Anandamide is an endogenous compound that acts as an agonist at cannabinoid receptors. It is inactivated via intracellular degradation after its uptake into cells by a carrier-mediated process that depends upon a concentration gradient. The fate of anandamide in those cells containing an amidase called fatty-acid amide hydrolase (FAAH) is hydrolysis to arachidonic acid and ethanolamine. The active site nucleophilic serine of FAAH is inactivated by a variety of inhibitors including methylarachidonylfluorophosphonate (MAFP) and palmitylsulfonyl fluoride. In the current report, the net uptake of anandamide in cultured neuroblastoma (N18) and glioma (C6) cells, which contain FAAH, was decreased by nearly 50% after 6 min of incubation in the presence of MAFP. Uptake in laryngeal carcinoma (Hep2) cells, which lack FAAH, is not inhibited by MAFP. Free anandamide was found in all MAFP-treated cells and in control Hep2 cells, whereas phospholipid was the main product in N18 and C6 control cells when analyzed by TLC. The intracellular concentration of anandamide in N18, C6, and Hep2 cells was up to 18-fold greater than the extracellular concentration of 100 nM, which strongly suggests that it is sequestered within the cell by binding to membranes or proteins. The accumulation of anandamide and/or its breakdown products was found to vary among the different cell types, and this correlated approximately with the amount of FAAH activity, suggesting that the breakdown of anandamide is in part a driving force for uptake. This was shown most clearly in Hep2 cells transfected with FAAH. The uptake in these cells was 2-fold greater than in vector-transfected or untransfected Hep2 cells. Therefore, it appears that FAAH inhibitors reduce anandamide uptake by cells by shifting the anandamide concentration gradient in a direction that favors equilibrium. Because inhibition of FAAH increases the levels of extracellular anandamide, it may be a useful target for the design of therapeutic agents.Endocannabinoids, such as anandamide (arachidonyl ethanolamide) and 2-arachidonyl glycerol, are endogenous ligands that bind to the cannabinoid receptors (1-3). Emerging evidence suggests that they are involved in many physiological phenomena such as pain, locomotion, memory, learning, blood pressure, immunity, sleep, reproduction, mood, perception, response to stress, and so forth (for review see Ref. 4). ⌬ 9 -Tetrahydrocannabinol, the active component of marijuana, appears to mimic many of the physiological and pharmacological effects of the endogenous cannabinoids, in some cases to an extreme degree (e.g. a marijuana "high"). Anandamide is transported into the neuroblastoma, glioma, brain neuron, brain astrocyte, cerebellar granule cells, leukocyte, macrophage, leukemia, and lymphoma cells in culture (5-10). The driving force for uptake is substrate concentration (facilitated diffusion) rather than an active cotransport system (11,12). The transport appears to be carrier-mediated, and specific transport inhibitors have been described (9,11,(13...

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The effect of cannabinoids on the induction of cytokine mRNA by rat microglial cells was examined. Exposure of neonatal rat cortical microglial cells to the exogenous cannabinoid δ9‐tetrahydrocannabinol (THC) resulted in reduced amounts of lipopolysaccharide (LPS)‐induced mRNAs for IL‐1α, IL‐1β, IL‐6, and TNF‐α. Of these cytokine mRNAs, the response of that for IL‐6 was exquisitely sensitive to THC. Similarly, exposure of microglial cells to the putative endogenous cannabinoid anandamide before LPS treatment resulted in a decrease in cytokine mRNA levels, but not to the same extent as that caused by THC; however, when methanandamide, the non‐hydrolyzable analog of anandamide was tested, its ability to inhibit cytokine mRNA expression was comparable to that of THC. Exposure of microglial cells to either of the paired enantiomers CP55,940 or CP56,667 resulted in similar inhibition of LPS‐induced cytokine mRNA expression. A comparable inhibitory outcome was obtained when the paired enantiomers levonantradol and dextronantradol were employed. Neither the CB1‐selective antagonist SR141716A nor the CB2‐selective antagonist SR144528 was able to reverse the inhibition of cytokine mRNA expression by levonantradol. The CB2 antagonist, however, when administered alone augmented the production of cytokine mRNAs. Collectively, these studies demonstrate that cannabinoids can modulate levels of cytokine mRNA in rat microglial cells; however, the inhibition of cytokine mRNA expression is apparently not mediated through either the CB1 or CB2 cannabinoid receptors. GLIA 29:58–69, 2000. © 2000 Wiley‐Liss, Inc.

Free-living amebae belonging to the genus Acanthamoeba are the causative agents of granulomatous amebic encephalitis, a chronic progressive disease of the central nervous system, and of amebic keratitis, a chronic eye infection. Granulomatous amebic encephalitis occurs more frequently in immunocompromised patients while keratitis occurs in healthy individuals. The recent increased incidence in Acanthamoeba infections is due in part to infection in patients with acquired immune deficiency syndrome, while that for keratitis is due to the increased use of contact lenses. Understanding the mechanism of host resistance to Acanthamoeba is essential since the amebae are resistant to many therapeutic agents. Studies in our laboratory as well as from others have demonstrated that macrophages from immunocompetent animals are important effector cells against Acanthamoeba. We have demonstrated also that microglial cells, resident macrophages of the brain, elicit cytokines in response to A. castellanii. Neonatal rat cortical microglia from Sprague-Dawley rats co-cultured with A. castellanii produced mRNA for the inflammatory cytokines, interleukin 1alpha, interleukin 1beta, and tumor necrosis factor alpha. In addition, scanning and transmission electron microscopy revealed that microglia ingested and destroyed A. castellanii in vitro. These results implicate macrophages as playing an effector role against Acanthamoeba and suggest immune modulation as a potential alternative therapeutic mode of treatment for these infections.