UHRF1 depletion causes a G2/M arrest, activation of DNA damage response and apoptosis

The ECS (endocannabinoid system) plays an important role in the onset of obesity and metabolic disorders, implicating central and peripheral mechanisms predominantly via CB1 (cannabinoid type 1) receptors. CB1 receptor antagonist/inverse agonist treatment improves cardiometabolic risk factors and insulin resistance. However, the relative contribution of peripheral organs to the net beneficial metabolic effects remains unclear. In the present study, we have identified the presence of the endocannabinoid signalling machinery in skeletal muscle and also investigated the impact of an HFD (high-fat diet) on lipid-metabolism-related genes and endocannabinoid-related proteins. Finally, we tested whether administration of the CB1 inverse agonist AM251 restored the alterations induced by the HFD. Rats were fed on either an STD (standard/low-fat diet) or an HFD for 10 weeks and then treated with AM251 (3 mg/kg of body weight per day) for 14 days. The accumulated caloric intake was progressively higher in rats fed on the HFD than the STD, resulting in a divergence in body weight gain. AM251 treatment reduced accumulated food/caloric intake and body weight gain, being more marked in rats fed on the HFD. CB2 (cannabinoid type 2) receptor and PPARα (peroxisome-proliferator-activated receptor α) gene expression was decreased in HFD-fed rats, whereas MAGL (monoglyceride lipase) gene expression was up-regulated. These data suggest an altered endocannabinoid signalling as a result of the HFD. AM251 treatment reduced CB2 receptor, PPARγ and AdipoR1 (adiponectin receptor 1) gene expression in STD-fed rats, but only partially normalized the CB2 receptor in HFD-fed rats. Protein levels corroborated gene expression results, but also showed a decrease in DAGL (diacylglycerol) β and DAGLα after AM251 treatment in STD- and HFD-fed rats respectively. In conclusion, the results of the present study indicate a diet-sensitive ECS in skeletal muscle, suggesting that blockade of CB1 receptors could work towards restoration of the metabolic adaption imposed by diet.     

The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB₁and CB₂receptors in the mouse hippocampus

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.     

Combined stimulation of glucagon-like peptide-1 receptor and inhibition of cannabinoid CB1 receptor act synergistically to reduce food intake and body weight in the rat

Pharmacological activation of the glucagon-like peptide-1 (GLP-1) receptor and inhibition of the cannabinoid CB1 receptor were found to reduce food intake and body weight in humans and animals. Since earlier studies revealed that endocannabinoids may interact with other neurotransmitters to affect feeding behavior, we have examined whether a stable GLP-1 agonist, exendin-4 and a CB1 receptor antagonist, AM 251, may reciprocally enhance their inhibitory effects on food consumption in the rat. Additionally, we have tested whether the blockade of the GLP-1 receptor by exendin (9-39) modifies AM 251-dependent effects on energy balance. In a dose-response study, male Wistar rats were injected intraperitoneally with either 1.5-6.0 μg/kg exendin-4, 0.5-2 mg/kg AM 251, 80-320 μg/kg exendin (9-39) or their vehicle and the daily food and water intake as well as body weight changes were monitored two days before and two days after the injection. Exendin-4 at a dose of 3.0 and 6.0 μg/kg and AM 251 at a dose 2 mg/kg decreased significantly 24-hour food intake and body weight. Therefore, in the next study, the effects of lower doses of exendin-4 (1.5 μg/kg) and AM 251 (1.0 mg/kg) administered alone or together on food consumption were compared. As opposed to being injected alone, the co-administration of the two resulted in a marked decrease in both daily food intake and body weight. Exendin (9-39) did not modify the suppressory effect of the highest AM 251 dose on food consumption. Apparently, the effect of AM 251 on the appetite is not mediated by GLP-1. The concomitant stimulation of GLP-1 receptor and blockade of CB1 receptor, however, may act synergistically to inhibit appetite in the rat.     

The role of central CB2 cannabinoid receptors on food intake in neonatal chicks

The endocannabinoids (ECBs) have diverse physiological functions including the regulation of food intake and metabolism. In mammals, ECBs regulate feeding primarily through the CB1 receptors within the brain whereas the CB2 receptors are primarily involved in the regulation of immune function by direct action on peripheral immune cells and central glia. The central effect of ECBs on feeding behavior has not been studied in non-mammalian species. Therefore, the present study investigated the effect of CB65, a selective CB2 receptors agonist, on food intake in the neonatal chicks. In addition, the effect of astressin, a CRF receptor antagonist, on CB65-induced food intake was also investigated. Intracerebroventricular injection of the CB65 (1.25 μg) increased the food intake at 30- and 60-min post-injection significantly as compared to the control group. Pretreatment with a selective CB2 receptor antagonist, AM630, but not astressin, significantly attenuated the CB65-induced food intake. These results suggested that CB2 receptor agonists act on the brain to induce food intake.     

Cannabinoid CB1 receptor activation does not prevent the toxicity of glutamate towards embryonic chick telencephalon primary cultures

Cannabinoids, as a result of their ability to activate cannabinoid CB1 receptors, have been shown to possess neuroprotective properties in vivo. In vitro studies into neuroprotective effects mediated by CB1 receptors have in general used primary neuronal cultures derived from embryonic rodents. In the present study, we have investigated whether embryonic chick telencephalon primary cultures in serum-free medium are a useful alternative for such in vitro studies. The CB agonist CP 55940 reduced the cAMP response to 5 microM forskolin by 40 and 50% at concentrations of 3 nM and 30 nM, respectively. This reduction was blocked by the CB1 receptor antagonist AM251, indicating the presence of functional CB1 receptors in the cultures. Incubation of the cultures with glutamate (100 microM or 1 mM) for 1 h followed by medium change and incubation for 24 h produced a release of the cytoplasmic enzyme lactate dehydrogenase into the medium. This release was prevented by MK-801 confirming the central role of NMDA receptors in the glutamate toxicity. However, 3-30 nM CP 55940 did not produce any neuroprotection in this model regardless as to whether dibutyryl cyclic AMP was added to the culture medium. The endocannabinoid anandamide was also without effect when added either per se or together with the related N-acyl ethanolamines palmitoylethanolamide, oleoylethanolamide and stearoylethanolamide (at relative concentrations matching those seen in rat brain after excitotoxic insult). It is concluded that embryonic chick neurons in primary serum-free culture are not a useful model for the study of neuroprotective effects mediated by CB1 receptors in vitro.     

Reciprocal inhibition of G-protein signaling is induced by CB(1) cannabinoid and GABA(B) receptor interactions in rat hippocampal membranes

Cannabinoid CB(1) and the metabotropic GABA(B) receptors have been shown to display similar pharmacological effects and co-localization in certain brain regions. Previous studies have reported a functional link between the two systems. As a first step to investigate the underlying molecular mechanism, here we show cross-inhibition of G-protein signaling between GABA(B) and CB(1) receptors in rat hippocampal membranes. The CB(1) agonist R-Win55,212-2 displayed high potency and efficacy in stimulating guanosine-5'-O-(3-[(35)S]thio)triphosphate, [(35)S]GTPgammaS binding. Its effect was completely blocked by the specific CB(1) antagonist AM251 suggesting that the signaling was via CB(1) receptors. The GABA(B) agonists baclofen and SKF97541 also elevated [(35)S]GTPgammaS binding by about 60%, with potency values in the micromolar range. Phaclofen behaved as a low potency antagonist with an ED(50) approximately 1mM. However, phaclofen at low doses (1 and 10nM) slightly but significantly attenuated maximal stimulation of [(35)S]GTPgammaS binding by the CB(1) agonist R-Win55,212-2. The observation that higher concentrations of phaclofen had no such effect rule out the possibility of its direct action on CB(1) receptors. The pharmacologically inactive stereoisomer S-Win55,212-3 had no effect either alone or in combination with phaclofen establishing that the interaction is stereospecific in hippocampus. The specific CB(1) antagonist AM251 at a low dose (1 nM) also inhibited the efficacy of G-protein signaling of the GABA(B) receptor agonist SKF97541. Cross-talk of the two receptor systems was not detected in either spinal cord or cerebral cortex membranes. It is speculated that the interaction might occur via an allosteric interaction between a subset of GABA(B) and CB(1) receptors in rat hippocampal membranes. Although the exact molecular mechanism of the reciprocal inhibition between CB(1) and GABA(B) receptors will have to be explored by future studies it is intriguing that the cross-talk might be involved in balance tuning the endocannabinoid and GABAergic signaling in hippocampus.     

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.     

Inverse agonism and neutral antagonism at cannabinoid CB1 receptors

There are at least two types of cannabinoid receptor, CB1 and CB2, both G protein coupled. CB1 receptors are expressed predominantly at nerve terminals and mediate inhibition of transmitter release whereas CB2 receptors are found mainly on immune cells, one of their roles being to modulate cytokine release. Endogenous cannabinoid receptor agonists also exist and these "endocannabinoids" together with their receptors constitute the "endocannabinoid system". These discoveries were followed by the development of a number of CB1- and CB2-selective antagonists that in some CB1 or CB2 receptor-containing systems also produce "inverse cannabimimetic effects", effects opposite in direction from those produced by cannabinoid receptor agonists. This review focuses on the CB1-selective antagonists, SR141716A, AM251, AM281 and LY320135, and discusses possible mechanisms by which these ligands produce their inverse effects: (1) competitive surmountable antagonism at CB1 receptors of endogenously released endocannabinoids, (2) inverse agonism resulting from negative, possibly allosteric, modulation of the constitutive activity of CB1 receptors in which CB1 receptors are shifted from a constitutively active "on" state to one or more constitutively inactive "off" states and (3) CB1 receptor-independent mechanisms, for example antagonism of endogenously released adenosine at A1 receptors. Recently developed neutral competitive CB1 receptor antagonists, which are expected to produce inverse effects through antagonism of endogenously released endocannabinoids but not by modulating CB1 receptor constitutive activity, are also discussed. So too are possible clinical consequences of the production of inverse cannabimimetic effects, there being convincing evidence that released endocannabinoids can have "autoprotective" roles.     

The cannabinoid receptor agonist CP-55,940 and ethyl arachidonate interfere with [(3)H]batrachotoxinin A 20 alpha-benzoate binding to sodium channels and inhibit sodium channel function

Recent investigations in our laboratory showed that voltage-gated sodium channels (VGSCs) in brain are sensitive to inhibition by various synthetic cannabinoids and endocannabinoids. The present experiments examined the effects of the cannabinoid-1 (CB1) receptor agonist CP-55,940 and ethyl arachidonate on [(3)H]batrachotoxinin A 20 alpha-benzoate ([(3)H]BTX-B]) binding and VGSC-dependent depolarization of the nerve membrane in synaptoneurosomes isolated from mouse whole brain. CP-55,940 acted as a full inhibitor of [(3)H]BTX-B binding and its IC(50) was established at 22.3 microM. At its maximum effect concentration, ethyl arachidonate achieved partial (approximately 70%) inhibition and was less effective than CP-55,940 as an inhibitor of binding (IC(50)=262.7 microM). The potent CB1 receptor antagonist AM251 (2 microM) had no significant effect on the displacement of [(3)H]BTX-B by either compound (P>0.05). Scatchard analyses showed that CP-55,940 and ethyl arachidonate reduce the binding of [(3)H]BTX-B by lowering its B(max) but ethyl arachidonate also increased the K(d) of radioligand binding. In kinetic experiments, CP-55,940 and ethyl arachidonate were found to boost the dissociation of [(3)H]BTX-B from VGSCs to rates that exceed the maximum velocity achievable by veratridine, indicating they operate as allosteric inhibitors of [(3)H]BTX-B binding. Neither compound was effective at changing the initial rate of association of [(3)H]BTX-B with sodium channels. CP-55,940 and ethyl arachidonate inhibited veratridine-dependent (TTX-suppressible) depolarization of the plasma membrane of synaptoneurosomes with IC(50)s of 3.2 and 50.1 microM respectively. These inhibitory effects were again not influenced by 2 microM AM251. Our data demonstrate that the potent cannabinoid receptor agonist CP-55,940 and the ethyl ester of arachidonic acid have the ability to associate with VGSCs and inhibit their function independently of effects on CB1 receptors. Binding data comparisons using mouse brain preparations indicate CP-55,940 is approximately 10,000 times more potent as a CB1 receptor ligand than a sodium channel ligand while ethyl arachidonate shows a much smaller differential. Ethyl arachidonate has been shown previously to be the principal metabolite of ethanol in the brains of intoxicated individuals and effects of this ester on VGSCs and CB1 receptors may contribute to the depressant effects of alcohol.     

Topically administered CB(2)-receptor agonist,JWH-133, does not decrease intraocular pressure (IOP) in normotensive rabbits

Recent IOP and receptor localization studies suggest that the IOP effects of cannabinoids are mediated via ocular CB(1) receptors. However, it is not yet known whether CB(2) receptor agonists decrease IOP. In this study, the IOP-lowering effects of the CB(2) receptor agonist JWH-133 were studied in normotensive rabbits, and compared with CP55,940. JWH-133 and CP55,940 were dissolved in aqueous hydroxypropyl-beta-cyclodextrin (HP-beta-CD) solutions and propylene glycol. The eye drops (25 microl) were administered unilaterally to the rabbit eye, and IOPs were measured at fixed time intervals. JWH-133, dissolved in either HP-beta-CD (doses = 10 microg and 25 microg) or propylene glycol (dose = 62.5 microg), did not have any effect on IOP when compared to vehicle treatments. In contrast, CP55,940 formulated in HP-beta-CD (doses = 25 microg and 62.5 microg) or propylene glycol (dose = 62.5 microg) reduced IOP significantly compared to vehicle treatments. The results suggest that topically administered CB(2) receptor agonist, JWH-133, does not decrease IOP in normotensive rabbits at the doses and formulations used, and thus, CB(2) receptor agonists may not be useful as cannabinoid-based IOP-lowering therapeutics.     

Compounds acting at the endocannabinoid and/or endovanilloid systems reduce hyperkinesia in a rat model of Huntington's disease

We have recently reported that the administration of AM404, an inhibitor of the endocannabinoid re-uptake process, which also has affinity for the vanilloid VR1 receptors, is able to reduce hyperkinesia, and causes recovery from neurochemical deficits, in a rat model of Huntington's disease (HD) generated by bilateral intrastriatal injections of 3-nitropropionic acid (3NP). In the present study, we wanted to explore the mechanism(s) by which AM404 produces its antihyperkinetic effect in 3NP-lesioned rats by employing several experimental approaches. First, we tried to block the effects of AM404 with selective antagonists for the CB1 or VR1 receptors, i.e. SR141716A and capsazepine, respectively. We found that the reduction caused by AM404 of the increased ambulation exhibited by 3NP-lesioned rats in the open-field test was reversed when the animals had been pre-treated with capsazepine but not with SR141716A, thus suggesting a major role of VR1 receptors in the antihyperkinetic effects of AM404. However, despite the lack of behavioral effects of the CB1 receptor antagonist, the pretreatment with this compound abolished the recovery of neurochemical [gamma-aminobutyric acid (GABA) and dopamine] deficits in the caudate- putamen caused by AM404, as also did capsazepine. In a second group of studies, we wanted to explore the potential antihyperkinetic effects of various compounds which, compared to AM404, exhibit more selectivity for either the endovanilloid or the endocannabinoid systems. First, we tested VDM11 or AM374, two selective inhibitors or the endocannabinoid re-uptake or hydrolysis, respectively. Both compounds were mostly unable to reduce hyperkinesia in 3NP-lesioned rats, although VDM11 produced a certain motor depression, and AM374 exhibited a trend to stimulate ambulation, in control rats. We also tested the effects of selective direct agonists for VR1 (capsaicin) or CB1 (CP55,940) receptors. Capsaicin exhibited a strong antihyperkinetic activity and, moreover, was able to attenuate the reductions in dopamine and GABA transmission provoked by the 3NP lesion, whereas CP55,940 had also antihyperkinetic activity but was unable to cause recovery of either dopamine or GABA deficits in the basal ganglia. In summary, our data indicate a major role for VR1 receptors, as compared to CB1 receptors, in the antihyperkinetic effects and the recovery of neurochemical deficits caused in 3NP-lesioned rats by compounds that activate both CB1 and VR1 receptors, either directly or via manipulation of the levels of endogenous agonists.     

BML-190 and AM251 act as inverse agonists at the human cannabinoid CB2 receptor: signalling via cAMP and inositol phosphates

The aminoalkylindole BML-190 and diarylpyrazole AM251 ligands have previously been shown to bind to cannabinoid CB(2) and CB(1) receptors, respectively. In HEK-293 cells stably expressing the human CB(2) receptor, BML-190 and AM251 potentiated the forskolin-stimulated accumulation of cAMP. Moreover, the CB(2) receptor can interact productively with 16z44, a promiscuous G alpha(16/z) chimera. BML-190 and AM251 reduce the basal levels of inositol phosphate production in cells expressing the CB(2) receptor and 16z44. These results demonstrate that BML-190 and AM251 act as inverse agonists at the human CB(2) receptor acting via G alpha(i/o) and G alpha(q) family-coupled pathways.     

Staphylococcal enterotoxin induces emesis through increasing serotonin release in intestine and it is downregulated by cannabinoid receptor 1

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are the most recognizable bacterial superantigenic toxins causing food poisoning in humans throughout the world. However, it remains unclear how SEs induce emesis and its emetic signal pathway. We investigated a mechanism of SEA-induced emesis using a small emetic animal model, house musk shrew. SEA-induced emesis in the animals was inhibited by a 5-hydroxytryptamine (5-HT) synthesis inhibitor and a 5-HT(3) receptor antagonist. SEA could increase 5-HT release in the small intestine. Pre-treatment with 5,7-dihydroxytryptamine (5,7-DHT) markedly inhibited SEA-induced emesis. SEA-induced emesis was also abolished by surgical vagotomy. Furthermore, cannabinoid (CB) receptor agonists inhibited SEA-induced emesis, and the action was reversed by a CB1 antagonist. Both 5-HT release and CB1 receptor expression were found in the mucosal and myenteric plexus of the intestine. Moreover, a CB1 receptor agonist significantly decreased the 5-HT release in the intestine. These results demonstrate that SEA induces 5-HT release in intestine, rather than in brain, and that the 5-HT(3) receptors on vagal afferent neurons are essential for SEA-stimulated emesis. In addition, SEA-induced emesis is downregulated by the CB system through decreasing 5-HT release in intestine.     

Cannabinoid-1 receptor antagonists reduce caloric intake by decreasing palatable diet selection in a novel dessert protocol in female rats

Although many feeding protocols induce obesity, few use multiple foods to analyze diet selection within a single group of animals. To this end, we describe a protocol using time-limited access to a dessert that induces hyperphagia and body weight gain while allowing simple analysis of diet selection. Female retired breeder Sprague-Dawley rats were provided with ad libitum access to standard moist chow (1.67 kcal/g) and daily 8-h nocturnal access to either a sugar gel (SG; 0.31 kcal/g) or sugar fat whip (SFW; 7.35 kcal/g) for 15 days, and food intake and body weight were measured daily. Rats given SFW reduced moist chow intake but not enough to compensate for the large amount of calories consumed from SFW, and thus gained weight. We use this SFW overconsumption protocol to investigate the hypothesis that cannabinoid (CB)1 receptor antagonists reduce caloric intake by selectively decreasing consumption of palatable foods. In two experiments, female retired breeder Sprague-Dawley rats were injected with either Rimonabant (1 mg/kg ip) or vehicle (equal parts polyethylene glycol and saline, 1 ml/kg ip) for 7 days, or one of three doses of AM251 (0.3, 1.0, or 3.0 mg/kg ip), or vehicle for 15 days; food intake and body weight were measured daily. Both Rimonabant and AM251 decreased 24-h caloric intake, but the reduction was specific to a decrease in SFW consumption. This supports the hypothesis that these CB1 receptor antagonists impact feeding by modulating the perception of palatability.     

Dopaminergic Modulation of Effort-Related Choice Behavior as Assessed by a Progressive Ratio Chow Feeding Choice Task: Pharmacological Studies and the Role of Individual Differences

Mesolimbic dopamine (DA) is involved in behavioral activation and effort-related processes. Rats with impaired DA transmission reallocate their instrumental behavior away from food-reinforced tasks with high response requirements, and instead select less effortful food-seeking behaviors. In the present study, the effects of several drug treatments were assessed using a progressive ratio (PROG)/chow feeding concurrent choice task. With this task, rats can lever press on a PROG schedule reinforced by a preferred high-carbohydrate food pellet, or alternatively approach and consume the less-preferred but concurrently available laboratory chow. Rats pass through each ratio level 15 times, after which the ratio requirement is incremented by one additional response. The DA D₂ antagonist haloperidol (0.025–0.1 mg/kg) reduced number of lever presses and highest ratio achieved but did not reduce chow intake. In contrast, the adenosine A_2A antagonist MSX-3 increased lever presses and highest ratio achieved, but decreased chow consumption. The cannabinoid CB1 inverse agonist and putative appetite suppressant AM251 decreased lever presses, highest ratio achieved, and chow intake; this effect was similar to that produced by pre-feeding. Furthermore, DA-related signal transduction activity (pDARPP-32(Thr34) expression) was greater in nucleus accumbens core of high responders (rats with high lever pressing output) compared to low responders. Thus, the effects of DA antagonism differed greatly from those produced by pre-feeding or reduced CB1 transmission, and it appears unlikely that haloperidol reduces PROG responding because of a general reduction in primary food motivation or the unconditioned reinforcing properties of food. Furthermore, accumbens core signal transduction activity is related to individual differences in work output.     

CP55,940 increases intracellular Ca2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.     

Regulation of peripheral cannabinoid receptor CB2 phosphorylation by the inverse agonist SR 144528. Implications for receptor biological responses.

We recently demonstrated that the selective cannabinoid receptor antagonist SR 144528 acts as an inverse agonist that blocks constitutive mitogen-activated protein kinase activity coupled to the spontaneous autoactivated peripheral cannabinoid receptor (CB2) in the Chinese hamster ovary cell line stably transfected with human CB2. In the present report, we studied the effect of SR 144528 on CB2 phosphorylation. The CB2 phosphorylation status was monitored by immunodetection using an antibody specific to the COOH-terminal CB2 which can discriminate between phosphorylated and non-phosphorylated CB2 isoforms at serine 352. We first showed that CB2 is constitutively active, phosphorylated, and internalized at the basal level. By blocking autoactivated receptors, inverse agonist SR 144528 treatment completely inhibited this phosphorylation state, leading to an up-regulated CB2 receptor level at the cell surface, and enhanced cannabinoid agonist sensitivity for mitogen-activated protein kinase activation of Chinese hamster ovary-CB2 cells. After acute agonist treatment, serine 352 was extensively phosphorylated and maintained in this phosphorylated state for more than 8 h after agonist treatment. The cellular responses to CP-55,940 were concomitantly abolished. Surprisingly, CP-55,940-induced CB2 phosphorylation was reversed by SR 144528, paradoxically leading to a non-phosphorylated CB2 which could then be fully activated by CP-55,940. The process of CP-55,940-induced receptor phosphorylation followed by SR 144528-induced receptor dephosphorylation kept recurring many times on the same cells, indicating that the agonist switches the system off but the inverse agonist switches the system back on. Finally, we showed that autophosphorylation and CP-55, 940-induced serine 352 CB2 phosphorylation involve an acidotropic GRK kinase, which does not use Gibetagamma. In contrast, SR 144528-induced CB2 dephosphorylation was found to involve an okadaic acid and calyculin A-sensitive type 2A phosphatase.     

Expression in Escherichia coli and characterisation of the human central CB1 and peripheral CB2 cannabinoid receptors

The human central CB1 and peripheral CB2 cannabinoid receptors were expressed in Escherichia coli as fusion proteins with the maltose-binding protein at their amino-termini and a hexa-histidine/Flag tag at their carboxyl-termini. Western blot analysis of the expressed proteins revealed considerable degradation of the CB1 fusion, which failed to bind either the cannabinoid agonist CP 55,940 or the CB1-specific antagonist SR 141716A. In contrast, the CB2 fusion was well-expressed and bound several cannabinoids with affinities comparable to those observed in mammalian expression systems. This revised version was published online in November 2006 with corrections to the Cover Date.     

Dual Neuroprotective and Neurotoxic Effects of Cannabinoid Drugs in Vitro

Either protective or toxic effects of cannabinoids on cell survival have been reported extensively in the literature; however, the factors that determine the direction of the effect are still obscured. In this study we have used the neuroblastoma cell line N18TG2 that expresses CB1 cannabinoid receptors to investigate several factors that may determine the consequences of exposure to cannabinoid agonists. Cells that were grown under optimal, stressful, or differentiating conditions were exposed to cannabinoid agonists and then assayed for cell viability by measuring MTT, LDH, and caspase-3 activity. Various cannabinoid agonists (CP 55,940, ∆9-THC, HU-210, and WIN 55,212-2) failed to affect cell viability when the cells were grown under optimal conditions. On the other hand, the same agonists significantly reduced cell viability when the cells were grown under stressful conditions (glucose- and serum-free medium), while enhancing the viability of cells grown in differentiation medium (0.5% serum and 1.5% DMSO). The toxic/protective profile was not dependent on the type or the concentration of the cannabinoid agonist that was applied. The cannabinoid agonist CP 55,940 similarly affected the non-neuronal HEK-293 cells that were grown under stressful conditions only when they expressed CB1 receptors. Our results shed light on the conflicting reports regarding the protective or toxic effects of cannabinoids in vitro and indicate that cannabinoids may activate different intracellular signaling mechanisms, depending on the state of the cell, thus leading to different physiological consequences.