Neuroprotective effect of (-)Delta9-tetrahydrocannabinol and cannabidiol in N-methyl-D-aspartate-induced retinal neurotoxicity: involvement of peroxynitrite. (17/1474)

In glaucoma, the increased release of glutamate is the major cause of retinal ganglion cell death. Cannabinoids have been demonstrated to protect neuron cultures from glutamate-induced death. In this study, we test the hypothesis that glutamate causes apoptosis of retinal neurons via the excessive formation of peroxynitrite, and that the neuroprotective effect of the psychotropic Delta9-tetrahydroxycannabinol (THC) or nonpsychotropic cannabidiol (CBD) is via the attenuation of this formation. Excitotoxicity of the retina was induced by intravitreal injection of N-methyl-D-aspartate (NMDA) in rats, which also received 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (TEMPOL,a superoxide dismutase-mimetic), N-omega-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), THC, or CBD. Retinal neuron loss was determined by TDT-mediated dUTP nick-end labeling assay, inner retinal thickness, and quantification of the mRNAs of ganglion cell markers. NMDA induced a dose- and time-dependent accumulation of nitrite/nitrate, lipid peroxidation, and nitrotyrosine (foot print of peroxynitrite), and a dose-dependent apoptosis and loss of inner retinal neurons. Treatment with L-NAME or TEMPOL protected retinal neurons and confirmed the involvement of peroxynitrite in retinal neurotoxicity. The neuroprotection by THC and CBD was because of attenuation of peroxynitrite. The effect of THC was in part mediated by the cannabinoid receptor CB1. These results suggest the potential use of CBD as a novel topical therapy for the treatment of glaucoma.  (+info)

Addicting drugs utilize a synergistic molecular mechanism in common requiring adenosine and Gi-beta gamma dimers. (18/1474)

The mesolimbic dopamine system and cAMP-dependent/protein kinase A (PKA) pathways are strongly implicated in addictive behaviors. Here we determine the role of dopamine D2 receptors (D2) in PKA signaling responses to delta-opioid (DOR) and cannabinoid (CB1) receptors. We find in NG108-15/D2 cells and in cultured primary neurons that a brief exposure to saturating concentrations of DOR and CB1 agonists increases cAMP, promotes PKA C alpha translocation and increases cAMP-dependent gene expression. Activation of PKA signaling is mediated by Gi-beta gamma dimers. Importantly, subthreshold concentrations of DOR or CB1 agonists with D2 agonists, which are without effect when added separately, together activate cAMP/PKA signaling synergistically. There is also synergy between DOR or CB1 with ethanol, another addicting agent. In all instances, synergy requires adenosine activation of adenosine A2 receptors and is mediated by beta gamma dimers. Synergy by this molecular mechanism appears to confer hypersensitivity to opioids and cannabinoids while simultaneously increasing the sensitivity of D2 signaling when receptors are expressed on the same cells. This mechanism may account, in part, for drug-induced activation of medium spiny neurons in the nucleus accumbens.  (+info)

Mediation of amphetamine-induced long-term depression of synaptic transmission by CB1 cannabinoid receptors in the rat amygdala. (19/1474)

The amygdala is thought to mediate memory consolidation of amphetamine-induced conditioned place preference, a behavioral paradigm that requires memory for an association between environmental cues and the affective state produced by the drug treatment. Here we show that amphetamine induces long-term synaptic depression (LTD) in the amygdala. Amphetamine LTD is not affected by dopamine, serotonin 1A, and norepinephrine alpha2 receptor antagonists but is blocked by the cannabinoid CB1 receptor antagonist AM251. It is mimicked by the CB1 agonist WIN55212-2 and facilitated and partially occluded by endocannabinoid uptake inhibitor AM404. Both amphetamine and WIN55212-2 LTDs are associated with an increase in the ratio of paired-pulse facilitation and a decrease in the frequency but not the amplitude of miniature EPSCs. They are also sensitive to block by P/Q type calcium channel blocker and occluded by each other, indicating that these two forms of synaptic plasticity share a common underlying mechanism. Loading postsynaptic neuron with calcium chelator blocked amphetamine LTD in some but not all neurons tested. However, in the presence of AM404, amphetamine LTD was present in all neurons recorded. These results suggest that amphetamine-induced endocannabinoid release depends on a rise in intracellular calcium and the incomplete block of LTD in some neurons may be attributable to the spillover of endocannabinoid from nearby cells. The finding that endocannabinoids underlie the synaptic actions of amphetamine may open a new avenue for the treatment of psychostimulants addiction.  (+info)

Anandamide content and interaction of endocannabinoid/GABA modulatory effects in the NTS on baroreflex-evoked sympathoinhibition. (20/1474)

Cannabinoids have been shown to modulate central autonomic regulation and baroreflex control of blood pressure (BP). The presence of cannabinoid CB(1) receptors on fibers in the nucleus tractus solitarius (NTS) suggests that some presynaptic modulation of transmitter release could occur in this region, which receives direct afferent projections from arterial baroreceptors and cardiac mechanoreceptors. This study, therefore, was performed to determine the mechanism(s) of effects of microinjection of an endocannabinoid, arachidonylethanolamide (anandamide, AEA), into the NTS on baroreflex sympathetic nerve responses produced by phenylephrine-induced pressure changes in anesthetized rats. AEA prolonged reflex inhibition of renal sympathetic nerve activity (RSNA), suggesting an increase in baroreflex sensitivity. This effect of AEA was blocked by prior microinjection of SR-141716 to block cannabinoid CB(1) receptors. To determine whether this baroreflex enhancement by AEA involved a GABA(A) mechanism, the baroreflex response to AEA was tested after prior blockade of postsynaptic GABA(A) receptors by bicuculline, which would eliminate any effects due to modulation of GABA activity. After bicuculline, which alone prolonged the baroreflex inhibition of RSNA, AEA shortened the duration of RSNA inhibition, suggesting a possible presynaptic inhibition of glutamate release previously obscured by a more dominant GABA(A) effect. To support a possible physiological role for AEA, AEA concentration in the NTS was measured after a phenylephrine-induced increase in BP. AEA content in the NTS was increased significantly over that in normotensive animals. These results support the hypothesis that AEA content is increased by brief periods of hypertension and suggest that AEA can modulate the baroreflex through activation of CB(1) receptors within the NTS, possibly modulating effectiveness of GABA and/or glutamate neurotransmission.  (+info)

An optimized approach to study endocannabinoid signaling: evidence against constitutive activity of rat brain adenosine A1 and cannabinoid CB1 receptors. (21/1474)

At nanomolar concentrations, SR141716 and AM251 act as specific and selective antagonists of the cannabinoid CB1 receptor. In the micromolar range, these compounds were shown to inhibit basal G-protein activity, and this is often interpreted to implicate constitutive activity of the CB1 receptors in native tissue. We show here, using [35S]GTPgammaS binding techniques, that micromolar concentrations of SR141716 and AM251 inhibit basal G-protein activity in rat cerebellar membranes, but only in conditions where tonic adenosine A1 receptor signaling is not eliminated. Unlike lipophilic A1 receptor antagonists (potency order DPCPX>>N-0840 approximately cirsimarin>caffeine), adenosine deaminase (ADA) was not fully capable in eliminating basal A1 receptor-dependent G-protein activity. Importantly, all antagonists reduced basal signal to the same extent (20%), and the response evoked by the inverse agonist DPCPX was not reversed by the neutral antagonist N-0840. These data indicate that rat brain A1 receptors are not constitutively active, but that an ADA-resistant adenosine pool is responsible for tonic A1 receptor activity in brain membranes. SR141716 and AM251, at concentrations fully effective in reversing CB1-mediated responses (10-6 m), did not reduce basal G-protein activity, indicating that CB1 receptors are not constitutively active in these preparations.4 At higher concentrations (1-2.5 x 10-5 m), both antagonists reduced basal G-protein activity in control and ADA-treated membranes, but had no effect when A1 receptor signaling was blocked with DPCPX. Moreover, the CB1 antagonists right-shifted A1 agonist dose-response curves without affecting maximal responses, suggesting competitive mode of antagonist action. The CB1 antagonists did not affect muscarinic acetylcholine or GABAB receptor signaling. When further optimizing G-protein activation assay for the labile endocannabinoid 2-arachidonoylglycerol (2-AG), we show, by using HPLC, that pretreatment of cerebellar membranes with methyl arachidonoyl fluorophosphonate (MAFP) fully prevented enzymatic degradation of 2-AG and concomitantly enhanced the potency of 2-AG. In contrast to previous claims, MAFP exhibited no antagonist activity at the CB1 receptor.6 The findings establish an optimized method with improved signal-to-noise ratio to assess endocannabinoid-dependent G-protein activity in brain membranes, under assay conditions where basal adenosinergic tone and enzymatic degradation of 2-AG are fully eliminated.  (+info)

D2 dopamine receptors modulate Galpha-subunit coupling of the CB1 cannabinoid receptor. (22/1474)

CB(1) cannabinoid (CB(1)) and D(2) dopamine (D(2)) receptors are known to couple to the G protein Galpha(i/o). It has been reported that concurrent activation of D(2) receptors and CB(1) receptors, in primary striatal neuronal culture, promotes functional CB(1) receptor coupling to Galpha(s) resulting in elevations in intracellular cyclic AMP levels. We now report that in the absence of D(2) receptors, acute activation of CB(1) receptors inhibits cyclic AMP accumulation, whereas the presence of D(2) receptors promotes CB(1)-stimulated cAMP accumulation, presumably through Galpha(s). This Galpha(s) subunit switching was not prevented by pertussis toxin treatment and occurred in the presence and absence of D(2) receptor activation. Thus, coexpression of the D(2) receptor with the CB(1) receptor was sufficient to switch the coupling of the CB(1) receptors from Galpha(i/o) to Galpha(s). Persistent activation of D(2) receptors resulted in heterologous sensitization of adenylate cyclase to subsequent stimulation by forskolin, whereas the persistent activation of CB(1) receptors did not. Additional studies in human embryonic kidney cells cotransfected with D(2) and CB(1) receptors revealed that persistent activation (18 h) of D(2) receptors induced a switch of CB(1) receptor coupling from Galpha(s) to Galpha(i/o). This D(2) receptor-induced effect allowed for CB(1) receptor-mediated inhibition of cyclic AMP accumulation. The present studies suggest D(2) receptors may have a significant modulatory role in determining the G protein coupling specificity of CB(1) receptors.  (+info)

Anandamide initiates Ca(2+) signaling via CB2 receptor linked to phospholipase C in calf pulmonary endothelial cells. (23/1474)

The endocannabinoid anandamide has been reported to affect neuronal cells, immune cells and smooth muscle cells via either CB1 or CB2 receptors. In endothelial cells, the receptors involved in activating signal transduction are still unclear, despite the fact that anandamide is produced in this cell type. The present study was designed to explore in detail the effect of this endocannabinoid on Ca2+ signaling in single cells of a calf pulmonary endothelial cell line. Anandamide initiated a transient Ca2+ elevation that was prevented by the CB2 receptor antagonist SR144528, but not by the CB1 antagonist SR141716A. These data were confirmed by molecular identification of the bovine CB2 receptor in these endothelial cells by partial sequencing. The phospholipase C inhibitor 1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5d ione and the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenylborate prevented Ca2+ signaling in response to anandamide. Using an improved cameleon probe targeted to the endoplasmic reticulum (ER), fura-2 and ratiometric-pericam, which is targeted to the mitochondria, anandamide was found to induce Ca2+ depletion of the ER accompanied by the activation of capacitative Ca2+ entry (CCE) and a transient elevation of mitochondrial Ca2+. These data demonstrate that anandamide stimulates the endothelial cells used in this study via CB2 receptor-mediated activation of phospholipase C, formation of inositol 1,4,5-trisphosphate, Ca2+ release from the ER and subsequent activation of CCE. Moreover, the cytosolic Ca2+ elevation was accompanied by a transient Ca2+ increase in the mitochondria. Thus, in addition to its actions on smooth muscle cells, anandamide also acts as a powerful stimulus for endothelial cells.  (+info)

Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation. (24/1474)

Mammalian fertility absolutely depends on synchronized development of the blastocyst to the stage when it is competent to implant, and the uterus to the stage when it is receptive to implantation. However, the molecular basis for the reciprocal interaction between the embryo and the uterus remains largely unexplored. One potentially important mechanism involves signaling between an evolutionarily conserved G protein-coupled protein cannabinoid receptor, CB1, that is expressed at high levels on the surface of the trophectoderm and anandamide (N-arachi-donoylethanolamine), an endocannabinoid ligand found to be produced at higher levels by the uterus before implantation and then down-regulated at the time of implantation. Using genetic, pharmacological, and physiological approaches, we show here that anandamide within a very narrow range regulates blastocyst function and implantation by differentially modulating mitogen-activated protein kinase signaling and Ca2+ channel activity via CB1 receptors. Anandamide at a low concentration (7 nM) induces extracellular regulated kinase phosphorylation and nuclear translocation in trophectoderm cells without influencing Ca2+ channels, and renders the blastocyst competent for implantation in the receptive uterus. In contrast, anandamide at a higher concentration (28 nM) inhibits Ca2+ channel activity and blastocyst competency for implantation without influencing mitogen-activated protein kinase signaling. Besides uncovering a potentially important regulatory mechanism for synchronizing blastocyst and uterine competency to implantation, this observation has high clinical relevance, because elevated levels of anandamide induce spontaneous pregnancy loss in women.  (+info)