the anal compartment. 6. These results indicate that sodium rhein (i) activates excitatory cholinergic nerves on circular smooth muscle presumably through a facilitation of Ca2+ entry through the N-type Ca2+ channel, (ii) has a direct inhibitory effect on circular smooth muscle and (iii) does not affect enteric ascending neuroneural transmission. Nitric oxide could have a modulatory excitatory role on sodium rhein-induced changes of agonist-induced contractions and an inhibitory modulator role on sodium rhein-induced changes of electrically-induced contractions.     

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.     

No evidence for a significant non-nitrergic, hyperpolarising factor contribution to field stimulation-induced relaxation of the mouse anococcygeus

1. The aim of the study was to determine whether a nerve-derived hyperpolarizing factor (NDHF) might contribute to non-adrenergic, non-cholinergic (NANC) relaxations of the mouse anococcygeus when low concentrations of contractile agent are used to raise tone and low frequencies of field stimulation applied; such a non-nitrergic NDHF has been proposed to contribute to NANC relaxations of the rat anococcygeus and guinea-pig taenia coli. 2. Phenylephrine (0.1-100 microM) produced concentration-related contractions of the mouse isolated anococcygeus muscle; 0.2 microM phenylephrine (EC26) was used to raise tone in subsequent experiments. 3. Field stimulation (0.5, 1.0 and 5.0 Hz) produced frequency-dependent relaxations of phenylephrine-induced tone. In the presence of the nitric oxide synthase inhibitor L-NG-nitro-arginine (L-NOARG; 100 microM), the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 microM), or a combination of these two drugs, relaxations to field stimulation were abolished at all frequencies studied. Relaxations to sodium nitroprusside (0.01-5 microM) were unaffected by L-NOARG but strongly inhibited by ODQ; neither enzyme inhibitor affected relaxations to 8-Br-cyclic GMP (10 microM). 4. Nifedipine (1 microM) reduced the contractile response to 0.2 microM phenylephrine by 38%; however, it had no effect on NANC relaxations. 5. It is concluded that NANC relaxations of the mouse anococcygeus are purely nitrergic and that there is no significant contribution from a putative NDHF.     

Frequency potentiation in the medial cortex of young turtle brains in vitro

The effects of norfloxacin and enoxacin were examined on spontaneous motor activity in the guinea-pig isolated ileum. Micromolar concentrations of both compounds caused a biphasic response consisting of relaxation followed by transient contraction. Relaxation to norfloxacin, which was unaffected by phentolamine, propranolol and hyoscine (each at 1 microM), was partially sensitive to tetrodotoxin (1 microM). This indicates that the response is partly mediated by non-adrenergic non-cholinergic (NANC) inhibitory nerves, and partly related to a direct action on the smooth muscle. Apamin (0.1 microM) and suramin (300 microM) inhibited norfloxacin-induced relaxations to an extent similar to that of tetrodotoxin. Conversely, NG-nitro-L-arginine (300 microM) was ineffective. In the presence of theophylline (100 microM) and 3-isobutyl-1-methylxanthine (10 microM), norfloxacin caused relaxation less effective than when added alone. Based on this observation, the NANC component of the relaxation apparently depends on ATP release, whereas the direct component might be due, at least in part, to phosphodiesterase inhibition. Norfloxacin-induced contractions were neurogenic and cholinergic in nature. They were reduced by indomethacin or S-ketoprofen (both at 0.01-1 microM) and suramin (300 microM), suggesting involvement of local prostaglandin production probably induced by ATP release. Previous findings revealed that norfloxacin acted as a non-competitive antagonist at enteric GABAA receptors. In this study the same property was shared by enoxacin against the contractile response to 3-aminopropane sulphonic acid (3-APS), a GABAA receptor agonist. In conclusion, fluoroquinolones exert inhibitory and excitatory effects in the guinea-pig ileum. These are mediated by ATP, prostaglandin and acetylcholine release that might underlie, at least in part, the alterations of gastrointestinal motility observed after fluoroquinolone administration. Furthermore, isolated intestinal preparations might be useful to predict the GABAA-antagonist potential of this class of compounds.     

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.     

Effects of noise stress on EFS-mediated cholinergic and inhibitory NANC responses in tracheae from normal and sensitized guinea-pigs

1 The aim of the present research was to study the cholinergic and inhibitory non-adrenergic-non-cholinergic (NANC) responses obtained with electrical field stimulation (EFS) of tracheal tissues from sham- and noise-exposed guinea-pigs. A comparison was also made between normal and ovalbumin (OA)-sensitized animals. 2 In proximal tracheae pretreated with indomethacin (3 microM), propranolol (1 microM), alpha-chymotrypsin (2 U ml-1) and L-NAME (0.1 mM), frequency-dependent responses to EFS (0.1 ms width; 20 V, 0.1-100 Hz, 15 s train duration) were obtained, both contractile and relaxing in nature. The contractile responses were abolished by atropine (1 microM), and did not vary significantly between sham- and noise-exposed guinea-pigs, or between normal and sensitized animals. The NANC relaxing responses, present in spite of the pre-treatment of the tissues with L-NAME and alpha-chymotrypsin, and almost completely abolished by tetrodotoxin (TTX) treatment (10 microM), appeared to be enhanced in noise-exposed guinea-pigs, with respect to sham-exposed animals, but only when the animals were not OA-sensitized. 3 In distal tracheae contracted with histamine (10 microM), the study of the whole inhibitory NANC response (pre-treatment with propranolol, but not with alpha-chymotrypsin and L-NAME), which was mainly TTX-sensitive, revealed a statistically non-significant difference between sham- and noise-exposed guinea-pigs, both normal and OA-sensitized. When distal tracheae were preincubated with alpha-chymotrypsin (2 U ml-1) and L-NAME (0.1 mM), in addition to propranolol, a significant residual inhibitory NANC response to EFS was observed. Surprisingly, in this case, similarly to the evidence obtained in proximal tracheae, a significantly enhanced response was revealed in noise-exposed guinea-pigs with respect to sham-exposed animals. 4 The noise-induced enhancement of the relaxant response disappeared when the tissues were pretreated with the A2 purinergic antagonist 3,7-dimethyl-1-propargylxanthine (DMPX, 1 microM), while it persisted in the presence of the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nM). 5 The above data indicate that, while not modifying the cholinergic and the whole inhibitory NANC response to EFS, noise stress selectively influences an inhibitory component of the NANC system in guinea-pig trachea with a mechanism probably involving an enhanced neurally mediated release of adenosine, which relaxes the smooth muscle via A2 receptors. This effect appears to be lacking or masked in sensitized guinea-pigs.     

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.     

Increase in meso-prefrontal dopaminergic activity after stimulation of CB1 receptors by cannabinoids

The intravenous administration of the psychoactive constituent of marijuana, delta9-tetrahydrocannabinol (delta9-THC) (62.5-1000 microg/kg), and the synthetic cannabinoid agonist WIN 55212,2 (WIN) (62.5-500 microg/kg), produced a dose-related increase in the firing rate and burst firing in the majority of antidromically identified meso-prefrontal dopaminergic neurons. In a restricted number of neurons (n=4), WIN administration did not increase firing rate but produced an increment of bursting activity. These effects of the cannabinoids were reversed by the intravenous administration of SR 141716 A, a selective cannabinoid antagonist (1 mg/kg), per se ineffective to modify the electrical activity of dopaminergic neurons. The results indicate that stimulation of cannabinoid CB1 receptors produces an activation of meso-prefrontal dopaminergic transmission. Considering that supranormal stimulation of D1 dopamine receptors in the prefrontal cortex has been shown to impair working memory, the present results suggest that the negative effects of cannabinoids on cognitive processes might be related to the activation of dopaminergic transmission in the prefrontal cortex.     

Cannabinoid modulation of rat pup ultrasonic vocalizations

The present study investigated the effects of the cannabinoid receptor agonist CP 55,940 (1-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl) phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) and the cannabinoid receptor antagonist SR 141716A (N-(piperidin-l-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1 H-pyrazole-3-carboxamide hydrochloride) on ultrasonic vocalizations, body temperature and activity in 11-13-day-old rat pups. Testing occurred in a 5-min session 30 min following drug administration. CP 55,940 produced a dose-dependent decrease in ultrasonic vocalizations, with a 1000-micrograms/kg dose causing an almost complete inhibition of calls. Doses of 100 and 1000 micrograms/kg of CP 55,940, but not 10 micrograms/kg, caused significant hypothermia in the pups and the 1000 micrograms/kg dose also inhibited activity. The cannabinoid receptor antagonist SR 141716A (20 mg/kg) reversed the effects of 1000 micrograms/kg CP 55,940 on ultrasonic vocalizations and body temperature, but the benzodiazepine receptor antagonist flumazenil (20 mg/kg), the dopamine D1 receptor antagonist SCH 23390 (0.5 mg/kg) and the opioid receptor antagonist naloxone (1 mg/kg) did not. When administered alone, SR 141716A (20 mg/kg) increased pup ultrasonic vocalizations without affecting body temperature or activity. These results indicate that cannabinoids modulate ultrasonic vocalization production in rat pups in a manner that is independent of hypothermia. The increase in ultrasonic vocalizations produced by SR 141716A is one of the first reported behavioural effects of this drug and suggests that the endogenous cannabinoid ligand anandamide may be involved in the regulation of ultrasonic vocalizations.     

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.     

A comparison of EDHF-mediated and anandamide-induced relaxations in the rat isolated mesenteric artery

1. Relaxation of the methoxamine-precontracted rat small mesenteric artery by endothelium-derived hyperpolarizing factor (EDHF) was compared with relaxation to the cannabinoid, anandamide (arachidonylethanolamide). EDHF was produced in a concentration- and endothelium-dependent fashion in the presence of NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) by either carbachol (pEC50 [negative logarithm of the EC50] = 6.19 +/- 0.01, Rmax [maximum response] = 93.2 +/- 0.4%; n = 14) or calcium ionophore A23187 (pEC50 = 6.46 +/- 0.02, Rmax = 83.6 +/- 3.6%; n = 8). Anandamide responses were independent of the presence of endothelium or L-NAME (control with endothelium: pEC50 = 6.31 +/- 0.06, Rmax = 94.7 +/- 4.6%; n = 10; with L-NAME: pEC50 = 6.33 +/- 0.04, Rmax = 93.4 +/- 6.0%; n = 4). 2. The selective cannabinoid receptor antagonist, SR 141716A (1 microM) caused rightward shifts of the concentration-response curves to both carbachol (2.5 fold) and A23187 (3.3 fold). It also antagonized anandamide relaxations in the presence or absence of endothelium giving a 2 fold shift in each case. SR 141716A (10 microM) greatly reduced the Rmax values for EDHF-mediated relaxations to carbachol (control, 93.2 +/- 0.4%; SR 141716A, 10.7 +/- 2.5%; n = 5; P < 0.001) and A23187 (control, 84.8 +/- 2.1%; SR 141716A, 3.5 +/- 2.3%; n = 6; P < 0.001) but caused a 10 fold parallel shift in the concentration-relaxation curve for anandamide without affecting Rmax. 3. Precontraction with 60 mM KCl significantly reduced (P < 0.01; n = 4 for all) relaxations to 1 microM carbachol (control 68.8 +/- 5.6% versus 17.8 +/- 7.1%), A23187 (control 71.4 +/- 6.1% versus 3.9 +/- 0.45%) and anandamide (control 71.1 +/- 7.0% versus 5.2 +/- 3.6%). Similar effects were seen in the presence of 25 mM K+. Incubation of vessels with pertussis toxin (PTX; 400 ng ml-1, 2 h) also reduced (P < 0.01; n = 4 for all) relaxations to 1 microM carbachol (control 63.5 +/- 7.5% versus 9.0 +/- 3.2%), A23187 (control 77.0 +/- 5.8% versus 16.2 +/- 7.1%) and anandamide (control 89.8 +/- 2.2% versus 17.6 +/- 8.7%). 4. Incubation of vessels with the protease inhibitor phenylmethylsulphonyl fluoride (PMSF; 200 microM) significantly potentiated (P < 0.01), to a similar extent (approximately 2 fold), relaxation to A23187 (pEC50: control, 6.45 +/- 0.04; PMSF, 6.74 +/- 0.10; n = 4) and anandamide (pEC50: control, 6.31 +/- 0.02; PMSF, 6.61 +/- 0.08; n = 8). PMSF also potentiated carbachol responses both in the presence (pEC50: control, 6.25 +/- 0.01; PMSF, 7.00 +/- 0.01; n = 4; P < 0.01) and absence (pEC50: control, 6.41 +/- 0.04; PMSF, 6.88 +/- 0.04; n = 4; P < 0.001) of L-NAME. Responses to the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) were also potentiated by PMSF (pEC50: control, 7.51 +/- 0.06; PMSF, 8.00 +/- 0.05, n = 4, P < 0.001). 5. EDHF-mediated relaxation to carbachol was significantly attenuated by the K+ channel blocker tetraethylammonium (TEA; 1 mM) (pEC50: control, 6.19 +/- 0.01; TEA, 5.61 +/- 0.01; n = 6; P < 0.01). In contrast, TEA (1 mM) had no effect on EDHF-mediated relaxation to A23187 (pEC50: control, 6.47 +/- 0.04; TEA, 6.41 +/- 0.02, n = 4) or on anandamide (pEC50: control, 6.28 +/- 0.06; TEA, 6.09 +/- 0.02; n = 5). TEA (10 mM) significantly (P < 0.01) reduced the Rmax for anandamide (control, 94.3 +/- 4.0%; 10 mM TEA, 60.7 +/- 4.4%; n = 5) but had no effect on the Rmax to carbachol or A23187. 6. BaCl2 (100 microM), considered to be selective for blockade of inward rectifier K+ channels, had no significant effect on relaxations to carbachol or A23187, but caused a small shift in the anandamide concentration-response curve (pEC50: control, 6.39 +/- 0.01; Ba2+, 6.20 +/- 0.01; n = 4; P < 0.01). BaCl2 (1 mM; which causes non-selective block of K+ channels) significantly (P < 0.01) attenuated relaxations to all three agents (pEC50 values: carbachol, 5.65 +/- 0.02; A23187, 5.84 +/- 0.04; anandamide, 5.95 +/- 0.02; n = 4 for each). 7. Apamin (1mu M), a selective blocker of small conductance, Ca2+-activated, K+ channels (SKCa), 4-aminopyridine (1mM), a blocker of delayed rectifier, voltage-dependent, K+ channels (Kv), and ciclazindol (10mu M), an inhibitor of Kv and adenosine 5'-triphosphate (ATP)-sensitive K+ channels (KATP), significantly reduced EDHF-mediated relaxations to carbachol, but had no significant effects on A23187 or anandamide responses. 8. Glibenclamide (10mu M), a KATP inhibitor and charybdotoxin (100 or 300nM), a blocker of several K+ channel subtypes, had no significant effect on relaxations to any of the agents. Iberiotoxin (50nM), an inhibitor of large conductance, Ca2+-activated, K+ channels (BKCa), had no significant effect on the relaxation responses, either alone or in combination with apamin (1muM). Also, a combination of apamin (1muM) with either glibenclamide (10muM) or 4-aminopyridine (1mM) did not inhibit relaxation to carbachol significantly more than apamin alone. Neither combination had any significant effect on relaxation to A23187 or anandamide. 9. A combination of apamin (1muM) with charybdotoxin (100nM) abolished EDHF-mediated relaxation to carbachol, but had no significant effect on that to A23187. Apamin (1muM) and charybdotoxin (300nM) together consistently inhibited the response to A23187, while apamin (1muM) and ciclazindol (10muM) together inhibited relaxations to both carbachol and A23187. None of these toxin combinations had any significant effect on relaxation to anandamide. 10. It was concluded that the differential sensitivity to K+ channel blockers of EDHF-mediated responses to carbachol and A23187 might be due to actions on endothelial generation of EDHF, as well as its actions on the vascular smooth muscle, and suggests care must be taken in choosing the means of generating EDHF when making comparative studies. Also, the relaxations to EDHF and anandamide may involve activation of cannabinoid receptors, coupled via PTX-sensitive G-proteins to activation of K+ conductances. The results support the hypothesis that EDHF is an endocannabinoid but relaxations to EDHF and anandamide show differential sensitivity to K+ channel blockers, therefore it is likely that anandamide is not identical to EDHF in the small rat mesenteric artery.     

Effects of mercuric chloride and methyl mercury on cholinergic neuromuscular transmission in the guinea-pig ileum

The effects of mercuric chloride (HgCl2) and methyl mercury (MeHg) were examined on basal mechanical activity and electrically-induced neurogenic cholinergic contractions (twitch contractions) in longitudinal muscle-myenteric plexus strips from guinea-pig distal ileum. Both compounds at 0.33 microM slightly enhanced the amplitude of twitch contractions in approximately 50% preparations. This effect was probably due to facilitation of acetylcholine (ACh) release since 0.1 and 1 microM mercurials increased electrically-evoked tritium outflow from [3H]choline preloaded muscle layer with attached myenteric plexus. Conversely, higher mercury concentrations inhibited twitch contractions (HgCl2 IC50 = 21.3 +/- 6.4 microM; MeHg IC50 = 45.1 +/- 5.5 microM), as well as contractions to exogenous ACh (0.1 microM) in resting preparations, and concomitantly increased the basal tone. The former effects possibly reflected an antimuscarinic activity of mercury, while the latter was related to alterations of calcium homeostasis in the effector cells. Indeed, the effect of HgCl2 on basal tone was antagonized by the Ca2+ entry blocker nifedipine (3, 10, 30 nM), indicating Hg-induced facilitation of Ca2+ influx through voltage-dependent channels. On the whole, our results suggest that cholinergic neuromuscular transmission and Ca(2+)-dependent mechanisms underlying smooth muscle contractility are targets for mercury toxicity in the intestine.     

Role of nitric oxide on cholinergic component of bronchial tone in pig

In vitro studies demonstrated that stimulation of intrinsic nerves of airway smooth muscle results in a predominantly contractile response, followed by a relaxant response which involves cholinergic, adrenergic and non-adrenergic non-cholinergic (NANC) nerve activation. Thus, in this paper it is determined whether endogenous nitric oxide (NO) modulates cholinergic neurotransmission in isolated pig airway smooth muscle. Bronchial rings were suspended in organ baths for isometric measurement of tension and the contractions were induced using electrical field stimulation (EFS) techniques. Then, the effects of L-NG-nitroarginine (L-NOARG, 10 microM), an inhibitor of NO synthase, and L-arginine (L-ARG, 1 mM), a precursor of NO synthesis, were evaluated. The cholinergic contractions induced by electrical field stimulation (EFS: 60 V, 2 ms, 60 Hz) of pig lobar bronchial preparations increased (29%) in the presence of L-NOARG (10 microM). This effect may be released by nerves in pig large airways during EFS.     

Effects of exogenous phytohormones on plastid tRNA modifications in ragi coleoptiles

The influence of adenosine and selective A1 and A2 agonists and antagonists was investigated on the cholinergic and the excitatory non-cholinergic (e-NC) contractions induced by electrical field stimulation in the guinea-pig bronchi. Adenosine (10 nM-1 mM) induced a concentration-dependent inhibition of the e-NC contraction (EC50 = 90 +/- 14 microM), whereas the cholinergic peak was only slightly affected. Preincubation of the tissue with the adenosine uptake blocker dipyridamole (10 microM) significantly shifted the concentration-inhibition curve to adenosine to the left (EC50 = 10 +/- 1 microM), suggesting an interaction with extracellular adenosine receptors of A1 and/or A2 subtype. To characterize the receptor type involved in this effect, selective adenosine derivatives were studied. The agonist to both A1 and A2 adenosine receptors, 5'-N-ethylcarboxamidoadenosine (NECA) was more potent than the selective A1 agonist, (-)-R-6-phenylisopropyladenosine (R-PIA), in inhibiting the e-NC contraction (EC50 = 0.10 +/- 0.04 and 0.60 +/- 0.12 microM, respectively, with a maximal inhibition of 70 and 45%, respectively). The concentration-response curve to NECA was shifted to the right by the A2 receptor selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (10 microM) (EC50 = 1.4 +/- 0.5 microM) as well as by the specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (10 microM) (EC50 = 0.7 +/- 0.3 microM). The inhibitory effect induced by the association of both antagonists, DPCPX and DMPX, was considerably potentiated (EC50 > 22 +/- 2.5 microM). The effect of R-PIA was also shifted to the right by DPCPX (EC50 = 8.2 +/- 1.6 microM) but was not modified by DMPX. The contractile response to exogenous substance P was unaffected by NECA pretreatment (0.3 microM). Altogether, these results suggest that adenosine-induced inhibition of e-NC contraction of guinea-pig bronchi is mediated through activation of both A1 and A2 adenosine receptors linked to inhibition of the release of neuropeptides from C-fibre nerve endings.