Antiproliferative mechanism of a cannabinoid agonist by cell cycle arrest in human gastric cancer cells

For gastric cancers, the antineoplastic activity of cannabinoids has been investigated in only a few reports and knowledge regarding the mechanisms involved is limited. We have reported previously that treatment of gastric cancer cells with a cannabinoid agonist significantly decreased cell proliferation and induced apoptosis. Here, we evaluated the effects of cannabinoids on various cellular mediators involved in cell cycle arrest in gastric cancer cells. AGS and MKN-1 cell lines were used as human gastric cancer cells and WIN 55,212-2 as a cannabinoid agonist. Cell cycles were analyzed by flow cytometry and western blotting. Treatment with WIN 55,212-2 arrested the cell cycle in the G0/G1 phase. WIN 55,212-2 also upregulated phospho-ERK1/2, induced Kip1/p27 and Cip1/WAF1/p21 expression, decreased cyclin D1 and cyclin E expression, decreased Cdk 2, Cdk 4, and Cdk 6 expression levels, and decreased phospho-Rb and E2F-1 expression. ERK inhibitor decreased the proportion of G0/G1 phase which was induced by WIN 55,212-2. Inhibition of pAKT led to cell cycle arrest in gastric cancer cells. Cell cycle arrest preceded apoptotic response. Thus, this cannabinoid agonist can reduce gastric cancer cell proliferation via G1 phase cell cycle arrest, which is mediated via activation of the MAPK pathway and inhibition of pAKT.     

Inhibition of autophagy and enhancement of endoplasmic reticulum stress increase sensitivity of osteosarcoma Saos‐2 cells to cannabinoid receptor agonist WIN55,212‐2

WIN55,212‐2, a cannabinoid receptor agonist, can activate cannabinoid receptors, which has proven anti‐tumour effects in several tumour types. Studies showed that WIN can inhibit tumour cell proliferation and induce apoptosis in diverse cancers. However, the role and mechanism of WIN in osteosarcoma are still unclear. In this study, we examined the effect of WIN55,212‐2 on osteosarcoma cell line Saos‐2 in terms of cell viability and apoptosis. Meanwhile, we further explored the role of endoplasmic reticulum stress and autophagy in apoptosis induced by WIN55,212‐2. Our results showed that the cell proliferation of Saos‐2 was inhibited by WIN55,212‐2 in a dose‐dependent and time‐dependent manner. WIN55,212‐2‐induced Saos‐2 apoptosis through mitochondrial apoptosis pathway. Meanwhile, WIN55,212‐2 can induce endoplasmic reticulum stress and autophagy in Saos‐2 cells. Inhibition of autophagy and enhancement of endoplasmic reticulum stress increased apoptosis induced by WIN55,212‐2 in Saos‐2 cells. These findings indicated that WIN55,212‐2 in combination with autophagic inhibitor or endoplasmic reticulum stress activator may shed new light on osteosarcoma treatment. Copyright © 2016 John Wiley & Sons, Ltd.     

Cannabinoids Inhibit Acid-Sensing Ion Channel Currents in Rat Dorsal Root Ganglion Neurons

Local acidosis has been found in various pain-generating conditions such as inflammation and tissue injury. Cannabinoids exert a powerful inhibitory control over pain initiation via peripheral cognate receptors. However, the peripheral molecular targets responsible for the antinociceptive effects of cannabinoids are still poorly understood. Here, we have found that WIN55,212-2, a cannabinoid receptor agonist, inhibits the activity of native acid-sensing ion channels (ASICs) in rat dorsal root ganglion (DRG) neurons. WIN55,212-2 dose-dependently inhibited proton-gated currents mediated by ASICs. WIN55,212-2 shifted the proton concentration–response curve downwards, with an decrease of 48.6±3.7% in the maximum current response but with no significant change in the EC₅₀ value. The inhibition of proton-gated current induced by WIN55,212-2 was almost completely blocked by the selective CB1 receptor antagonist AM 281, but not by the CB2 receptor antagonist AM630. Pretreatment of forskolin, an AC activator, and the addition of cAMP also reversed the inhibition of WIN55,212-2. Moreover, WIN55,212-2 altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. Finally, WIN55,212-2 attenuated nociceptive responses to injection of acetic acid in rats. These results suggest that WIN55,212-2 inhibits the activity of ASICs via CB1 receptor and cAMP dependent pathway in rat primary sensory neurons. Thus, cannabinoids can exert their analgesic action by interaction with ASICs in the primary afferent neurons, which was novel analgesic mechanism of cannabinoids.     

Estimation of the hypothermic component in neuroprotection provided by cannabinoids following cerebral ischemia

Cannabinoids have neuroprotective potentials, and the expression of endocannabinoids as well as cannabinoid receptors is induced after cerebral ischemia. They also induce hypothermia by lowering the hypothalamic set point. We have estimated the significance of such hypothermia in ischemic neuroprotection following systemic administration of WIN 55,212-2, a synthetic cannabinoid receptor agonist. Results showed that WIN 55,212-2 significantly reduced infarct volumes of rats subjected to focal cerebral ischemia (middle cerebral artery occlusion) and significantly decreased ischemic CA1 damage in rats subjected to global cerebral ischemia (two-vessel occlusion). A significant (approximately 50%) part of this neuroprotection was provided by WIN 55,212-2 induced hypothermia (33.7+/-1.1 degrees C/34.9+/-1.6 degrees C), because prevention of hypothermia by maintaining body core temperatures between 37.0 and 38.0 degrees C dissolved the neuroprotective effect into a hypothermic component and an unidentified component. Finally, the ability of WIN 55,212-2 to reduce levels of the proinflammatory cytokine IFNgamma in the infarcted hemisphere of rats subjected to focal cerebral ischemia required hypothermia. For the cannabinoid WIN 55,212-2, we have isolated and directly demonstrated that hypothermia is only part of, although significant, cannabinoid mediated neuroprotection in both global and focal cerebral ischemia. We conclude that cannabinoids are reliable candidates for drug-induced hypothermia and neuroprotection. These neuroprotective effects of cannabinoids could provide the basis for potential therapeutic uses of cannabinoids and/or endocannabinoids in stroke.     

WIN 55,212-2, Agonist of Cannabinoid Receptors,Prevents Amyloid β1-42 Effects on Astrocytes in Primary Culture

Alzheimer´s disease (AD), a neurodegenerative illness involving synaptic dysfunction with extracellular accumulation of Aβ_1-42 toxic peptide, glial activation, inflammatory response and oxidative stress, can lead to neuronal death. Endogenous cannabinoid system is implicated in physiological and physiopathological events in central nervous system (CNS), and changes in this system are related to many human diseases, including AD. However, studies on the effects of cannabinoids on astrocytes functions are scarce. In primary cultured astrocytes we studied cellular viability using MTT assay. Inflammatory and oxidative stress mediators were determined by ELISA and Western-blot techniques both in the presence and absence of Aβ_1-42 peptide. Effects of WIN 55,212-2 (a synthetic cannabinoid) on cell viability, inflammatory mediators and oxidative stress were also determined. Aβ_1-42 diminished astrocytes viability, increased TNF-α and IL-1β levels and p-65, COX-2 and iNOS protein expression while decreased PPAR-γ and antioxidant enzyme Cu/Zn SOD. WIN 55,212-2 pretreatment prevents all effects elicited by Aβ_1-42. Furthermore, cannabinoid WIN 55,212-2 also increased cell viability and PPAR-γ expression in control astrocytes. In conclusion cannabinoid WIN 55,212-2 increases cell viability and anti-inflammatory response in cultured astrocytes. Moreover, WIN 55,212-2 increases expression of anti-oxidant Cu/Zn SOD and is able to prevent inflammation induced by Aβ_1-42 in cultured astrocytes. Further studies would be needed to assess the possible beneficial effects of cannabinoids in Alzheimer''s disease patients.     

Effects of cannabinoid treatment on Chagas disease pathogenesis: balancing inhibition of parasite invasion and immunosuppression

Trypanosoma cruzi invades heart cells via a calcium-dependent, G protein-mediated mechanism, leading to severe cardiac inflammation considered by some to be autoimmune in nature. Cannabinoids inhibit calcium flux and G protein signalling; as potent immunosuppressive agents, they are effective in the treatment of autoimmune disease but contraindicated for the treatment of infections. We compared the action of the synthetic cannabinoid R(+)WIN55,212 and its inactive isomer S(-)WIN55,212 on cardiac myoblast invasion: R(+)WIN55,212 inhibited invasion by over 85%. We then tested for efficacy in modulating pathogenesis in mice by assaying parasite burden in heart and blood, cellular and humoral immunity to parasite and self antigens, and mortality. R(+)WIN55,212 significantly reduced cardiac inflammation but led to considerably increased parasitaemia. Cardiac parasitosis and mortality were not significantly different in treatment and control groups. We conclude that cannabinoids can block cardiac cell puncture repair mechanisms, thereby inhibiting trypanosome invasion as predicted by the mode of drug action, but, also inhibit immune cell effector functions, offsetting the benefit of inhibition parasite cell invasion. Refined use of cannabinoids may prove therapeutic in the future, but our results raise concern about the effect of cannabis use on those chronically infected by T. cruzi and on heart cell homeostasis generally.     

PPARγ mediates the effects of WIN55,212-2, an synthetic cannabinoid, on the proliferation and apoptosis of the BEL-7402 hepatocarcinoma cells

Cannabis sativa has long been used as a traditional medicine in China. Among its effective compounds are cannabinoids. This study determined the effect of WIN55,212-2 (WIN), a synthetic cannabinoid, on the BEL-7402 human hepatocellular carcinoma (HCC) cell line. The results showed that WIN could decrease the proliferation of BEL-7402 cells. Moreover, WIN could cause apoptosis of the cells via up-regulation of Bax expression, down-regulation of Bcl-2 expression, induction of the mitochondrial membrane potential, increase of caspase-3, -8 and -9 activities, and induction of the cleavage of caspase-3 and poly-ADP-ribose polymerase (PARP). The WIN-induced apoptosis was accompanied by the up-regulation of PPARγ expression, the activation of PPARγ DNA binding activity, and a down-regulation of PPARγ target oncogene c-myc. Conversely, the effects of WIN could be attenuated by PPARγ antagonist GW9662, and the WIN induced PPARγ expression was partially attenuated by AM630, a cannabinoid receptor-2 antagonist, whereas the WIN-induced reduction of c-myc expression was partially restored by GW9662. Collectively, our results suggest that WIN can decrease the proliferation and cause apoptosis of the BEL-7402 cells via a mitochondrial-caspase pathway and mediated by PPARγ. These results may provide a basis for the application of WIN in HCC treatment.     

Role of receptor internalization in the agonist-induced desensitization of cannabinoid type 1 receptors

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid-rafts- and clathrin-coated-pits-mediated pathways were involved in agonist-induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Δ⁹-THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212-2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist-induced CB1 receptor desensitization. Furthermore, co-expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co-expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212-2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Δ⁹-THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212-2, CP 55940 or 2-arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid-mediated effects.     

The Endocannabinoid/Endovanilloid N-Arachidonoyl Dopamine (NADA) and Synthetic Cannabinoid WIN55,212-2 Abate the Inflammatory Activation of Human Endothelial Cells

Although cannabinoids, such as Δ⁹-tetrahydrocannabinol, have been studied extensively for their psychoactive effects, it has become apparent that certain cannabinoids possess immunomodulatory activity. Endothelial cells (ECs) are centrally involved in the pathogenesis of organ injury in acute inflammatory disorders, such as sepsis, because they express cytokines and chemokines, which facilitate the trafficking of leukocytes to organs, and they modulate vascular barrier function. In this study, we find that primary human ECs from multiple organs express the cannabinoid receptors CB₁R, GPR18, and GPR55, as well as the ion channel transient receptor potential cation channel vanilloid type 1. In contrast to leukocytes, CB₂R is only minimally expressed in some EC populations. Furthermore, we show that ECs express all of the known endocannabinoid (eCB) metabolic enzymes. Examining a panel of cannabinoids, we demonstrate that the synthetic cannabinoid WIN55,212-2 and the eCB N-arachidonoyl dopamine (NADA), but neither anandamide nor 2-arachidonoylglycerol, reduce EC inflammatory responses induced by bacterial lipopeptide, LPS, and TNFα. We find that endothelial CB₁R/CB₂R are necessary for the effects of NADA, but not those of WIN55,212-2. Furthermore, transient receptor potential cation channel vanilloid type 1 appears to counter the anti-inflammatory properties of WIN55,212-2 and NADA, but conversely, in the absence of these cannabinoids, its inhibition exacerbates the inflammatory response in ECs activated with LPS. These data indicate that the eCB system can modulate inflammatory activation of the endothelium and may have important implications for a variety of acute inflammatory disorders that are characterized by EC activation.     

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.     

CB2 Receptor Agonists Protect Human Dopaminergic Neurons against Damage from HIV-1 gp120

Despite the therapeutic impact of anti-retroviral therapy, HIV-1-associated neurocognitive disorder (HAND) remains a serious threat to AIDS patients, and there currently remains no specific therapy for the neurological manifestations of HIV-1. Recent work suggests that the nigrostriatal dopaminergic area is a critical brain region for the neuronal dysfunction and death seen in HAND and that human dopaminergic neurons have a particular sensitivity to gp120-induced damage, manifested as reduced function (decreased dopamine uptake), morphological changes, and reduced viability. Synthetic cannabinoids inhibit HIV-1 expression in human microglia, suppress production of inflammatory mediators in human astrocytes, and there is substantial literature demonstrating the neuroprotective properties of cannabinoids in other neuropathogenic processes. Based on these data, experiments were designed to test the hypothesis that synthetic cannabinoids will protect dopaminergic neurons against the toxic effects of the HIV-1 protein gp120. Using a human mesencephalic neuronal/glial culture model, which contains dopaminergic neurons, microglia, and astrocytes, we were able to show that the CB₁/CB₂ agonist WIN55,212-2 blunts gp120-induced neuronal damage as measured by dopamine transporter function, apoptosis and lipid peroxidation; these actions were mediated principally by the CB₂ receptor. Adding supplementary human microglia to our cultures enhances gp120-induced damage; WIN55,212-2 is able to alleviate this enhanced damage. Additionally, WIN55,212-2 inhibits gp120-induced superoxide production by purified human microglial cells, inhibits migration of human microglia towards supernatants generated from gp120-stimulated human mesencephalic neuronal/glial cultures and reduces chemokine and cytokine production from the human mesencephalic neuronal/glial cultures. These data suggest that synthetic cannabinoids are capable of protecting human dopaminergic neurons from gp120 in a variety of ways, acting principally through the CB₂ receptors and microglia.     

Modulation of cannabinoid agonist binding by 5-HT in the rat cerebellum

Cross-talk between cannabinoid CB1 and serotonin 5-HT receptors in rat cerebellar membranes was investigated using radioligand binding. In competition against the CB1 antagonist, [3 H]SR141716A, the agonist, WIN 55,212-2 yielded a biphasic isotherm. The majority of binding was to a high-affinity state that was significantly reduced by the GTP analogue, Gpp(NH)p. Interestingly, 5-HT enhanced the high-affinity binding constant of WIN 55,212-2 while attenuating the proportion of high-affinity binding. 5-HT also significantly reduced the proportion of high-affinity binding of the cannabinoid agonist, HU 210, but had no effect on the agonist, CP 55,940. The effect of 5-HT on WIN 55,212-2 binding was inhibited by the 5-HT2 receptor antagonist ritanserin as well as Gpp(NH)p, suggesting a dependence on the 5-HT2 receptor and on G protein-receptor interactions, respectively. Subsequent [3 H]WIN 55,212-2 dissociation kinetic experiments revealed that 5-HT promoted a slower-dissociating species of radiolabelled agonist-receptor complex. Our findings support a membrane-delimited cross-talk between two G protein-coupled receptors that are co-localized in certain cells of the central nervous system. Intriguingly, the cannabinoid agonist dependence of the 5-HT modulatory effect suggests that agonist-specific conformations of the CB1 receptor may also be important in determining the extent of this cross-talk.     

Inhibition of sea urchin fertilization by fatty acid ethanolamides and cannabinoids

The influence of saturated and unsaturated fatty acid ethanolamides as well as Δ⁹-tetrahydrocannabinol (Δ⁹-THC), WIN 55,212-2 and cannabinoid CB₁ receptor antagonist SR 141716 on sea urchin fertilization was studied. The ethanolamides of arachidonic, oleic and linoleic acids but not saturated fatty acid (C₁₄–C₂₀) derivatives inhibited fertilization when pre-incubated with sperm cells. Δ⁹-THC and WIN 55,212-2 also inhibited fertilization, Δ⁹-THC being ten times as potent as WIN 55,212-2. Selective cannabinoid CB₁ receptor antagonist SR 141716 also blocked fertilization and did not antagonize the action of Δ⁹-THC. The obtained results indicate that different unsaturated fatty acid ethanolamides may control sea urchin fertilization, and that sea urchin sperm cell cannabinoid receptor may differ from the known cannabinoid receptor subtypes.     

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.     

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.     

Inhibition of striatal dopamine release by CB1 receptor activation requires nonsynaptic communication involving GABA, H2O2, and KATP channels

The main psychoactive component of marijuana, Delta9-tetrahydrocannabinol (THC), acts in the CNS via type 1 cannabinoid receptors (CB1Rs). The behavioral consequences of THC or synthetic CB1R agonists include suppression of motor activity. One explanation for movement suppression might be inhibition of striatal dopamine (DA) release by CB1Rs, which are densely localized in motor striatum; however, data from previous studies are inconclusive. Here we examined the effect of CB1R activation on locally evoked DA release monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry in striatal slices. Consistent with previous reports, DA release evoked by a single stimulus pulse was unaffected by WIN55,212-2, a cannabinoid receptor agonist. However, when DA release was evoked by a train of stimuli, WIN55,212-2 caused a significant decrease in evoked extracellular DA concentration ([DA]o), implicating the involvement of local striatal circuitry, with similar suppression seen in guinea pig, rat, and mouse striatum. Pulse-train evoked [DA]o was not altered by either AM251, an inverse CB1R agonist, or VCHSR1, a neutral antagonist, indicating the absence of DA release regulation by endogenous cannabinoids with the stimulation protocol used. However, both CB1R antagonists prevented and reversed suppression of evoked [DA]o by WIN55,212-2. The effect of WIN55,212-2 was also prevented by picrotoxin, a GABAA receptor antagonist, and by catalase, a metabolizing enzyme for hydrogen peroxide (H2O2). Furthermore, blockade of ATP-sensitive K+ (KATP) channels by tolbutamide or glybenclamide prevented the effect of WIN55,212-2 on DA release. Together, these data indicate that suppression of DA release by CB1R activation within striatum occurs via a novel nonsynaptic mechanism that involves GABA release inhibition, increased generation of the diffusible messenger H2O2, and activation of KATP channels to inhibit DA release. In addition, the findings suggest a possible physiological substrate for the motor effects of cannabinoid agonist administration.