The novel endocannabinoid receptor GPR55 is activated by atypical cannabinoids but does not mediate their vasodilator effects.

BACKGROUND AND PURPOSE: Atypical cannabinoids are thought to cause vasodilatation through an as-yet unidentified 'CBx' receptor. Recent reports suggest GPR55 is an atypical cannabinoid receptor, making it a candidate for the vasodilator 'CBx' receptor. The purpose of the present study was to test the hypothesis that human recombinant GPR55 is activated by atypical cannabinoids and mediates vasodilator responses to these agents. EXPERIMENTAL APPROACH: Human recombinant GPR55 was expressed in HEK293T cells and specific GTPgammaS activity was monitored as an index of receptor activation. In GPR55-deficient and wild-type littermate control mice, in vivo blood pressure measurement and isolated resistance artery myography were used to determine GPR55 dependence of atypical cannabinoid-induced haemodynamic and vasodilator responses. KEY RESULTS: Atypical cannabinoids O-1602 and abnormal cannabidiol both stimulated GPR55-dependent GTPgammaS activity (EC50 approximately 2 nM), whereas the CB1 and CB2-selective agonist WIN 55,212-2 showed no effect in GPR55-expressing HEK293T cell membranes. Baseline mean arterial pressure and heart rate were not different between WT and GPR55 KO mice. The blood pressure-lowering response to abnormal cannabidiol was not different between WT and KO mice (WT 20+/-2%, KO 26+/-5% change from baseline), nor was the vasodilator response to abnormal cannabidiol in isolated mesenteric arteries (IC50 approximately 3 micro M for WT and KO). The abnormal cannabidiol vasodilator response was antagonized equivalently by O-1918 in both strains. CONCLUSIONS: These results demonstrate that while GPR55 is activated by atypical cannabinoids, it does not appear to mediate the vasodilator effects of these agents.

Role of group III metabotropic glutamate receptors in excitotoxin-induced cerebellar granule cell death.

The neuronal effects of the metabotropic glutamate receptor agonist (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid have been studied in cultured rat cerebellar granule cells, and compared with those of the endogenous excitotoxin glutamate, and the dietary excitotoxin beta-N-methylamino-L-alanine. Glutamate, beta-N-methylamino-L-alanine, and (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid all caused concentration-dependent cerebellar granule cell death over a 24-h exposure period. The metabotropic antagonist (RS)-alpha-methyl-4-carboxyphenylglycine reduced glutamate-, beta-N-methylamino-L-alanine-, and (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid-induced death by 50, 37, and 90%, respectively. (1S,3R)-Aminocyclopentane-1,3-dicarboxylic acid-induced death was unaffected by the group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid, increased by the group II antagonist ethylglutamic acid, and markedly decreased by the group III antagonist (RS)-alpha-methylserine-O-phosphate. Neither (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid nor the group I agonist (RS)-3,5-dihydroxyphenylglycine caused an increase in intracellular free calcium levels. The group III agonist L-(+)-2-amino-4-phosphonobutyric acid also induced concentration-dependent cerebellar granule cell death, and so it was suggested that the group III metabotropic glutamate receptors were responsible for (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid-induced death. Blocking these receptors with (RS)-alpha-methylserine-O-phosphate also prevented a proportion of glutamate- and beta-N-methylamino-L-alanine-induced death.

The dietary excitotoxins beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine induce necrotic- and apoptotic-like death of rat cerebellar granule cells.

The neurotoxic properties of the dietary excitotoxins beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine have been studied in rat cerebellar granule cells and compared with those of glutamate. Glutamate caused dose-dependent death of cerebellar granule cells after a 30-min exposure when viability was assessed 24 h later. Beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine, however, were toxic only after 24 or 48 h of exposure. The neurotoxic effects of beta-N-methylamino-L-alanine were blocked by D(-)-2-amino-5-phosphonopentanoic acid, and those of beta-N-oxalylamino-L-alanine were blocked by kynurenic acid, which demonstrated that these excitotoxins caused cerebellar granule cell death through the activation of glutamate receptors. The features of this death were examined morphologically (fluorescent dyes, electron microscopy) and biochemically (conventional agarose gel electrophoresis, effect of aurintricarboxylic acid). Characteristics of apoptosis were identified by transferring cerebellar granule cells from a high K+ (30 mM)- to a low K+ (10 mM)-containing medium. In cerebellar granule cells exposed to beta-N-methylamino-L-alanine or beta-N-oxalylamino-L-alanine (3 mM), hallmarks of necrotic- and apoptotic-like death were observed at various time points over a 72-h period. Therefore, in cerebellar granule cells, beta-N-methylamino-L-alanine and beta-N-oxalylamino-L-alanine induce death over 12-72 h of exposure via a mechanism that involves both necrotic- and apoptotic-like cell death.