Effects of cannabinoids on endogenous K+ and Ca2+ currents in HEK293 cells

Effects of cannabinoids on endogenous potassium and calcium currents in HEK293 cells were studied using the whole-cell variant of the patch-clamp technique. The cannabinoid agonists WIN 55,212-2, methanandamide, and anandamide (1 microM) decreased the calcium current by 53.1 +/- 2.6, 47.5 +/- 1.2, and 38.8 +/- 3.1%, respectively, after transfection of human CB1 cannabinoid receptor (hCB1) cDNA into HEK293 cells. The delayed rectifier-like current was not changed after application of these agonists, but the inward rectifier was increased by 94.0 +/- 3.6, 83.7 +/- 5.1, and 63.0 +/- 2.5% after application of WIN 55,212-2, methanandamide, and anandamide, respectively. The effects of the cannabinoid antagonists (AM251, AM281, and AM630) on the inward rectifier and calcium currents were the opposite of those seen with cannabinoid agonists; thus, these compounds act as inverse agonists in this preparation. These results suggest that endogenous inward rectifier and calcium currents are modulated by cannabinoids in HEK293 cells, and that some expressed receptors may be constitutively active.     

Cannabinoid pharmacology: implications for additional cannabinoid receptor subtypes

Delta(9)-Tetrahydrocannabinol (delta(9)-THC), the primary psychoactive constituent of marijuana (Cannabis sativa), is known to bind to two cannabinoid receptors: CB(1) receptors, located primarily in the brain, and CB(2) receptors, located primarily in the periphery. Recent research has suggested that other cannabinoids, including anandamide and WIN 55212-2, may also act at novel non-CB(1), non-CB(2) cannabinoid receptor(s). Anandamide produces a number of in vivo pharmacological effects in CB(1) knockout mice that are not produced by delta(9)-THC and cannot be explained by anandamide's rapid metabolism. In addition, in vitro anandamide and WIN 55212-2 stimulate [35S]GTPgammaS binding in both CB(1) knockout and wildtype mice while delta(9)-THC stimulates this binding only in wildtype mice. Although anandamide and vanilloid agonists share pharmacological effects, anandamide's actions in CB(1) knockout mice do not appear to be mediated by vanilloid VR(1) receptors. While not yet conclusive, these results suggest the possibility of additional cannabinoid receptors in the brain and periphery.     

Receptor-independent depression of DA and 5-HT uptake by cannabinoids in rat neocortex--involvement of Na(+)/K(+)-ATPase

There is evidence that cannabinoids modulate the reuptake of some neurotransmitters in the central nervous system. In this study, we investigated the effects of the synthetic cannabinoid receptor agonist WIN55212-2, the endocannabinoid anandamide and the chemically related arachidonic acid on serotonin (5-HT) and dopamine (DA) uptake into rat neocortical synaptosomes. At micromolar concentrations, anandamide and arachidonic acid produced steep inhibition curves with Hill coefficients above unity. WIN55212-2 inhibited both DA and 5-HT uptake with Hill coefficients near unity, also within the micromolar range. The effect of WIN55212-2 was not mediated by cannabinoid receptors, since the CB1 receptor antagonist AM251 failed to diminish uptake inhibition by WIN55212-2 and since the Ki estimates of WIN55212-2 were outside the range of the dissociation constants of WIN55212-2 at both CB1 and CB2 receptors. A 100-fold higher concentration of DA, respectively 5-HT, did not induce a shift to the right of the WIN55212-2 concentration-inhibition curves, suggesting a carrier-independent mechanism. The Na(+)/K(+)-ATPase inhibitor ouabain concentration dependently inhibited 5-HT uptake. Possible drug effects on commercial Na(+)/K(+)-ATPase and synaptosomal ATP consumption were investigated using an ATP bioluminescence assay. Ouabain inhibited both commercial and synaptosomal Na(+)/K(+)-ATPase. WIN55212-2 had no effect on commercial Na(+)/K(+)-ATPase, but inhibited synaptosomal ATP consumption. Anandamide produced a sharp decrease in the activity of commercial Na(+)/K(+)-ATPase and on synaptosomal ATP consumption. Presence of ouabain significantly reduced the inhibitory effect of anandamide on synaptosomal ATP consumption, whereas the effect of WIN55212-2 remained unchanged. Our results show that cannabinoids and arachidonic acid inhibit DA and 5-HT uptake into rat neocortical synaptosomes. This effect is neither cannabinoid receptor-mediated nor due to competitive inhibition of membrane transporters, but is partly effected by a decreased Na(+)/K(+)-ATPase activity.     

Anandamide-induced vasorelaxation in rabbit aortic rings has two components: G protein dependent and independent

The endogenous cannabinoid anandamide (arachidonylethanolamide) produces vasorelaxation in different vascular beds. In the present study, we found that anandamide and a metabolically stable analog, methanandamide, produced dose-dependent (10 nM-10 microM) vasorelaxation of approximately 80% in a rabbit aortic ring preparation in an endothelium-dependent manner. Non-endothelium-dependent vasorelaxation was observed to be a maximum of 20-22% at >10 microM methanandamide. The efficacious CB(1) receptor analogs desacetyllevonantradol (10 microM) and WIN55212-2 (10 microM) failed to produce vasorelaxation; however, the endothelium-dependent vasorelaxation evoked by methanandamide was partially (75%) blocked by the CB(1) receptor antagonist SR141716A. The VR(1) vanilloid receptor antagonist capsazepine or the calcitonin gene-related peptide (CGRP) antagonist CGRP-(8-37) partially attenuated (25%) the vasorelaxation in endothelium-intact preparations and greatly reduced the response in endothelium-denuded preparations. Pretreatment of aortic rings with N(G)-nitro-L-arginine methyl ester completely blocked the methanandamide-, capsaicin-, and CGRP-induced vasorelaxation. Pretreatment of aortic rings with pertussis toxin attenuated the methanandamide-induced vasorelaxation in endothelium-intact aortic rings, indicating the involvement of G(i/o) proteins in the vasorelaxation; however, pertussis toxin treatment failed to block the endothelium-independent response. Thus, in the rabbit aorta, methanandamide-induced vasorelaxation exhibits two components: 1) in endothelium-intact rings, an SR141716A-sensitive, non-CB(1) receptor component that requires pertussis toxin-sensitive G proteins and nitric oxide (NO) production; and 2) in endothelium-denuded rings, a component that is mediated by VR(1) vanilloid receptors and possibly by the subsequent release of CGRP that requires NO production but is independent of pertussis toxin-sensitive G proteins.     

Cannabinoid receptor agonists inhibit depolarization-induced calcium influx in cerebellar granule neurons.

Neuronal cannabinoid receptors (CB(1)) are coupled to inhibition of voltage-sensitive Ca(2+) channels (VSCCs) in several cell types. The purpose of these studies was to characterize the interaction between endogenous CB(1) receptors and VSCCs in cerebellar granule neurons (CGN). Ca(2+) transients were evoked by KCl-induced depolarization and imaged using fura-2. The CB(1) receptor agonists CP55940, Win 55212-2 and N-arachidonylethanolamine (anandamide) produced concentration-related decreases in peak amplitude of the Ca(2+) response and total Ca(2+) influx. Pre-treatment of CGN with pertussis toxin abolished agonist-mediated inhibition. The inhibitory effect of Win 55212-2 on Ca(2+) influx was additive with inhibition produced by omega-agatoxin IVA and nifedipine but not with omega-conotoxin GVIA, indicating that N-type VSCCs are the primary effector. Paradoxically, the CB(1) receptor antagonist, SR141716, also inhibited KCl-induced Ca(2+) influx into CGN in a concentration-related manner. SR141716 inhibition was pertussis toxin-insensitive and was not additive with the inhibition produced by Win 55212-2. Confocal imaging of CGN in primary culture demonstrate a high density of CB(1) receptor expression on CGN plasma membranes, including the neuritic processes. These data demonstrate that the CB(1) receptor is highly expressed by CGN and agonists serve as potent and efficacious inhibitory modulators of Ca(2+) influx through N-type VSCC.     

CB1-independent inhibition of dopamine transporter activity by cannabinoids in mouse dorsal striatum

Cannabinoid drugs are known to affect dopaminergic neurotransmission in the basal ganglia circuitry. In this study, we used in vitro and in vivo techniques to investigate whether cannabinoid agonists and antagonist could affect dopaminergic transmission in the striatum by acting at the dopamine transporter. Incubation of striatal synaptosomes with the cannabinoid agonists WIN55,212-2 or methanandamide decreased dopamine uptake (IC(50) = 2.0 micromol/L and 3.1 micromol/L, respectively). A similar inhibitory effect was observed after application of the inactive WIN55,212-2 isomer, S(-)WIN55,212-3. The CB(1) antagonist AM251 did not reverse WIN55,212-2 effect but rather mimicked it. WIN55,212-2 and AM251 partially displaced the binding of the cocaine analog [(3)H]WIN35,428, thus acting as dopamine transporter pseudo-substrates in the high micromolar range. High-speed chronoamperometry measurements showed that WIN55,212-2 (4 mg/kg, i.p.) caused significant release of endogenous dopamine via activation of CB(1) receptors, followed by a reduction of dopamine clearance. This reduction was CB(1)-independent, as it was mimicked by S(-)WIN55,212-3. Administration of AM251 (1 and 4 mg/kg, i.p.) increased the signal amplitude and reduced the clearance of dopamine pressure ejected into the striatum. These results indicate that both cannabinoid agonists and antagonists inhibit dopamine transporter activity via molecular targets other than CB(1) receptors.     

Cannabinoid–vanilloid receptor interactions in pain signaling

Agents that activate cannabinoid CB1 receptors for marijuana's active principal, THC, or vanilloid VR1 receptors for red chilli peppers' pungent ingredient, capsaicin, modulate pain perception. Stimulation of presynaptic CB1 leads to inhibition of glutamate release in the spinal cord, whereas VR1 stimulation causes release of substance P and CGRP from DRG neurons. VR1 undergoes rapid desensitization by its agonists, which makes VR1‐expressing neurons insensitive to subsequent stimulation and results in analgesia. Thus, both CB1 and VR1, which are coexpressed in several spinal and DRG neurons, are targets for analgesic drug development. CB1 and VR1 also share endogenous agonists, namely anandamide, NADA and some of their analogs, and may be regarded as metabotropic and ionotropic receptors for the same family of mediators, with opposing roles in pain perception. The development of ‘hybrid’ CB1/VR1 agonists as potent analgesics and the functional relationships between CB1 and VR1 in sensory neurons will be discussed.     

Characterization of VR1 within the BMBF-Leitproject: 'Molecular Pain Research'

Agents that activate cannabinoid CB1 receptors for marijuana's active principal, THC, or vanilloid VR1 receptors for red chilli peppers' pungent ingredient, capsaicin, modulate pain perception. Stimulation of presynaptic CB1 leads to inhibition of glutamate release in the spinal cord, whereas VR1 stimulation causes release of substance P and CGRP from DRG neurons. VR1 undergoes rapid desensitization by its agonists, which makes VR1-expressing neurons insensitive to subsequent stimulation and results in analgesia. Thus, both CB1 and VR1, which are coexpressed in several spinal and DRG neurons, are targets for analgesic drug development. CB1 and VR1 also share endogenous agonists, namely anandamide, NADA and some of their analogs, and may be regarded as metabotropic and ionotropic receptors for the same family of mediators, with opposing roles in pain perception. The development of 'hybrid' CB1/VR1 agonists as potent analgesics and the functional relationships between CB1 and VR1 in sensory neurons will be discussed.     

Cardiovascular pharmacology of anandamide

The fatty acid amide anandamide produces hypotension and a decrease in systemic vascular resistance in vivo. A drop in blood pressure is also seen with synthetic cannabinoid (CB) receptor agonists. The hypotensive responses to anandamide and synthetic cannabinoids are absent in CB1 receptor gene knockout mice. In isolated arteries and perfused vascular beds, anandamide induces vasodilator responses, which cannot be mimicked by synthetic cannabinoids. Instead, vanilloid receptors on perivascular sensory nerves play a key role in these effects of anandamide. Activation of vanilloid receptors by anandamide triggers the release of sensory neuropeptides such as the vasodilator calcitonin gene-related peptide (CGRP). Anandamide is detected in blood and in many cells of the cardiovascular system, and macrophage-derived anandamide may be involved in several hypotensive clinical conditions. Interestingly, cannabinoid and vanilloid receptors display an overlap in ligand recognition properties, and the frequently used CB1 receptor antagonist SR141716A also inhibits vanilloid receptor-mediated responses. The presence of anandamide in endothelial cells, neurones and activated macrophages (monocytes), and its ability to activate CB and vanilloid receptors make this lipid a potential bioregulator in the cardiovascular system.     

Paticipation of cannabinoid receptors in the regulation of cardiac rhythm and cardiac contractility

It has been found that i. v. administration of cannabinoid receptor (CB) agonists (HU-210, ACPA, anandamide, methanandamide) induced a decrease in the heart rate (HR) in anesthetized rats. Pretreatment with CB1 receptor antagonist SR141716A completely abolished a negative chronotropic effect of CB receptor agonist HU-210. The CB2 receptor antagonist SRI 44528 did not prevent a HU-210-induced decrease in the HR. Pretreatment with the ganglion blocker hexamethonium had no effect on the negative chronotropic action of HU-210. Addition of HU-210 (100 nM) to perfusion solution induced a decrease in the HR, left ventricular development pressure, rate of contractility and relaxation of isolated perfused rate heart without change in end diastolic pressure. These data suggest that cardiac CBI receptor activation induces a decrease in the HR both in vivo and in vitro. An occupancy of the same receptors mediates a negative inotropic effects of cannabinoids.     

Significance of cardiac cannabinoid receptors in regulation of cardiac rhythm,myocardial contractility,and electrophysiologic processes in heart

Intravenous administration of cannabinoid (CB) receptor agonists (HU-210, 0.1 mg/kg; ACPA, 0.125 mg/kg; methanandamide, 2.5 mg/kg; and anandamide, 2.5 mg/kg) induced bradycardia in chloralose-anesthetized rats irrespective of the solubilization method. Methanandamide, HU-210, and ACPA had no effect on the electrophysiological activity of the heart, while anandamide increased the duration of the QRS complex. The negative chronotropic effect of HU-210 was due to CB1 receptor activation since it was not observed after CB1 receptor blockade by SR141716A (1 mg/kg intravenously) but was present after pretreatment with CB2 receptor antagonist SR144528 (1 mg/kg intravenously). CB receptor antagonists SR141716A and SR144528 had no effect on cardiac rhythm or ECG indices. Hence, in the intact heart, endogenous CB receptor agonists are not involved in the regulation of cardiac rhythm and electrophysiological processes. The chronotropic effect of CBs was independent of the autonomic nervous system because it remained significant after autonomic ganglion blockade by hexamethonium (1 mg/kg intravenously). CB receptor activation by HU-210 (0.1 and 1 μM) in vitro decreased the rate and force of isolated heart contractions, the rates of contraction and relaxation, and end diastolic pressure. The negative chronotropic effect of HU-210 was less pronounced in vitro than in vivo. The maximum inotropic effect of HU-210 was reached at the concentration of 0.1 μM.     

大麻素抑制大鼠三叉神经节神经元ATP激活电流

本文在培养的大鼠三叉神经节(trigeminal ganglion,TG)神经元上采用全细胞膜片钳技术,探讨大麻素对大鼠TG神经元ATP激活电流(ATP-activated currents,IATP)的影响.结果显示(1)胞外给予ATP,大部分受检细胞(67/75,89.33%)可记录到一个内向电流,且具有剂量依赖性.该电流可被P2X嘌呤受体特异性拮抗剂PPADS所阻断.(2)预加WIN55212-2[大麻素受体1(cannabinoid receptor 1,CB1受体)激动剂]可对IATP产生抑制作用,此作用呈剂量依赖性,并可被CB1受体特异性拮抗剂AM281阻断.预加不同浓度的WIN55212-2(1x10-13、1x10-12、1x10-11、1x10-10、1x10-9和1x10-8mol/L)对IATP(1x10-4mol/L ATP)的抑制作用分别为(8.14±3.14)%、(20.11±2.72)%、(46.62±3.51)%、(72.16±5.64)%、(80.21±2.80)%和(80.59±3.55)%.(3)预加WIN55212-2后IATP的浓度-反应曲线明显下移;最大反应浓度时的IATP幅值减小了(58.02±4.21)%,而阈值基本不变;预加WIN55212.2前后曲线的EC50值非常接近(1.15x10-4mol/L vs 1.27x10-4 mol/L).(4)预加forskolin[腺苷酸环化酶(adenylyl cyclase,AC)激动剂]或8-Br-cAMP可以逆转WIN55212-2对IATP的抑制作用.以上结果表明,大麻素可能作用于CB1受体,通过抑制AC-cAMP-PKA途径发挥对IATP的抑制作用.     

Anandamide administration alone and after inhibition of fatty acid amide hydrolase (FAAH) increases dopamine levels in the nucleus accumbens shell in rats

Although endogenous cannabinoid systems have been implicated in the modulation of the rewarding effects of abused drugs and food, little is known about the direct effects of endogenous ligands for cannabinoid receptors on brain reward processes. Here we show for the first time that the intravenous administration of anandamide, an endogenous ligand for cannabinoid receptors, and its longer-lasting synthetic analog methanandamide, increase the extracellular dopamine levels in the nucleus accumbens shell of awake, freely moving rats, an effect characteristic of most drugs abused by humans. Anandamide produced two distinctly different effects on dopamine levels: (1) a rapid, transient increase that was blocked by the cannabinoid CB1 receptor antagonist rimonabant, but not by the vanilloid VR1 receptor antagonist capsazepine, and was magnified and prolonged by the fatty acid amide hydrolase (FAAH) enzyme inhibitor, URB597; (2) a smaller delayed and long-lasting increase, not sensitive to CB1, VR1 or FAAH blockade. Both effects were blocked by infusing either tetrodotoxin (TTX, 1 microm) or calcium-free Ringer's solution through the microdialysis probe, demonstrating that they were dependent on the physiologic activation of dopaminergic neurotransmission. Thus, these results indicate that anandamide, through the activation of the mesolimbic dopaminergic system, participates in the signaling of brain reward processes.     

Characterization of CB1 Receptors on Rat Neuronal Cell Cultures: Binding and Functional Studies Using the Selective Receptor Antagonist SR 141716A

Abstract: This study was undertaken to characterize further the central cannabinoid receptors in rat primary neuronal cell cultures from selected brain structures. By using [³H]SR 141716A, the specific CB1 receptor antagonist, we demonstrate in cortical neurons the presence of a high density of specific binding sites ( B _max = 139 ± 9 fmol/mg of protein) displaying a high affinity ( K _D = 0.76 ± 0.09 n M ). The two cannabinoid receptor agonists, CP 55940 and WIN 55212-2, inhibited in a concentration-dependent manner cyclic AMP production induced by either 1 µ M forskolin or isoproterenol with EC₅₀ values in the nanomolar range (4.6 and 65 n M with forskolin and 1.0 and 5.1 n M with isoproterenol for CP 55940 and WIN 55212-2, respectively). Moreover, in striatal neurons and cerebellar granule cells, CP 55940 was also able to reduce the cyclic AMP accumulation induced by 1 µ M forskolin with a potency similar to that observed in cortical neurons (EC₅₀ values of 3.5 and 1.9 n M in striatum and cerebellum, respectively). SR 141716A antagonized the CP 55940- and WIN 55212-2-induced inhibition of cyclic AMP accumulation, suggesting CB1 receptor-specific mediation of these effects on all primary cultures tested. Furthermore, CP 55940 was unable to induce mitogen-activated protein kinase activation in either cortical or striatal neurons. In conclusion, our results show nanomolar efficiencies for CP 55940 and WIN 55212-2 on adenylyl cyclase activity and no effect on any other signal transduction pathway investigated in primary neuronal cultures.     

The endocannabinoid system protects rat glioma cells against HIV-1 Tat protein-induced cytotoxicity. Mechanism and regulation

Cannabinoids modulate nitric oxide (NO) levels in cells of the central nervous system. Here we studied the effect of cannabinoid CB(1) and CB(2) receptor agonists on the release of NO and cell toxicity induced by the human immuno-deficiency virus-1 Tat protein (HIV-1 Tat) in rat glioma C6 cells. The CB(1) and CB(2) agonist WIN 55,212-2 inhibited the expression of inducible NO synthase (iNOS) and NO release caused by treatment of C6 cells with HIV-1 Tat and interferon-gamma (IFN-gamma). The effect of WIN 55,212-2 was uniquely due to CB(1) receptors, as shown by experiments carried out with selective CB(1) and CB(2) receptor agonists and antagonists. CB(1) receptor stimulation also inhibited HIV-1 Tat + IFN-gamma-induced and NO-mediated cell toxicity. Moreover, cell treatment with HIV-1 Tat + IFN-gamma induced a significant inhibition of CB(1), but not CB(2), receptor expression. This effect was mimicked by the NO donor GSNO, suggesting that the inhibition of CB(1) expression was due to HIV-1 Tat + IFN-gamma-induced NO overexpression. HIV-1 Tat + IFN-gamma treatment also induced a significant inhibition of the uptake of the endocannabinoid anandamide by C6 cells with no effect on anandamide hydrolysis. These findings show that the endocannabinoid system, through the modulation of the l-arginine/NO pathway, reduces HIV-1 Tat-induced cytotoxicity, and is itself regulated by HIV-1 Tat.     

Endogenous unsaturated C18 N-acylethanolamines are vanilloid receptor (TRPV1) agonists

The endogenous C18 N-acylethanolamines (NAEs) N-linolenoylethanolamine (18:3 NAE), N-linoleoylethanolamine (18:2 NAE), N-oleoylethanolamine (18:1 NAE), and N-stearoylethanolamine (18:0 NAE) are structurally related to the endocannabinoid anandamide (20:4 NAE), but these lipids are poor ligands at cannabinoid CB(1) receptors. Anandamide is also an activator of the transient receptor potential (TRP) vanilloid 1 (TRPV(1)) on primary sensory neurons. Here we show that C18 NAEs are present in rat sensory ganglia and vascular tissue. With the exception of 18:3 NAE in rat sensory ganglia, the levels of C18 NAEs are equal to or substantially exceed those of anandamide. At submicromolar concentrations, 18:3 NAE, 18:2 NAE, and 18:1 NAE, but not 18:0 NAE and oleic acid, activate native rTRPV(1) on perivascular sensory nerves. 18:1 NAE does not activate these nerves in TRPV(1) gene knock-out mice. Only the unsaturated C18 NAEs elicit whole cell currents and fluorometric calcium responses in HEK293 cells expressing hTRPV(1). Molecular modeling revealed a low energy cluster of U-shaped unsaturated NAE conformers, sharing several pharmacophoric elements with capsaicin. Furthermore, one of the two major low energy conformational families of anandamide also overlaps with the cannabinoid CB(1) receptor ligand HU210, which is in line with anandamide being a dual activator of TRPV(1) and the cannabinoid CB(1) receptor. This study shows that several endogenous non-cannabinoid NAEs, many of which are more abundant than anandamide in rat tissues, activate TRPV(1) and thus may play a role as endogenous TRPV(1) modulators.     

Gz mediates the long-lasting desensitization of brain CB1 receptors and is essential for cross-tolerance with morphine

Background

Although the systemic administration of cannabinoids produces antinociception, their chronic use leads to analgesic tolerance as well as cross-tolerance to morphine. These effects are mediated by cannabinoids binding to peripheral, spinal and supraspinal CB1 and CB2 receptors, making it difficult to determine the relevance of each receptor type to these phenomena. However, in the brain, the CB1 receptors (CB1Rs) are expressed at high levels in neurons, whereas the expression of CB2Rs is marginal. Thus, CB1Rs mediate the effects of smoked cannabis and are also implicated in emotional behaviors. We have analyzed the production of supraspinal analgesia and the development of tolerance at CB1Rs by the direct injection of a series of cannabinoids into the brain. The influence of the activation of CB1Rs on supraspinal analgesia evoked by morphine was also evaluated.

Results

Intracerebroventricular (icv) administration of cannabinoid receptor agonists, WIN55,212-2, ACEA or methanandamide, generated a dose-dependent analgesia. Notably, a single administration of these compounds brought about profound analgesic tolerance that lasted for more than 14 days. This decrease in the effect of cannabinoid receptor agonists was not mediated by depletion of CB1Rs or the loss of regulated G proteins, but, nevertheless, it was accompanied by reduced morphine analgesia. On the other hand, acute morphine administration produced tolerance that lasted only 3 days and did not affect the CB1R. We found that both neural mu-opioid receptors (MORs) and CB1Rs interact with the HINT1-RGSZ module, thereby regulating pertussis toxin-insensitive Gz proteins. In mice with reduced levels of these Gz proteins, the CB1R agonists produced no such desensitization or morphine cross-tolerance. On the other hand, experimental enhancement of Gz signaling enabled an acute icv administration of morphine to produce a long-lasting tolerance at MORs that persisted for more than 2 weeks, and it also impaired the analgesic effects of cannabinoids.

Conclusion

In the brain, cannabinoids can produce analgesic tolerance that is not associated with the loss of surface CB1Rs or their uncoupling from regulated transduction. Neural specific Gz proteins are essential mediators of the analgesic effects of supraspinal CB1R agonists and morphine. These Gz proteins are also responsible for the long-term analgesic tolerance produced by single doses of these agonists, as well as for the cross-tolerance between CB1Rs and MORs.     

Mechanisms for the coupling of cannabinoid receptors to intracellular calcium mobilization in rat insulinoma beta-cells

In RIN m5F rat insulinoma beta-cells, agonists at cannabinoid CB(1) receptors modulate insulin release. Here we investigated in these cells the effect of the activation of cannabinoid CB(1) and CB(2) receptors on intracellular Ca(2+) ([Ca(2+)](i)). The CB(1) agonist arachidonoyl-chloro-ethanolamide (ACEA), and the CB(2) agonist JWH133, elevated [Ca(2+)](i) in a way sensitive to the inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), U73122 (but not to pertussis toxin and forskolin), and independently from extracellular Ca(2+). PI-PLC-dependent Ca(2+) mobilization by ACEA was entirely accounted for by activation of inositol-1,3,4-phosphate (IP(3)) receptors on the endoplasmic reticulum (ER), whereas the effect of JWH133 was not sensitive to all tested inhibitors of IP(3) and ryanodine receptors. ACEA, but not JWH133, significantly inhibited the effect on [Ca(2+)](i) of bombesin, which acts via G(q/11)- and PI-PLC-coupled receptors in insulinoma cells. The endogenous CB(1) agonists, anandamide and N-arachidonoyldopamine, which also activate transient receptor potential vanilloid type 1 (TRPV1) receptors expressed in RIN m5F cells, elevated [Ca(2+)](i) in the presence of extracellular Ca(2+) in a way sensitive to both CB(1) and TRPV1 antagonists. These results suggest that, in RIN m5F cells, CB(1) receptors are coupled to PI-PLC-mediated mobilization of [Ca(2+)](i) and might inhibit bombesin signaling.     

Stage-specific excitation of cannabinoid receptor exhibits differential effects on mouse embryonic development

Anandamide (N-arachidonoylethanolamine), an arachidonic acid derivative, is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. We have previously demonstrated that preimplantation mouse embryos express mRNA for these receptors and that the periimplantation uterus contains the highest level of anandamide yet discovered in a mammalian tissue. We further demonstrated that 2-cell mouse embryos exposed to low levels of anandamide (7 nM) or other known cannabinoid agonists in culture exhibit markedly compromised embryonic development to blastocysts and that this effect is mediated by CB1-R. In contrast, the present study demonstrates that blastocysts exposed in culture to the same low levels of cannabinoid agonists exhibited accelerated trophoblast differentiation with respect to fibronectin-binding activity and trophoblast outgrowth. Again, these effects resulted from activation of embryonic CB1-R. There was a differential concentration-dependent effect of cannabinoids on the trophoblast, with an observed inhibition of differentiation at higher doses. These results provide evidence for the first time that cannabinoid effects are differentially executed depending on the embryonic stage and cannabinoid levels in the environment. Since uterine anandamide levels are lowest at the sites of implantation and highest at the interimplantation sites, the new findings imply that site-specific levels of anandamide and/or other endogenous ligands in the uterus may regulate implantation spatially by promoting trophoblast differentiation at the sites of blastocyst implantation.