Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death.

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD.

Microsomal omega-hydroxylated metabolites of N-arachidonoyl dopamine are active at recombinant human TRPV1 receptors.

N-Arachidonoyl dopamine (NADA) is an endogenous lipid that modulates signal transduction in neuronal and immune pathways. NADA activates the non-selective cation channel, transient receptor potential vanilloid type 1 (TRPV(1)) and cannabinoid receptor 1. That NADA is comprised of an arachidonic acid (AA) backbone suggests that it may be metabolized through many of the enzymes that act upon AA such as the other AA-derived signaling lipids, the endogenous cannabinoids. To investigate the metabolism of NADA through the cytochrome P450 (CYP450) metabolic pathway, we studied the in vitro rat liver microsomal production of hydroxylated metabolites and their activity at recombinant human TRPV(1) receptors. We showed that following microsomal activation in the presence of NADA, omega and (omega-1) hydroxylated metabolites (19- and 20-HETE-DA) were formed. These metabolites were active at recombinant human TRPV(1) receptors, inducing a dose-dependent calcium influx. Both metabolites exhibited lower potency compared to NADA. We conclude that CYP450 enzymes are capable of metabolizing this signaling lipid forming a larger family of potential neuromodulators.

Compartmentalization of endocannabinoids into lipid rafts in a dorsal root ganglion cell line.

BACKGROUND AND PURPOSE: N-arachidonoyl ethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids binding to the cannabinoid receptors CB1 and CB2 to modulate neuronal excitability and synaptic transmission in primary afferent neurons. To investigate the compartmentalization of the machinery for AEA and 2-AG signalling, we studied their partitioning into lipid raft fractions isolated from a dorsal root ganglion X neuroblastoma cell line (F-11). EXPERIMENTAL APPROACH: F-11 cells were homogenized and fractionated using a detergent-free OptiPrep density gradient. All lipids were partially purified from methanolic extracts of the fractions on solid phase cartridges and quantified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Protein distribution was determined by Western blotting. KEY RESULTS: Under basal conditions, the endogenous cannabinoid AEA was present in both lipid raft and specific non-lipid raft fractions as was one of its biosynthetic enzymes, NAPE-PLD. The 2-AG precursor 1-stearoyl-2-arachidonoyl-sn-glycerol (DAG), diacylglycerol lipase alpha (DAGLalpha), which cleaves DAG to form 2-AG, and 2-AG were all co-localized with lipid raft markers. CB1 receptors, previously reported to partition into lipid raft fractions, were not detected in F-11 membranes, but CB2 receptors were detected at high levels and partitioned into non-lipid raft fractions. CONCLUSIONS AND IMPLICATIONS: The biochemical machinery for the production of 2-AG via the putative diacylglycerol pathway is localized within lipid rafts, suggesting that 2-AG synthesis via DAG occurs within these microdomains. The observed co-localization of AEA, 2-AG, and their synthetic enzymes with the reported localization of CB1 raises the possibility of intrinsic-autocrine signalling within lipid raft domains and/or retrograde-paracrine signalling.

Latent inhibition in 35-day-old rats is not an "adult" latent inhibition: implications for neurodevelopmental models of schizophrenia.

RATIONALE: Latent inhibition (LI) refers to retarded conditioning to a stimulus as a consequence of its inconsequential preexposure. Amphetamine-induced disruption of LI and its potentiation by antipsychotic drugs (APDs) in the adult rat are well-established models of schizophrenia and antipsychotic drug action, respectively. It is not clear whether LI can be similarly modulated at prepubertal age. OBJECTIVES: In view of the notion that schizophrenia is a neurodevelopmental disorder whose overt expression depends on postpubertal brain maturational processes, we investigated whether several manipulations known to modulate LI in adult rats, including systemic administration of amphetamine and the atypical APD clozapine, are capable of producing the same effects in prepubertal (35-day-old) rats. METHODS: LI was measured in a thirst motivated conditioned emotional response (CER) procedure in which rats received 10 or 40 tone preexposures followed by 2 or 5 tone-footshock pairings. RESULTS: Like in adults, LI was present with 40 preexposures and 2 conditioning trials. In contrast to findings in adults, LI was resistant to disruption by amphetamine at a dose (1 mg/kg) that significantly increased locomotor activity, as well as by reducing the number of preexposures to ten, increasing the number of conditioning trials to five, or changing the context between preexposure and conditioning. Clozapine (5 mg/kg) and the selective 5HT2A antagonist M100907 (0.3 mg/kg) administered in conditioning were without an effect on "persistent" LI with extended conditioning, but were capable of disrupting LI when administered in the preexposure stage, as found in adults. CONCLUSION: The results point to functionality within brain systems regulating LI acquisition but not those regulating LI expression in periadolescent rats, further suggesting that postpubertal maturation of the latter systems may underlie schizophrenia-mimicking LI disruption reported in adult rats following perinatal manipulations and possibly disrupted LI observed in schizophrenia.