Anandamide, an endogenous cannabimimetic eicosanoid, was confirmed as an agonist for the rate and human cannabinoid receptor. Enzymatic synthesis of anandamide was demonstrated in rat and bovine membranes, and showed specific brain region and subcellular distribution. Synthesis was found to linear with increasing protein concentration, selective for arachidonic acid over other fatty acid substrates, and have a pH optimum of 9. Preliminary evidence indicates that anandamide formation was evoked by glutamate receptor stimulation, high potassium depolarization, phospholipase A2 activation, and calcium influx stimulation in metabolically labelled cerebellar granule cell cultures. A cannabinoid receptor antagonist was discovered by screening drugs by binding and functional inhibition of adenylate cyclase in cells expressing the cloned human cannabinoid receptor. Muscarinic receptor-stimulated calcium influx was shown to occur through a voltage-insensitive calcium channel. Electrophysiological characterization confirmed that the channel was calcium and second messenger independent and therefore should be classified as a receptor-operated calcium channel. Muscarinic receptor- stimulated arachidonic acid release was shown in A2058 human melanoma cells. The muscarinic receptor-mediated release of arachidonic acid was augmented by tumor necrosis factor-alpha (TNF-alpha), although TNF-alpha had no effect on its own. Muscarinic receptor-stimulated and TNF-alpha-- augmented arachidonic acid release required calcium influx and protein kinase C activation.
