Basalingappa L. Hungund

Cannabinoid CB1 receptor knockout mice exhibit markedly reduced voluntary alcohol consumption and lack alcohol-induced dopamine release in the nucleus accumbens

The mechanisms underlying predisposition to alcohol abuse and alcoholism are poorly understood. In this study, we evaluated the role of cannabinoid (CB1) receptors in (i) voluntary alcohol consumption, and (ii) acute alcohol‐induced dopamine (DA) release in the nucleus accumbens, using mice that lack the CB1 receptor gene (CB1–/–). CB1–/– mice exhibited dramatically reduced voluntary alcohol consumption, and completely lacked alcohol‐induced DA release in the nucleus accumbens, as compared to wild‐type mice. The gender difference, with female mice consuming significantly more alcohol than wild‐type male mice, was observed in wild‐type mice, whereas this gender difference was nonexistent in CB1 mutant male and female mice. There was also a significant gender difference, with the wild‐type, heterozygous, and mutant females consuming significantly more liquid and food than wild‐type, heterozygous and mutant males. However, the total volume of fluid consumption and food intake did not differ between wild‐type, heterozygous, and mutant mice. These results strongly suggest that the CB1 receptor system plays an important role in regulating the positive reinforcing properties of alcohol.

Chronic ethanol increases the cannabinoid receptor agonist anandamide and its precursor N-arachidonoylphosphatidylethanolamine in SK-N-SH cells.

Abstract : In an earlier study, we demonstrated that chronic ethanol (EtOH) exposure down‐regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane. In the present study, we investigated the effect of chronic EtOH on the formation of anandamide (AnNH), an endogenous cannabimimetic compound, and its precursor N‐arachidonoylphosphatidylethanolamine (N‐ArPE) in SK‐N‐SH cells that were prelabeled with [3H]arachidonic acid. The results indicate that exposure of SK‐N‐SH cells to EtOH (100 mM) for 72 h significantly increased levels of [3H]AnNH and [3H]N‐ArPE (p < 0.05) (1.43‐fold for [3H]AnNH and 1.65‐fold for [3H]N‐ArPE). Exposure of SK‐N‐SH cells to EtOH (100 mM, 24h) inhibited initially the formation of [3H]AnNH at 24 h, followed by a progressive increase, reaching a statistical significance level at 72 h (p < 0.05). [3H]N‐ArPE increased gradually to a statistically significant level after 48 and 72 h (p < 0.05). Incubation with exogenous ethanolamine (7 mM) and EtOH (100 mM, 72 h) did not result in an additive increase in the formation of [3H]AnNH. The formation of [3H]AnNH and [3H]N‐ArPE by EtOH was enhanced by the Ca2+ ionophore A23187 or by the depolarizing agent veratridine and the K+ channel blocker 4‐aminopyridine. Further, the EtOH‐induced formation of [3H]AnNH and [3H]N‐ArPE was inhibited by exogenous AnNH, whereas only [3H]AnNH formation was inhibited by the CB1 receptor antagonist SR141716A and pertussis toxin, suggesting that the CB1 receptor and Gi/o protein mediated the regulation of AnNH levels. The observed increase in the levels of these lipids in SK‐N‐SH cells may be a mechanism for neuronal adaptation and may serve as a compensatory mechanism to counteract the continuous presence of EtOH. The present observation taken together with our previous results indicate the involvement of the endocannabinoid system in mediating some of the pharmacological actions of EtOH and may constitute part of a common brain pathway mediating reinforcement of drugs of abuse including EtOH.

Chronic ethanol administration down-regulates cannabinoid receptors in mouse brain synaptic plasma membrane

The effects of chronic ethanol (EtOH) consumption on the central nervous system may be related in part to its action on biological membranes by altering various receptor functions. In the current study, we examined the effects of chronic EtOH (4 day inhalation) on cannabinoid receptors (CB1) labeled with [3H]CP55,940 in synaptic plasma membranes (SPM) isolated from mouse brain. Our results indicate the presence of a high level of CB1 receptors in controls (Bmax=12.0+/-0.3 pmol mg-1 protein) which decreased significantly (-58%) in SPM from mouse brain chronically exposed to EtOH. This effect occurs without any changes in the receptor affinity (Kd=2. 3+/-0.3 nM for control and 2.9+/-0.3 nM for EtOH group, P>0.05). Dissociation kinetic results showed a dissociation rate constant (K-1) of 0.09+/-0.01 min-1 for control and this dissociation rate constant decreased significantly in the chronic EtOH treated mice brain (0.05+/-0.01 min-1, P<0.05). The competition studies with anandamide resulted in a substantial decrease in [3H]CP55,940 binding in both the control and EtOH group, with a decrease (P<0.05) in the Ki values in the SPM of chronic EtOH exposed mice. Hill transformation analysis showed an nH close to one in control (0. 92+/-0.01). This did not change significantly after chronic EtOH (0. 95+/-0.01) administration, which indicates the existence of a single class of receptor for [3H]CP55,940 binding in SPM from control and EtOH treated mice. The observed down-regulation of CB1 receptors by chronic EtOH may indicate the involvement of cannabinoid receptors in EtOH tolerance and dependence.

Chronic ethanol inhibits the anandamide transport and increases extracellular anandamide levels in cerebellar granule neurons

Ethanol increases extracellular anandamide levels in neuronal cells. However, the molecular mechanisms by which this occurs are unknown. Chronic exposure of cerebellar granule neurons to ethanol increased the levels of anandamide accumulated in the cellular medium. Anandamide uptake was saturable and was inhibited (30% at 3 min) in response to chronic exposure to ethanol. Chronic ethanol treatment did not alter the K(m), but significantly decreased V(max) of anandamide transport (33%) (P<0.0001). Fatty acid amide hydrolase activity was not affected by chronic ethanol treatment. Anandamide transport processes are independent of cannabinoid CB1 receptor, as cannabinoid CB1 receptor knockout mice exhibited time-dependent anandamide transport and cannabinoid CB1 receptor antagonists did not alter the effects of chronic ethanol on anandamide transport. Furthermore, anandamide transport was inhibited by acute ethanol in a time- and dose-dependent manner. Interestingly, acute ethanol-induced inhibition of anandamide transport was abolished in neurons exposed to chronic ethanol, suggesting that the anandamide transport processes may play a role in the development of long-term cellular tolerance to ethanol.

The endocannabinoid 2-arachidonoyl glycerol induces death of hepatic stellate cells via mitochondrial reactive oxygen species

ABSTRACT The endocannabinoid system is an important regulator of hepatic fibrogenesis. In this study, we determined the effects of 2‐arachidonoyl glycerol (2‐AG) on hepatic stellate cells (HSCs), the main fibro‐genic cell type in the liver. Culture‐activated HSCs were highly susceptible to 2‐AG‐induced cell death with >50% cell death at 10 μM after 18 h of treatment. 2‐AG‐induced HSC death showed typical features of apoptosis such as PARP‐ and caspase 3‐cleavage and depended on reactive oxygen species (ROS) formation. Confocal microscopy revealed mitochondria as primary site of ROS production and demonstrated mitochon‐drial depolarization and increased mitochondrial permeability after 2‐AG treatment. 2‐AG‐induced cell death was independent of cannabinoid receptors but required the presence of membrane cholesterol. Primary hepatocytes were resistant to 2‐AG‐induced ROS induction and cell death but became susceptible after GSH depletion suggesting antioxidant defenses as a critical determinant of 2‐AG sensitivity. Hepatic levels of 2‐AG were significantly elevated in two models of experimental fibrogenesis and reached concentrations that are sufficient to induce death in HSCs. These findings suggest that 2‐AG may act as an antifibrogenic mediator in the liver by inducing cell death in activated HSCs but not hepatocytes.—Siegmund, S. V., Qian, T., de Minicis, S., Harvey‐White, J., Kunos, G., Vinod, K Y., Hungund, B., Schwabe, R. F. The endocannabinoid 2‐arachidonoyl glycerol induces death of hepatic stellate cells via mitochondrial reactive oxygen species. FASEB J. 21, 2798–2806 (2007)

Increased ethanol consumption and preference and decreased ethanol sensitivity in female FAAH knockout mice.

Previous studies have shown that mice lacking cannabinoid (CB1) receptor gene consume markedly reduced levels of ethanol. Mice lacking the enzyme fatty acid amidohydrolase (FAAH) are severely impaired in their ability to degrade anandamide (AEA) and therefore represent a unique animal model in which to examine the function of AEA in vivo on ethanol-drinking behavior. In the current study, FAAH(-/-) mice were tested for ethanol, saccharin or quinine consumption and preference. Ethanol-induced hypothermia, and sleep time were used to evaluate the sensitivity to acute effects of ethanol. Ethanol intake and preference were increased only in female FAAH(-/-) mice. No significant difference in saccharin or quinine consumption or preference was observed between genotypes. Female FAAH(-/-) mice were less sensitive to the hypothermic and sedative/hypnotic effects of acute ethanol. Supersensitivity to exogenous AEA was noted in both male and female FAAH(-/-) mice. Following voluntary ethanol consumption, CB1 receptor levels and function were down-regulated in male FAAH(+/+), FAAH(-/-), and female FAAH(+/+) mice but not in female FAAH(-/-) mice. Our results suggest that absence of an effect in male mice indicates a sex-linked mechanism that is secondary (or modulatory) to FAAH function. Thus, the data suggest that FAAH may be indirectly related to ethanol intake and sensitivity and central endocannabinoidergic-mediated pathways may regulate ethanol consumption.

Expression of aspartoacylase activity in cultured rat macroglial cells is limited to oligodendrocytes

N-acetyl-l-aspartate (NAA) is an important osmolyte in the vertebrate brain and eye, and its cyclical metabolism is accomplished in two separate compartments. In the brain, NAA is synthesized primarily in neurons, and after its regulated release, NAA is hydrolyzed by aspartoacylase, which is present in a glial-associated compartment. However, the precise nature of this hydrolytic compartment has remained obscure. It has been proposed that one role of aspartoacylase in the central nervous system (CNS) is as part of a molecular water pump (MWP) that uses the NAA intercompartmental cycle to remove nerve cell metabolic water against a water gradient and that oligodendrocytes comprise the second compartment in this metabolic sequence. The absence of aspartoacylase activity in Canavan disease (CD), a rare early onset genetic spongiform leukodystrophy, is associated with CNS edema, intramyelinic swelling and a progressive loss of oligdendrocytes. In order to evaluate the MWP hypothesis and its possible relationship to the etiology of CD further, both oligodendrocytes and astrocytes obtained from neonatal rat brain were grown in culture and tested for the presence of aspartoacylase activity. The results of this study show for the first time that aspartoacylase activity is expressed only in oligodendrocytes. The meaning of this observation in understanding the function of the NAA metabolic cycle is discussed.

Elevated levels of endocannabinoids and CB1 receptor-mediated G-protein signaling in the prefrontal cortex of alcoholic suicide victims

BACKGROUND Alcoholism is often comorbid with mood disorders and suicide. We recently reported an upregulation of CB(1) receptor-mediated signaling in the dorsolateral prefrontal cortex (DLPFC) of subjects with major depression who died by suicide. In the present study, we sought to determine whether the changes in depressed suicides would also be present in alcoholic suicides and whether the endocannabinoid (EC) system plays a role in suicide in alcoholism. METHODS The density of CB(1) receptor and its mediated [(35)S]GTP gamma S signaling were measured in the DLPFC of alcoholic suicides (AS) (n = 11) and chronic alcoholics (CA) (n = 11). The levels of ECs were measured by a liquid chromatograph/mass spectrometry. RESULTS The CB(1) receptor density was higher in AS compared with the CA group in the DLPFC. Western blot analysis confirmed a greater immunoreactivity of the CB(1) receptor in AS. The CB(1) receptor-mediated [(35)S]GTP gamma S binding indicated a greater signaling in AS. Higher levels of N-arachidonyl ethanolamide and 2-arachidonylglycerol were observed in the DLPFC of AS. CONCLUSIONS The elevated levels of ECs, CB(1) receptors, and CB(1) receptor-mediated [(35)S]GTP gamma S binding strongly suggest a hyperactivity of endocannabinoidergic signaling in AS. EC system may be a novel therapeutic target for the treatment of suicidal behavior.

Formation of fatty acid ethyl esters during chronic ethanol treatment in mice

Ethyl esters of long-chain fatty acids are formed in the liver and brain of mice after 1-6 days of ethanol intoxication. This observation extends the reports of Lange and co-workers who detected these compounds as unusual metabolites of ethanol in human tissues [E. A. Laposata and L. G. Lange, Science 231, 497 (1986)]. Ethyl esters of oleic and linoleic acids, and, in smaller amounts, ethyl esters of palmitic and stearic acids were found in the livers of mice that had been treated with ethanol by inhalation. In the brain, only the esters of unsaturated fatty acids were found, in lower amounts than in liver. All the fatty acid ethyl esters seemed to have reached steady-state levels in the tissues after 3 or 4 days of alcohol treatment. When incorporated into synaptosomal plasma membranes in vitro, in intramembrane concentrations estimated to resemble those observed in the mice, these esters reduced the fluorescence anisotropy, i.e. they disordered the membranes.

Effect of chronic ethanol exposure and its withdrawal on the endocannabinoid system

The present study investigated the effect of ethanol (EtOH) exposure and its withdrawal on the central endocannabinoid system utilizing an EtOH vapor inhalation model, which is known to produce functional tolerance and dependence to EtOH. Swiss Webster mice (n=24) were exposed to EtOH vapors for 72h. Mice were sacrificed after 72h following EtOH exposure (n=12) and 24h after its withdrawal (n=12). Radioligand binding assays were performed to measure the density of CB(1) receptor and CB(1) receptor agonist-stimulated [(35)S]GTPgammaS binding in crude synaptic membranes isolated from the cortex, hippocampus, striatum and cerebellum. The density of CB(1) receptor was significantly decreased (31-39%) in all the brain regions when compared to the control group. The CB(1) receptor-stimulated G(i/o) protein activation was also found to be decreased (29-40%) in these brain regions of EtOH exposed mice. Recovery of the CB(1) receptor density, in addition to, the CB(1) receptor-mediated G-protein activation was observed after 24h withdrawal from EtOH. The levels of cortical anandamide, which was significantly increased (147%) by EtOH exposure, returned to basal levels after 24h of withdrawal from EtOH exposure. A significant reduction (21%) in the activity of fatty acid amide hydrolase was found in the cortex of EtOH administered mice. Taken together, the neuroadaptation in the EC system may have a potential role in development of tolerance and dependence to EtOH.