Macrophages are the primary cellular targets of bacterial lipopolysaccharide (LPS), but the role of macrophage-derived cytokines in LPS-induced septic shock is uncertain. Recent evidence indicates that activation of peripheral CB1 cannabinoid receptors contributes to hemorrhagic hypotension and that macrophage-derived anandamide as well as unidentified platelet-derived substances may be contributing factors. Here we demonstrate that rat platelets contain the endogenous cannabinoid 2-arachidonyl glyceride (2-AG), as identified by reverse phase high-performance liquid chromatography, gas chromatography, and mass spectrometry, and that in vitro exposure of platelets to LPS (200 microg/ml) markedly increases 2-AG levels. LPS-stimulated, but not control, macrophages contain anandamide, which is undetectable in either control or LPS-stimulated platelets. Prolonged hypotension and tachycardia are elicited in urethane-anesthetized rats treated 1) with LPS (15 mg/kg i.v.); 2) with macrophages plus platelets isolated from 3 ml of blood from an LPS-treated donor rat; or 3) with rat macrophages or 4) platelets preincubated in vitro with LPS (200 microg/ml). In all four cases, the hypotension but not the tachycardia is prevented by pretreatment of the recipient rat with the CB1 receptor antagonist SR141716A (3 mg/kg i.v.), which also inhibits the hypotensive response to anandamide or 2-AG. The hypotension elicited by LPS-treated macrophages or platelets remains unchanged in the absence of sympathetic tone or after blockade of nitric oxide synthase. These findings indicate that platelets and macrophages generate different endogenous cannabinoids, and that both 2-AG and anandamide may be paracrine mediators of endotoxin-induced hypotension via activation of vascular CB1 receptors.

Anandamide [arachidonylethanolamide (AEA)] appears to be an endogenous agonist of brain cannabinoid receptors (CB 1 ), yet some of the neurobehavioral effects of this compound in mice are unaffected by a selective CB 1 antagonist. We studied the levels, pharmacological actions, and degradation of AEA in transgenic mice lacking the CB 1 gene. We quantified AEA and the other endocannabinoid, 2-arachidonoyl glycerol, in six brain regions and the spinal cord by isotope-dilution liquid chromatography-mass spectrometry. The distribution of endocannabinoids and their inactivating enzyme, fatty acid amide hydrolase, were found to overlap with CB 1 distribution only in part. In CB 1 knockout homozygotes (CB 1 Ϫ/Ϫ), the hippocampus and, to a lesser extent, the striatum exhibited lower AEA levels as compared with wild-type (CB 1 ϩ/ϩ) controls. These data suggest a ligand/receptor relationship between AEA and CB 1 in these two brain regions, where tonic activation of the receptor may tightly regulate the biosynthesis of its endogenous ligand. 2-Arachidonoyl glycerol levels and fatty acid amide hydrolase activity were unchanged in CB 1 Ϫ/Ϫ with respect to CB 1 ϩ/ϩ mice in all regions. AEA and ⌬ 9 -tetrahydrocannabinol (THC) were tested in CB 1 Ϫ/Ϫ mice for their capability of inducing analgesia and catalepsy and decreasing spontaneous activity. The effects of AEA, unlike THC, were not decreased in CB 1 Ϫ/Ϫ mice. AEA, but not THC, stimulated GTP␥S binding in brain membranes from CB 1 Ϫ/Ϫ mice, and this stimulation was insensitive to CB 1 and CB 2 antagonists. We suggest that non-CB 1 , non-CB 2 G protein-coupled receptors might mediate in mice some of the neurobehavioral actions of AEA. Key Words: CannabinoidAnandamide -2-Arachidonyl glycerol-Tetrahydrocannabinol-CB 1 receptor knockout-C57BL/6 mouse.

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