Characterization of the 5'-sequence of the mouse fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) is critical for degradation of several important fatty acid amides including anandamide, an endocannabinoid, as well as oleamide, a sleep-inducing factor. These compounds play roles in diverse physiological processes ranging from memory and learning to the regulation of blood pressure. The mechanisms that regulate FAAH expression have not been characterized. A 5'-region of the mouse FAAH with promoter activity was isolated from 1.8 kbp of genomic sequence. Characterization of +1 of transcription of FAAH by RNA ligase mediated-rapid amplification of cDNA ends showed that FAAH mRNA is transcribed from multiple transcription start sites lacking a TATA-box element. Functional analysis of the FAAH upstream sequence fused to a luciferase reporter gene revealed a FAAH-promoter construct with tissue specific activity. A 674-bp FAAH-promoter construct was active in N18TG2 (N18) neuroblastoma cells and C6 glioma cells, lines that have endogenous FAAH activity. The same 674-bp FAAH-promoter construct was not active in C2C12 or L6 myogenic cells, two lines that do not have FAAH activity.

The cellular uptake of anandamide is coupled to its breakdown by fatty-acid amide hydrolase.

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.

The fatty acid amide hydrolase (FAAH).

The topic of this review is fatty acid amide hydrolase (FAAH), one of the best-characterized enzymes involved in the hydrolysis of bioactive lipids such as anandamide, 2-arachidonoylglycerol (2-AG), and oleamide. Herein, we discuss the nomenclature, the various assays that have been developed, the relative activity of the various substrates and the reversibility of the enzyme reactions catalyzed by FAAH. We also describe the cloning of the enzyme from rat and subsequent cDNA isolation from mouse, human, and pig. The proteins and the mRNAs from different species are compared. Cloning the enzyme permitted the purification and characterization of recombinant FAAH. The conserved regions of FAAH are described in terms of sequence and function, including the amidase domain which contains the serine catalytic nucleophile, the hydrophobic domain important for self association, and the proline rich domain region, which may be important for subcellular localization. The distribution of FAAH in the major organs of the body is described as well as regional distribution in the brain and its correlation with cannabinoid receptors. Since FAAH is recognized as a drug target, a large number of inhibitors have been synthesized and tested since 1994 and these are reviewed in terms of reversibility, potency, and specificity for FAAH and cannabinoid receptors.

The increasing importance of Acanthamoeba infections.

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.

Cannabinoids inhibit LPS-inducible cytokine mRNA expression in rat microglial cells.

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 delta(9)-tetrahydrocannabinol (THC) resulted in reduced amounts of lipopolysaccharide (LPS)-induced mRNAs for IL-1alpha, IL-1beta, IL-6, and TNF-alpha. 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 CB(1)-selective antagonist SR141716A nor the CB(2)-selective antagonist SR144528 was able to reverse the inhibition of cytokine mRNA expression by levonantradol. The CB(2) 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 CB(1) or CB(2) cannabinoid receptors.

Combination chemotherapy with doxorubicin and mitomycin C in non-small cell bronchogenic carcinoma. Severe pulmonary toxicity from q 3 weekly mitomycin C.

Forty-five patients with advanced, measurable, or evaluable non-small bronchogenic carcinoma (NSCBC) were treated with doxorubicin and mitomycin C combination chemotherapy. The first 27 patients received doxorubicin 50 mg/m2 I.V. every 3 weeks and mitomycin C 10 mg/m2 I.V. every 3 weeks. Because of severe cardiopulmonary toxicity in seven patients, with four otherwise unexplained deaths, the next 18 patients were treated with the mitomycin C dose reduced to 10 mg/m2 every 6 weeks. Overall, 11 patients (25%) responded, with one complete and 10 partial remissions. Eight responses (30%) were observed in the patients who received mitomycin C every 3 weeks and three responses (17%) were found in those given mitomycin C every 6 weeks (p less than 0.5), with no cardiopulmonary toxicity in the latter group. The median survival was 21 weeks for the entire group of patients, with the group receiving mitomycin C every 3 weeks living a median of 15.5 weeks and those given mitomycin C every 6 weeks surviving 35.5 weeks (p less than 0.025). We conclude that there is a higher tumor response rate but more cardiopulmonary toxicity and shorter survival among the group receiving mitomycin C every 3 weeks compared to those receiving mitomycin C every 6 weeks. Future studies should consider this toxicity of mitomycin C administered on an every-3-week schedule.