Enzymatic formation of prostamide F2alpha from anandamide involves a newly identified intermediate metabolite, prostamide H2

Prostaglandin F2alpha 1-ethanolamide (prostamide F2alpha) is a potent ocular hypotensive agent in animals and represents a new class of fatty acid amide compounds. Accumulated evidence indicated that anandamide, an endogenous bioactive ligand for cannabinoid receptors, may serve as a common substrate to produce all prostamides, including prostamide F2alpha. After incubation of anandamide with cyclooxygenase 2 (COX-2), the reaction mixture was profiled by HPLC and an intermediate metabolite was discovered and characterized as a cyclic endoperoxide ethanolamide using HPLC-tandem mass spectrometry. Formation of prostamide F2alpha was also demonstrated when the intermediate metabolite was isolated and incubated with prostaglandin F synthase (PGF synthase). These results suggest that the biosynthesis of prostamide F2alpha proceeds in two consecutive steps: oxidation of anandamide to form an endoperoxide intermediate by COX-2, and reduction of the endoperoxide intermediate to form prostamide F2alpha by PGF synthase. This endoperoxide ethanolamide intermediate has been proposed as prostamide H2.     

Identification of prostamides,fatty acyl ethanolamines,and their biosynthetic precursors in rabbit cornea

Arachidonoyl ethanolamine (anandamide) and pros­taglandin ethanolamines (prostamides) are biologically active derivatives of arachidonic acid. Although available through different precursor phospholipids, there is considerable overlap between the biosynthetic pathways of arachidonic acid-derived eicosanoids and anandamide-derived prostamides. Prostamides exhibit physiological actions and are involved in ocular hypotension, smooth muscle contraction, and inflammatory pain. Although topical application of bimatoprost, a structural analog of prostaglandin F_2α ethanolamide (PGF_2α-EA), is currently a first-line treatment for ocular hypertension, the endogenous production of prostamides and their biochemical precursors in corneal tissue has not yet been reported. In this study, we report the presence of anandamide, palmitoyl-, stearoyl-, α-linolenoyl docosahexaenoyl-, linoleoyl-, and oleoyl-ethanolamines in rabbit cornea, and following treatment with anandamide, the formation of PGF_2α-EA, PGE₂-EA, PGD₂-EA by corneal extracts (all analyzed by LC/ESI-MS/MS). A number of N-acyl phosphatidylethanolamines, precursors of anandamide and other fatty acyl ethanolamines, were also identified in corneal lipid extracts using ESI-MS/MS. These findings suggest that the prostamide and fatty acid ethanolamine pathways are operational in the cornea and may provide valuable insight into corneal physiology and their potential influence on adjacent tissues and the aqueous humor.     

An anatomical and temporal portrait of physiological substrates for fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) regulates amidated lipid transmitters, including the endocannabinoid anandamide and its N-acyl ethanolamine (NAE) congeners and transient receptor potential channel agonists N-acyl taurines (NATs). Using both the FAAH inhibitor PF-3845 and FAAH(-/-) mice, we present a global analysis of changes in NAE and NAT metabolism caused by FAAH disruption in central and peripheral tissues. Elevations in anandamide (and other NAEs) were tissue dependent, with the most dramatic changes occurring in brain, testis, and liver of PF-3845-treated or FAAH(-/-) mice. Polyunsaturated NATs accumulated to very high amounts in the liver, kidney, and plasma of these animals. The NAT profile in brain tissue was markedly different and punctuated by significant increases in long-chain NATs found exclusively in FAAH(-/-), but not in PF-3845-treated animals. Suspecting that this difference might reflect a slow pathway for NAT biosynthesis, we treated mice chronically with PF-3845 for 6 days and observed robust elevations in brain NATs. These studies, taken together, define the anatomical and temporal features of FAAH-mediated NAE and NAT metabolism, which are complemented and probably influenced by kinetically distinguishable biosynthetic pathways that produce these lipids in vivo.     

Inactivation of fatty acid amide hydrolase protects against ischemic reperfusion injury-induced renal fibrogenesis

Although cannabinoid receptors (CB) are recognized as targets for renal fibrosis, the roles of endogenous cannabinoid anandamide (AEA) and its primary hydrolytic enzyme, fatty acid amide hydrolase (FAAH), in renal fibrogenesis remain unclear. The present study used a mouse model of post-ischemia-reperfusion renal injury (PIR) to test the hypothesis that FAAH participates in the renal fibrogenesis. Our results demonstrated that PIR showed upregulated expression of FAAH in renal proximal tubules, accompanied with decreased AEA levels in kidneys. Faah knockout mice recovered the reduced AEA levels and ameliorated PIR-triggered increases in blood urea nitrogen, plasma creatinine as well as renal profibrogenic markers and injuries. Correspondingly, a selective FAAH inhibitor, PF-04457845, inhibited the transforming growth factor-beta 1 (TGF-β1)–induced profibrogenic markers in human proximal tubular cell line (HK-2 cells) and mouse primary cultured tubular cells. Knockdown of FAAH by siRNA in HK-2 cells had similar effects as PF-04457845. Tubular cells isolated from Faah^?/? mice further validated the protection against TGF-β1–induced damages. The CB 1 or CB2 receptor antagonist and exogenous FAAH metabolite arachidonic acid failed to reverse the protective effects of FAAH inactivation in HK-2 cells. However, a substrate-selective inhibitor of AEA-cyclooxygenase-2 (COX-2) pathway significantly suppressed the anti-profibrogenic actions of FAAH inhibition. Further, the AEA-COX-2 metabolite, prostamide E2 exerted anti-fibrogenesis effect. These findings suggest that FAAH activation and the consequent reduction of AEA contribute to the renal fibrogenesis, and that FAAH inhibition protects against fibrogenesis in renal cells independently of CB receptors via the AEA-COX-2 pathway by the recovery of reduced AEA.     

9 alpha,11 beta-prostaglandin F2 formation in various bovine tissues. Different isozymes of prostaglandin D2 11-ketoreductase, contribution of prostaglandin F synthetase and its cellular localization

9 alpha,11 beta-prostaglandin F2 was formed from prostaglandin D2 by its 11-ketoreductases in 100,000 x g supernatants of various bovine tissues in the presence of an NADPH-generating system. The reductase activities were high in liver (51.09 nmol/h/mg of protein), lung (24.99), and spleen (14.20); moderate in heart and pancreas (3.09-3.61); weak in stomach, intestine, colon, kidney, uterus, adrenal gland, and thymus (0.11-2.63); and undetectable in brain, retina, carotid artery, and blood (less than 0.10). No formation of prostaglandin F2 alpha from prostaglandin D2 was detected in all tissues. In immunotitration analyses with a polyclonal antibody specific for prostaglandin F synthetase, the reductase activities in lung and spleen showed identical titration curves to that of the purified synthetase and decreased to less than 15% of the initial activity under the condition of antibody excess. Prostaglandin F synthetase-immunoreactive protein in these two tissues showed peptide fingerprints identical to that of the purified enzyme after partial digestion with Staphylococcus aureus V8 protease. The antibody was partially cross-reactive to the reductase in liver (about 20% of that to the synthetase) but not to the reductase(s) in other tissues. The Km value for prostaglandin D2 of the reductase activity was the same in lung and spleen as that of the purified prostaglandin F synthetase (120 microM) but differed in liver (6 microM), heart, and pancreas (15 microM). The predominant distribution of prostaglandin F synthetase in lung and spleen was confirmed by radioimmunoassay (2.8 and 1.0 micrograms/mg protein, respectively) and Northern blot analyses. In immunoperoxidase staining, this enzyme was localized in alveolar interstitial cells and nonciliated epithelial cells in lung, histiocytes and/or dendritic cells in spleen, and a few interstitial cells in kidney and adrenal cortex.     

Hemodynamic profile, responsiveness to anandamide, and baroreflex sensitivity of mice lacking fatty acid amide hydrolase

The endocannabinoid anandamide exerts neurobehavioral, cardiovascular, and immune-regulatory effects through cannabinoid receptors (CB). Fatty acid amide hydrolase (FAAH) is an enzyme responsible for the in vivo degradation of anandamide. Recent experimental studies have suggested that targeting the endocannabinergic system by FAAH inhibitors is a promising novel approach for the treatment of anxiety, inflammation, and hypertension. In this study, we compared the cardiac performance of FAAH knockout (FAAH-/-) mice and their wild-type (FAAH+/+) littermates and analyzed the hemodynamic effects of anandamide using the Millar pressure-volume conductance catheter system. Baseline cardiovascular parameters, systolic and diastolic function at different preloads, and baroreflex sensitivity were similar in FAAH-/- and FAAH+/+ mice. FAAH-/- mice displayed increased sensitivity to anandamide-induced, CB1-mediated hypotension and decreased cardiac contractility compared with FAAH(+/+) littermates. In contrast, the hypotensive potency of synthetic CB1 agonist HU-210 and the level of expression of myocardial CB1 were similar in the two strains. The myocardial levels of anandamide and oleoylethanolamide, but not 2-arachidonylglycerol, were increased in FAAH-/- mice compared with FAAH+/+ mice. These results indicate that mice lacking FAAH have a normal hemodynamic profile, and their increased responsiveness to anandamide-induced hypotension and cardiodepression is due to the decreased degradation of anandamide rather than an increase in target organ sensitivity to CB1 agonists.     

Synthesis of prostaglandin F ethanolamide by prostaglandin F synthase and identification of Bimatoprost as a potent inhibitor of the enzyme: new enzyme assay method using LC/ESI/MS

Prostaglandin (PG) D(2) ethanolamide (prostamide D(2)) was reduced to 9alpha,11beta-PGF(2) ethanolamide (9alpha,11beta-prostamide F(2)) by PGF synthase, which also catalyzes the reduction of PGH(2) and PGD(2) to PGF(2alpha) and 9alpha,11beta-PGF(2), respectively. These enzyme activities were measured by a new method, the liquid chromatographic-electrospray ionization-mass spectrometry (LC/ESI/MS) technique, which could simultaneously detect the substrate and all products. PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2) were separated on a TSKgel ODS 80Ts column, ionized by electrospray, and detected in the negative mode. Selected ion monitoring (SIM) of m/z 353 ([M-H](-)), 353 ([M-H](-)), 351 ([M-H](-)), 333 ([M-H-H(2)O](-)), 456 ([M+59](-)), and m/z 358 ([M-37](-)) was used for quantifying PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2), respectively. The detection limit for PGF(2alpha) and 9alpha,11beta-PGF(2) was 0.01pmol; that for PGH(2) and PGD(2), 0.1pmol; and that for prostamide D(2) and 9alpha,11beta-prostamide F(2), 0.5 and 0.03pmol, respectively. The LC/ESI/MS technique for measuring PGF synthase activity showed higher sensitivity than other methods. Using this method, we found that Bimatoprost, the ethyl amide analog of 17-phenyl-trinor PGF(2alpha) and an anti-glaucoma agent, inhibited all three reductase activities of PGF synthase when used at a low concentration. These results suggest that Bimatoprost also behaves as a potent PGF synthase inhibitor in addition to having prostamide-like activity.     

The endocannabinoid arachidonyl ethanolamide (anandamide) increases pulmonary arterial pressure via cyclooxygenase-2 products in isolated rabbit lungs

Several cannabinoids elicit systemic vasodilation, mainly via CB1 cannabinoid and vanilloid receptors. However, effects in the pulmonary circulation are unknown. Using the isolated, ventilated, buffer-perfused rabbit lung, we have shown that the endocannabinoids arachidonyl ethanolamide (anandamide) and 2-arachidonyl glycerol (2-AG) dose-dependently increase pulmonary arterial pressure (+19.9 +/- 3.4 mmHg, 5 microM, and +39.5 +/- 10.8 mmHg, 0.4 microM, respectively). 2-AG induced lung edema. The CB1 receptor antagonist AM-251 (0.1 and 5 microM) and the VR1 vanilloid receptor antagonist capsazepine (10 microM) failed to reduce anandamide's effects. The metabolically stable anandamide and 2-AG analogs R-methanandamide and noladin ether, Delta9-tetrahydrocannabinol, and the synthetic cannabinoid HU-210, which is no arachidonic acid product, were without effect. The unspecific cyclooxygenase (COX) inhibitor aspirin (100 microM, P < 0.001) and the specific COX-2 inhibitor nimesulide (10 microM, P < 0.01) completely prevented pulmonary hypertension after 5 microM anandamide. COX-2 RNA was detected in rabbit lungs. The synthetic thromboxane receptor antagonist SQ 29,548 was without effect, but the specific EP1 prostanoid receptor antagonist SC-19220 (100 microM) inhibited the pressure increase after anandamide (P < 0.05). PCR analysis detected fatty acid amidohydrolase (FAAH), an enzyme that degrades endocannabinoids, in rabbit lung tissue. Furthermore, the specific FAAH inhibitor methyl arachidonyl fluorophosphonate (0.1 microM) blocked pressure effects of anandamide (P < 0.01). Finally, anandamide (99 +/- 55 pmol/g) and 2-AG (19.6 +/- 8.4 nmol/g) were found in native lungs. We conclude that anandamide increases pulmonary arterial pressure via COX-2 metabolites following enzymatic degradation by FAAH into arachidonic acid products.     

Decreased age-related cardiac dysfunction, myocardial nitrative stress, inflammatory gene expression, and apoptosis in mice lacking fatty acid amide hydrolase

Recent studies have uncovered important cross talk between inflammation, generation of reactive oxygen and nitrogen species, and lipid metabolism in the pathogenesis of cardiovascular aging. Inhibition of the endocannabinoid anandamide metabolizing enzyme, the fatty acid amide hydrolase (FAAH), is emerging as a promising novel approach for the treatment of various inflammatory disorders. In this study, we have investigated the age-associated decline of cardiac function and changes in inflammatory gene expression, nitrative stress, and apoptosis in FAAH knockout (FAAH(-/-)) mice and their wild-type (FAAH(+/+)) littermates. Additionally, we have explored the effects of anandamide on TNF-alpha-induced ICAM-1 and VCAM-1 expression and monocyte-endothelial adhesion in human coronary artery endothelial cells (HCAECs). There was no difference in the cardiac function (measured by the pressure-volume conductance catheter system) between 2- to 3-mo-old (young) FAAH(-/-) and FAAH(+/+) mice. In contrast, the aging-associated decline in cardiac function and increased myocardial gene expression of TNF-alpha, gp91phox, matrix metalloproteinase (MMP)-2, MMP-9, caspase-3 and caspase-9, myocardial inducible nitric oxide synthase protein expression, nitrotyrosine formation, poly (ADP-ribose)polymerase cleavage and caspase-3/9 activity, observed in 28- to 31-mo-old (aging) FAAH(+/+) mice, were largely attenuated in knockouts. There was no difference in the myocardial cannabinoid CB(1) and CB(2) receptor gene expression between young and aging FAAH(-/-) and FAAH(+/+) mice. Anandamide dose dependently attenuated the TNF-alpha-induced ICAM-1 and VCAM-1 expression, NF-kappaB activation in HCAECs, and the adhesion of monocytes to HCAECs in a CB(1)- and CB(2)-dependent manner. These findings suggest that pharmacological inhibition of FAAH may represent a novel protective strategy against chronic inflammation, oxidative/nitrative stress, and apoptosis associated with cardiovascular aging and atherosclerosis.     

Comparison of prostaglandin F2alpha,bimatoprost (prostamide), and butaprost (EP2 agonist) on Cyr61 and connective tissue growth factor gene expression

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.     

Molecular characterization of a novel type of prostamide/prostaglandin F synthase, belonging to the thioredoxin-like superfamily

Prostaglandin F (PGF) ethanolamide (prostamide F) synthase, which catalyzed the reduction of prostamide H(2) to prostamide F(2alpha), was found in mouse and swine brain. The enzyme was purified from swine brain, and its amino acid sequence was defined. The mouse enzyme consisted of a 603-bp open reading frame coding for a 201-amino acid polypeptide with a molecular weight of 21,669. The amino acid sequence placed the enzyme in the thioredoxin-like superfamily with Cys(44) being the active site. The enzyme expressed in Escherichia coli as well as the native enzyme catalyzed not only the reduction of prostamide H(2) to prostamide F(2alpha) but also that of PGH(2) to PGF(2alpha). The V(max) and K(m) values for prostamide H(2) were about 0.25 micromol/min.mg of protein and 7.6 microm, respectively, and those for PGH(2) were about 0.69 micromol/min.mg of protein and 6.9 microm, respectively. Neither PGE(2) nor PGD(2) served as a substrate for this synthase. Based on these data, we named the enzyme prostamide/PGF synthase. Although the enzyme showed a broad specificity for reductants, reduced thioredoxin preferentially served as a reducing equivalent donor for this enzyme. Moreover, Northern and Western blot analyses in addition to the prostamide F synthase activity showed that the enzyme was mainly distributed in the brain and spinal cord, and the immunohistochemical study in the spinal cord showed that the enzyme was found mainly in the cytosol. These results suggest that prostamide/PGF synthase may play an important functional role in the central nervous system.     

A FAAH-regulated class of N-acyl taurines that activates TRP ion channels

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that catabolizes several bioactive lipids in vivo. Most of the physiological substrates of FAAH characterized to date belong to the N-acyl ethanolamine (NAE) class of fatty acid amides, including the endocannabinoid anandamide, the anti-inflammatory lipid N-palmitoyl ethanolamine, and the satiating factor N-oleoyl ethanolamine. We recently identified a second structural class of fatty acid amides regulated by FAAH in vivo: the N-acyl taurines (NATs). Global metabolite profiling revealed high concentrations of long chain (> or = C20) saturated NATs in the central nervous system (CNS) of FAAH(-/-) mice. Here, we use metabolite profiling to characterize the FAAH-NAT system in peripheral mouse tissues. Livers and kidneys of FAAH(-/-) mice possessed dramatic elevations in NATs, which, in contrast to those detected in the CNS, were enriched in polyunsaturated acyl chains (e.g., C20:4, C22:6). Peripheral NATs rose more than 10-fold within 1 h following pharmacological inactivation of FAAH and reached levels up to approximately 5000 pmol/g tissue (C22:6 in kidney), implicating a constitutive and highly active pathway for NAT metabolism in which FAAH plays an integral part. Interestingly, NATs were found to activate multiple members of the transient receptor potential (TRP) family of calcium channels, including TRPV1 and TRPV4, which are both expressed in kidney. The dramatic elevation in endogenous levels of NATs following acute or chronic inactivation of FAAH, in conjunction with the pharmacological effects of these lipids on TRP channels, suggests the existence of a second major lipid signaling system regulated by FAAH in vivo.     

Vasoactive properties of dihomo-gamma-linolenic acid and series one prostaglandins in a freshwater teleost, Channa maculata

1. Prostaglandins A1, B1, E1 and F1 alpha (2-120 micrograms/kg), arachidonic acid and dihomo-gamma-linolenic acid (0.1-2 mg/kg) were injected intravenously into Channa maculata and changes in arterial blood pressure were recorded. 2. Injection of PGF1 alpha had no significant effect on arterial blood pressure. Injection of PGA1 and PGE1 was followed by dose-dependent hypotension whereas injection of PGB1 elicited significant dose-dependent increase in arterial blood pressure. 3. Both dihomo-gamma-linolenic acid and arachidonic acid were also depressor agents but dihomo-gamma-linolenic acid was more potent. 4. A single bolus intravenous injection of indomethacin (5 mg/kg) or 4 daily intraperitoneal injections (4 x 10 mg/kg) significantly lowered arterial blood pressure. One hour after pre-treatment of indomethacin, the vascular effects of both prostaglandin precursors were abolished. 5. It appears that the vascular effects of prostaglandins in Channa maculata are qualitatively different from those reported for mammals.     

Two pathways for GM2(NeuGc) expression in mice: genetic analysis

We have reported that WHT/Ht mice express neither GM2(NeuGc) nor GM1(NeuGc) in the liver or erythrocytes due to a defect on the Ggm-2 gene, which was demonstrated to control the activity of UDP-GalNAc:GM3(NeuGc) N-acetylgalactosaminyltransferase in mouse liver, and, in addition, WHT/Ht mice do not express a detectable amount of GM2(NeuGc) but do express GM1(NeuGc) in tissues other than the liver and erythrocytes, such as the spleen, thymus, heart, lung, kidney, and testis [Nakamura et al. (1988) J. Biochem. 103, 201-208]. In order to determine whether the phenotype of WHT/Ht mice exhibiting an undetectable amount of GM2(NeuGc) in these tissues is genetically controlled or not, we analyzed the expression of gangliosides in the progeny obtained on backcross mating between (BALB/c X WHT/Ht)F1 and WHT/Ht mice, and in a GM2(NeuGc) congenic mouse, WHT.C. Concerning the expression of GM2(NeuGc) in the liver, lung, and kidney, 102 backcross mice could be segregated into two types. One type expressed a detectable amount of GM2(NeuGc) in the liver, lung, and kidney, and the other type did not. The ratio of the numbers of mice exhibiting these two types was 42: 60, indicating that the two phenotypes were genetically determined by the involvement of a single autosomal gene. Recombination as to GM2(NeuGc) expression in the liver, lung, and kidney was not detected among the 102 backcross mice. Analysis of the GM2(NeuGc) congenic mouse indicated that a detectable amount of GM2(NeuGc) was expressed in the liver, erythrocytes, lung, kidney, heart, spleen, and small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrolysis of prostaglandin glycerol esters by the endocannabinoid-hydrolyzing enzymes, monoacylglycerol lipase and fatty acid amide hydrolase

Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variety of prostaglandin-EA's and prostaglandin-G's nearly as diverse as those derived from arachidonic acid. Thus, COX-2 may regulate endocannabinoid levels in neurons during retrograde signaling or produce novel endocannabinoid metabolites for receptor activation. Endocannabinoid-metabolizing enzymes are important regulators of their action, so we tested whether PG-G levels may be regulated by monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH). We found that PG-Gs are poor substrates for purified MGL and FAAH compared to 2-AG and/or AEA. Determination of substrate specificity demonstrates a 30-100- and 150-200-fold preference of MGL and FAAH for 2-AG over PG-Gs, respectively. The substrate specificity of AEA compared to those of PG-Gs was approximately 200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH.     

Distribution of prostaglandin biosynthetic pathways in organs and tissues of the fetal lamb

Five prostaglandins, i.e. prostaglandins E2, F2alpha and D2, 6-keto-prostaglandin F1alpha and thromboxane B2, were measured by mass spectrometry. Homogenates of fetal lamb brain, lung, liver, spleen and kidney and the ductus arteriosus, aorta and pulmonary artery formed different amounts of each product. Although the main prostaglandin in the fetal organs was prostaglandin E2, arterial tissue formed mostly 6-keto-prostaglandin F1alpha. These results demonstrate significant differences between organs and tissues in the relative direction of the 'prostaglandin synthetase' enzyme complex.     

Induction and suppression of cytochrome P450 isoenzymes and generation of oxygen radicals by procymidone in liver,kidney and lung of CD1 mice

Although chronic administration of procymidone (a widely used dicarboximide fungicide) leads to an increased incidence of liver tumors in mice, short-term genotoxicity studies proved negative. As cytochrome P450 (CYP) induction has been linked to non-genotoxic carcinogenesis, we investigated whether procymidone administration causes induction of CYP-dependent monooxygenases in liver, kidney and lung microsomes of male Swiss Albino CD1 mice after single or repeated (daily for three consecutive days) i.p. treatment with either 400 or 800 (1/10 or 1/20 of the DL(50)) mgkg(-1) b.w. procymidone. CYP content and CYP3A1/2, 1A1, 1A2, 2B1/2, 2E1, 2A, 2D9 and 2C11 supported oxidations were studied using either the regio- and stereo-selective hydroxylation of testosterone as multibiomarker or highly specific substrates as probes of various CYPs. While a single dose was uneffective, multiple procymidone administration lead to marked inductions of various monooxygenases: CYP3A1/2 in liver and lung (as measured by N-demethylation of aminopyrine and testosterone 6 beta-hydroxylase); CYP2E1 in liver (p-nitrophenol hydroxylation); CYP1A1 in liver and kidney (deethylation of ethoxyresorufin). Several hydroxylations were induced in the liver, including the CYP2A-linked 7 alpha (14-fold) as well as 6 alpha (22-fold), 6 beta, 16 beta and 2 beta hydroxylases. The pattern of inductions/suppressions recorded in the three different tissues suggests that procymidone exerts complex effects on the CYP profile. Tissue-specific trends included a large number of inductions in the liver and suppressions in the lung. The main inductions were corroborated by immunoblotting analyses and Northern blotting showed that inductions of CYP3A1/2, CYP2E1 and CYP1A1/2 were paralleled by increased mRNA levels. It was also found that CYP over-expression generates large amounts of reactive oxygen species (ROS), especially in liver. These data may explain why in vitro short-term genotoxicity studies on procymidone were negative, whereas in vivo long-term carcinogenesis studies turned out positive: long-term CYP induction (e.g. oxygen centered free radicals over-production) can have a co-carcinogenic and/or promoting potential.     

Fatty acid amide hydrolase determines anandamide-induced cell death in the liver

The endocannabinoid anandamide (AEA) induces cell death in many cell types, but determinants of AEA-induced cell death remain unknown. In this study, we investigated the role of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in AEA-induced cell death in the liver. Primary hepatocytes expressed high levels of FAAH and were completely resistant to AEA-induced cell death, whereas primary hepatic stellate cells (HSCs) expressed low levels of FAAH and were highly sensitive to AEA-induced cell death. Hepatocytes that were pretreated or with the FAAH inhibitor URB597 isolated from FAAH(-/-) mice displayed increased AEA-induced reactive oxygen species (ROS) formation and were susceptible to AEA-mediated death. Conversely, overexpression of FAAH in HSCs prevented AEA-induced death. Since FAAH inhibition conferred only partial AEA sensitivity in hepatocytes, we analyzed additional factors that might regulate AEA-induced death. Hepatocytes contained significantly higher levels of glutathione (GSH) than HSCs. Glutathione depletion by dl-buthionine-(S,R)-sulfoximine rendered hepatocytes susceptible to AEA-mediated ROS production and cell death, whereas GSH ethyl ester prevented ROS production and cell death in HSCs. FAAH inhibition and GSH depletion had additive effects on AEA-mediated hepatocyte cell death resulting in almost 70% death after 24 h at 50 microm AEA and lowering the threshold for cell death to 500 nm. Following bile duct ligation, FAAH(-/-) mice displayed increased hepatocellular injury, suggesting that FAAH protects hepatocytes from AEA-induced cell death in vivo. In conclusion, FAAH and GSH are determinants of AEA-mediated cell death in the liver.     

Regulation of gene expression of epithelial calcium channels in intestine and kidney of mice by 1alpha,25-dihydroxyvitamin D3

In wild-type (VDR(+/+)) mice, ECaC2 expression was confirmed in the intestine and kidney, while ECaC1 expression was exclusively confined to the kidney. Both mRNAs expression of ECaC1 and ECaC2 in the kidney and ECaC2 mRNA expression in the intestine increased time- and dose-dependently in response to 1alpha,25(OH)(2)D(3) injection in VDR(+/+) mice, but not in VDR(-/-) mice. The mRNA levels of ECaC2 in the intestine of VDR(-/-) mice were remarkably reduced when compared to VDR(+/+) mice, while no significant differences were observed in both mRNA levels of ECaC1 and ECaC2 in the kidney between VDR(+/+) mice and VDR(-/-) mice. In the primary renal tubular cells (PRTC) isolated from VDR(+/+) mice, both ECaCs mRNA expression increased in response to 1alpha,25(OH)(2)D(3) treatment, but not in the PRTC of VDR(-/-) mice. PTH increased both ECaCs mRNA expression in the PRTC of VDR(+/+) mice. These results suggest that 1alpha,25(OH)(2)D(3) directly modulates the gene expression of ECaC1 and ECaC2 together with PTH in the kidney of mice. 1alpha,25(OH)(2)D(3) also modulates the gene expression of ECaC2 in the intestine of mice, however, further studies are needed to elucidate the direct action of 1alpha,25(OH)(2)D(3) on the expression of ECaC2 in the intestine.