Soluble epoxide hydrolase inhibitor promotes immunomodulation to inhibit bone resorption.

BACKGROUND AND OBJECTIVE: Soluble epoxide hydrolase (sEH) is an enzyme in the arachidonate cascade which converts epoxy fatty acids (EpFAs), such as epoxyeicosatrienoic acids (EETs) produced by cytochrome P450 enzymes, to dihydroxy-eicosatrienoic acids. In the last 20 years with the development of inhibitors to sEH it has been possible to increase the levels of EETs and other EpFAs in in vivo models. Recently, studies have shown that EETs play a key role in blocking inflammation in a bone resorption process, but the mechanism is not clear. In the current study we used the sEH inhibitor (1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea [TPPU]) to investigate the immunomodulatory effects in a mouse periodontitis model. MATERIAL AND METHODS: Mice were infected on days 0, 2, and 4 with Aggregatibacter actinomycetemcomitans and divided into groups (n = 6) that were treated orally, daily for 15 days, with 1 mg/kg of TPPU. Then, the mice were killed and their jaws were analyzed for bone resorption using morphometry. Immunoinflammatory markers in the gingival tissue were analyzed by microarray PCR or western blotting. RESULTS: Infected mice treated with TPPU showed lower bone resorption than infected mice without treatment. Interestingly, infected mice showed increased expression of sEH; however, mice treated with TPPU had a reduction in expression of sEH. Besides, several proinflammatory cytokines and molecular markers were downregulated in the gingival tissue in the group treated with 1 mg/kg of TPPU. CONCLUSION: The sEH inhibitor, TPPU, showed immunomodulatory effects, decreasing bone resorption and inflammatory responses in a bone resorption mouse model.

Pharmacokinetics and antinociceptive effects of the soluble epoxide hydrolase inhibitor t-TUCB in horses with experimentally induced radiocarpal synovitis.

This study determined the pharmacokinetics, antinociceptive, and anti-inflammatory effects of the soluble epoxide hydrolase (sEH) inhibitor t-TUCB (trans-4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid) in horses with lipopolysaccharide (LPS)-induced radiocarpal synovitis. A total of seven adult healthy mares (n = 4-6/treatment) were administered 3 µg LPS into one radiocarpal joint and t-TUCB intravenously (i.v.) at 0 (control), 0.03, 0.1, 0.3, and 1 mg/kg in a blinded, randomized, crossover design with at least 3 weeks washout between. Two investigators independently assigned pain scores (at rest, walk and trot) and lameness scores before and up to 48 hr after t-TUCB/LPS. Responses to touching the joint skin to assess tactile allodynia, plasma, and synovial fluid (SF) t-TUCB concentrations were determined before and up to 48 hr after t-TUCB/LPS. Blood and SF were collected for clinical laboratory evaluations before and up to 48 hr after t-TUCB/LPS. Areas under the curves of pain and lameness scores were calculated and compared between control and treatments. Data were analyzed using repeated measures ANOVA with Dunnett or Bonferroni post-test. p < .05 was considered significant. Data are mean ± SEM. Compared to control, pain, lameness, and tactile allodynia were significantly lower with 1 mg/kg t-TUCB, but not the other doses. For 0.1, 0.3, and 1 mg/kg t-TUCB treatments, plasma terminal half-lives were 13 ± 3, 13 ± 0.5, and 24 ± 5 hr, and clearances were 68 ± 15, 48 ± 5, and 14 ± 1 ml hr-1  kg-1 . The 1 mg/kg t-TUCB reached the SF at high concentrations. There were no important anti-inflammatory effects. In conclusion, sEH inhibition with t-TUCB may provide analgesia in horses with inflammatory joint pain.

Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa.

Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared with controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment.

High salt intake increases blood pressure in normal rats: putative role of 20-HETE and no evidence on changes in renal vascular reactivity.

UNLABELLED: Background/Aims . High salt (HS) intake may elevate blood pressure (BP), also in animals without genetic salt sensitivity. The development of salt-dependent hypertension could be mediated by endogenous vasoactive agents; here we examined the role of vasodilator epoxyeicosatrienoic acids (EETs) and vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE). METHODS: In conscious Wistar rats on HS diet systolic BP (SBP) was examined after chronic elevation of EETs using 4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (c-AUCB), a blocker of soluble epoxide hydrolase, or after inhibition of 20-HETE with 1-aminobenzotriazole (ABT). Thereafter, in acute experiments the responses of renal artery blood flow (Transonic probe) and renal regional perfusion (laser-Doppler) to intrarenal acetylcholine (ACh) or norepinephrine were determined. RESULTS: HS diet increased urinary 20-HETE excretion. The SBP increase was not reduced by c-AUCB but prevented by ABT until day 5 of HS exposure. Renal vasomotor responses to ACh or norepinephrine were similar on standard and HS diet. ABT but not c-AUCB abolished the responses to ACh. Conclusions . 20-HETE seems to mediate the early-phase HS diet-induced BP increase while EETs are not engaged in the process. Since HS exposure did not alter renal vasodilator responses to Ach, endothelial dysfunction is not a critical factor in the mechanism of salt-induced blood pressure elevation.

In vitro glucuronidation of the antibacterial triclocarban and its oxidative metabolites.

Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is widely used as an antibacterial in bar soaps. During use of these soaps, a significant portion of TCC is absorbed by humans. For the elimination from the body, glucuronidation plays a key role in both biliary and renal clearance. To investigate this metabolic pathway, we performed microsomal incubations of TCC and its hydroxylated metabolites 2'-OH-TCC, 3'-OH-TCC, and 6-OH-TCC. Using a new liquid chromatography-UV-mass spectrometry method, we could show a rapid glucuronidation for all OH-TCCs by the uridine-5'-diphosphate-glucuronosyltransferases (UGT) present in liver microsomes of humans (HLM), cynomolgus monkeys (CLM), rats (RLM), and mice (MLM). Among the tested human UGT isoforms, UGT1A7, UGT1A8, and UGT1A9 showed the highest activity for the conjugation of hydroxylated TCC metabolites followed by UGT1A1, UGT1A3, and UGT1A10. Due to this broad pattern of active UGTs, OH-TCCs can be efficiently glucuronidated in various tissues, as shown for microsomes from human kidney (HKM) and intestine (HIM). The major renal metabolites in humans, TCC-N-glucuronide and TCC-N'-glucuronide, were formed at very low conversion rates (<1%) by microsomal incubations. Low amounts of N-glucuronides were generated by HLM, HIM, and HKM, as well as by MLM and CLM, but not by RLM, according to the observed species specificity of this metabolic pathway. Among the human UGT isoforms, only UGT1A9 had activity for the N-glucuronidation of TCC. These results present an anomaly where in vivo the predominant urinary metabolites of TCC are N and N'-glucuronides, but these compounds are slowly produced in vitro.

Bioactivation of leukotoxins to their toxic diols by epoxide hydrolase.

Leukotoxin is a linoleic acic oxide produced by leukocytes and has been associated with the multiple organ failure and adult respiratory distress syndrome seen in some severe burn patients. Leukotoxin has been reported to be toxic when injected into animals intravenously. Herein, we report that this lipid is not directly cytotoxic in at least two in vitro systems. Using a baculovirus expression system we demonstrate that leukotoxin is only cytotoxic in the presence of epoxide hydrolases. In addition, it is the diol metabolite that proves toxic to pulmonary alveolar epithelial cells, suggesting a critical role for the diol in leukotoxin-associated respiratory disease. In vivo data also support the toxicity of leukotoxin diol. For the first time we demonstrate that soluble epoxide hydrolase can bioactivate epoxides to diols that are apparently cytotoxic. Thus leukotoxin should be regarded as a protoxin corresponding to the more toxic diol. This clearly has implications for designing new clinical interventions.

Phage anti-immunocomplex assay for clomazone: two-site recognition increasing assay specificity and facilitating adaptation into an on-site format.

The impact of the use of herbicides in agriculture can be minimized by compliance with good management practices that reduce the amount used and their release into the environment. Simple tests that provide real time on-site information about these chemicals are a major aid for these programs. In this work, we show that phage anti-immunocomplex assay (PHAIA), a method that uses phage-borne peptides to detect the formation of antibody-analyte immunocomplexes, is an advantageous technology to produce such field tests. A monoclonal antibody to the herbicide clomazone was raised and used in the development of conventional competitive and noncompetitive PHAIA immunoassays. The sensitivity attained with the PHAIA format was over 10 times higher than that of the competitive format. The cross-reactivity of the two methods was also compared using structurally related compounds, and we observed that the two-site binding of PHAIA "double-checks" the recognition of the analyte, thereby increasing the assay specificity. The positive readout of the noncompetitive PHAIA method allowed adaptation of the assay into a rapid and simple format where as little as 0.4 ng/mL clomazone (more than 10-fold lower than the proposed standard) in water samples from a rice field could be easily detected by simple visual inspection.

Soluble epoxide hydrolase inhibition modulates vascular remodeling.

The soluble epoxide hydrolase enzyme (SEH) and vascular remodeling are associated with cardiovascular disease. Although inhibition of SEH prevents smooth muscle cell proliferation in vitro, the effects of SEH inhibition on vascular remodeling in vivo and mechanisms of these effects remain unclear. Herein we determined the effects of SEH antagonism in an endothelium intact model of vascular remodeling induced by flow reduction and an endothelium denuded model of vascular injury. We demonstrated that chronic treatment of spontaneously hypertensive stroke-prone rats with 12-(3-adamantan-1-yl-ureido) dodecanoic acid, an inhibitor of SEH, improved the increment of inward remodeling induced by common carotid ligation to a level that was comparable with normotensive Wistar Kyoto rats. Similarly, mice with deletion of the gene responsible for the production of the SEH enzyme (Ephx2(-/-)) demonstrated enhanced inward vascular remodeling induced by carotid ligation. However, the hyperplastic response induced by vascular injury that denudes the endothelium was unabated by SEH inhibition or Ephx2 gene deletion. These results suggest that SEH inhibition or Ephx2 gene deletion antagonizes neointimal formation in vivo by mechanisms that are endothelium dependent. Thus SEH inhibition may have therapeutic potential for flow-induced remodeling and neointimal formation.

Electrochemistry-mass spectrometry unveils the formation of reactive triclocarban metabolites.

Triclocarban (3,4,4'-trichlorocarbanilide, TCC) is a widely used antibacterial agent in personal care products and is frequently detected as an environmental pollutant in waste waters and surface waters. In this study, we report novel reactive metabolites potentially formed during biotransformation of TCC. The oxidative metabolism of TCC has been predicted using an electrochemical cell coupled online to liquid chromatography and electrospray ionization mass spectrometry. The electrochemical oxidation unveils the fact that hydroxylated metabolites of TCC may form reactive quinone imines. Moreover, a so-far unknown dechlorinated and hydroxylated TCC metabolite has been identified. The results were confirmed by in vitro studies with human and rat liver microsomes. The reactivity of the newly discovered quinone imines was demonstrated by their covalent binding to glutathione and macromolecules, using ß-lactoglobulin A as a model protein. The results regarding the capability of the electrochemical cell to mimic the oxidative metabolism of TCC are discussed. Moreover, the occurrence of reactive metabolites is compared with findings from earlier in vivo studies and their relevance in vivo is argued.

Assessment of dioxin-like soil contamination in Mexico by enzyme-linked immunosorbent assay.

In this work, we describe the results of a preliminary soil assessment program for the detection of dioxins at different sites in Mexico performed by immunoassay. We studied five different sectors considered relevant sources of dioxins: Anaversa and Tekchem industrial areas where organochlorine pesticides were manufactured and released by accidental explosions, secondary smelters, brick kilns, and rural dwellings. In the context of the Agency for Toxic Substances and Disease Registry (ATSDR) guidelines, only the brick kilns sites can be considered as low-risk areas. The dioxin concentrations detected in the vicinity of the Anaversa and Tekchem chemical plants and secondary smelters exceed the screening level of 0.05 ppb set by the ATSDR, and therefore further site-specific studies are needed. The dioxin levels found in all soot samples from indigenous dwellings where wood is used for indoor cooking were above the evaluation level. Considering that the studied areas are representative examples of dioxin sources in less developed countries, our work demonstrates the useful application of dioxin immunoassays as a tool for dioxin screening for environmental assessment programs in developing countries.

1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a soluble epoxide hydrolase inhibitor, lowers L-NAME-induced hypertension through suppression of angiotensin-converting enzyme in rats.

OBJECTIVE: This study evaluated the efficacy of the soluble epoxide hydrolase (sEH) inhibitor, TPPU on chronic NG-Nitro L-arginine methyl ester (L-NAME)-induced hypertension in rats and its effects on plasma Angiotensin II (Ang II), cardiac Angiotensin-converting enzyme (ACE) and Angiotensin II receptor type 1 (AT1R) expressions. MATERIALS AND METHODS: Forty Sprague Dawley rats were divided into 5 groups. Two groups served as control and received orally either vehicle or TPPU (3 mg/kg) for five weeks. The other three groups were given L-NAME (50 mg/kg/day) in drinking water for five weeks. Two weeks after the L-NAME treatment, animals received orally either saline or TPPU (3 mg/kg/day) or lisinopril (10 mg/kg/day) daily for 3 weeks. Blood pressure (BP) was measured weekly. At the end of the experiment, plasma Ang II, cardiac ACE and AT1R protein and gene expressions were determined. RESULTS: L-NAME caused a significant increase in BP of the animals. TPPU and lisinopril resulted in normalization of L-NAME-induced hypertension. They also caused a significant reduction in Ang II and ACE protein and gene expressions compared to L-NAME and vehicle-treated animals. CONCLUSIONS: This study demonstrates that TPPU effectively lowers L-NAME-induced hypertension in rats. The mechanism of its antihypertensive effect is likely mediated by the suppression of ACE gene and protein expression, leading to a lower Ang II level.

Cytosolic and microsomal epoxide hydrolases: differential properties in mammalian liver.

The epoxide hydrolase activities of the 100,000 g pellet (microsomal) and 100,00 g soluble (cystosolic) fractions of mouse, rat, and guinea pig liver were measured with three closely related compounds used as substrates. Differences between the species in the distribution of the cytosolic and microsomal hydrolases and in their substrate specificities and pH optima demonstrate why epoxide hydrolase activity in the cytosolic fraction was not detected earlier in spie of intensive work on the microsomal epoxide hydrolase.

Transition state analogs as ligands for affinity purification of juvenile hormone esterase.

Insect juvenile hormones are metabolized in numerous species of caterpillars by low abundance, highly specific esterases. Because of their role in regulating and possibly disrupting juvenile hormone titer and thus insect metamorphosis, they are of interest to developmental biologists as well as scientists interested in selective insect control. However, the enzymes have defied attempts to purify and characterize them. Juvenile hormone esterase activity can be inhibited by a variety of 3-substituted 1,1,1-trifluoropropanone sulfides. These apparent transition state analogs were used as ligands and eluting agents to purify juvenile hormone esterase from four insect species from 500-fold to over 1000-fold in high yield. After elution from the affinity column, the enzymes were radiolabeled with paraoxon and analyzed by electrophoresis, and the results demonstrate a high degree of purity. Transition state analogs may be useful for the affinity purification of other enzymes.

A rapid luminescent assay for measuring cytochrome P450 activity in individual larval Culex pipiens complex mosquitoes (Diptera: Culicidae).

Multiple assays are available to measure P450 activity in insects, including mosquitoes; however, each of these assays has drawbacks in terms of the number of mosquitoes required, specificity, sensitivity, cost, and/or time required to prepare active enzyme homogenates. In this study, a commercially available luminescent assay, P450-Glo, was modified and evaluated to measure P450 activity from the gut of a single larva after removal of the gut contents. We also compared this assay to an earlier developed fluorescent assay. After optimization of assay conditions, the P450-Glo assay held considerable promise to be used as an effective, inexpensive, high-throughput, and sensitive screening assay to measure P450 activities in single mosquitoes. Furthermore, we tested the utility of the single gut assay using the pyrethroid resistant Marin strain of Culex pipiens pipiens form molestus and the pyrethroid sensitive CQ-1 strain of Cx. pipiens quinqefasciatus. We observed on average 1.8-fold higher levels of P450 activity in the resistant mosquitoes in comparison to the sensitive mosquitoes. Additionally, consistent with our previous findings, distribution plots of P450 activity showed 33% of individual Marin mosquitoes had higher P450 activities than the highest activity displayed by a CQ-1 mosquito. The assay platform is highly flexible in terms of choice of tissue, method of preparation, isozyme specificity, and sample quantity and thus could easily be adapted to be used for other arthropod species.

Addition of milk fat globule membrane-enriched supplement to a high-fat meal attenuates insulin secretion and induction of soluble epoxide hydrolase gene expression in the postprandial state in overweight and obese subjects.

CVD and associated metabolic diseases are linked to chronic inflammation, which can be modified by diet. The objective of the present study was to determine whether there is a difference in inflammatory markers, blood metabolic and lipid panels and lymphocyte gene expression in response to a high-fat dairy food challenge with or without milk fat globule membrane (MFGM). Participants consumed a dairy product-based meal containing whipping cream (WC) high in saturated fat with or without the addition of MFGM, following a 12 h fasting blood draw. Inflammatory markers including IL-6 and C-reactive protein, lipid and metabolic panels and lymphocyte gene expression fold changes were measured using multiplex assays, clinical laboratory services and TaqMan real-time RT-PCR, respectively. Fold changes in gene expression were determined using the Pfaffl method. Response variables were converted into incremental AUC, tested for differences, and corrected for multiple comparisons. The postprandial insulin response was significantly lower following the meal containing MFGM (P < 0·01). The gene encoding soluble epoxide hydrolase (EPHX2) was shown to be more up-regulated in the absence of MFGM (P = 0·009). Secondary analyses showed that participants with higher baseline cholesterol:HDL-cholesterol ratio (Chol:HDL) had a greater reduction in gene expression of cluster of differentiation 14 (CD14) and lymphotoxin ß receptor (LTBR) with the WC+MFGM meal. The protein and lipid composition of MFGM is thought to be anti-inflammatory. These exploratory analyses suggest that addition of MFGM to a high-saturated fat meal modifies postprandial insulin response and offers a protective role for those individuals with higher baseline Chol:HDL.

High-throughput method for ranking the affinity of peptide ligands selected from phage display libraries.

The use of phage display peptide libraries allows rapid isolation of peptide ligands for any target selector molecule. However, due to differences in peptide expression and the heterogeneity of the phage preparations, there is no easy way to compare the binding properties of the selected clones, which operates as a major "bottleneck" of the technology. Here, we present the development of a new type of library that allows rapid comparison of the relative affinity of the selected peptides in a high-throughput screening format. As a model system, a phage display peptide library constructed on a phagemid vector that contains the bacterial alkaline phosphatase gene (BAP) was selected with an antiherbicide antibody. Due to the intrinsic switching capacity of the library, the selected peptides were transferred "en masse" from the phage coat protein to BAP. This was coupled to an optimized affinity ELISA where normalized amounts of the peptide-BAP fusion allow direct comparison of the binding properties of hundreds of peptide ligands. The system was validated by plasmon surface resonance experiments using synthetic peptides, showing that the method discriminates among the affinities of the peptides within 3 orders of magnitude. In addition, the peptide-BAP protein can find direct application as a tracer reagent.

Substituted 3-phenylpropenoates and related analogs: electron ionization mass spectral fragmentation and density functional theory calculations.

Analysis of ethyl 3-(2-chlorophenyl)propenoate by electron ionization mass spectrometry showed the distinct loss of an ortho chlorine. To characterize the structural requisites for the observed mass fragmentation, a series of 30 halogen-substituted 3-phenylpropenoate-related structures were examined. All ester-containing alkene derivatives exhibited loss of the distinctive chlorine from the 2-position of the phenyl ring. Analogous derivatives with the halogen (chlorine or bromine) in the para position did not evidence selective halogen loss. Results demonstrated that substituted 3-phenylpropenoates and their analogs fragment via the formation of a previously reported benzopyrylium intermediate. To understand the correlation between the intramolecular radical substitution and the abundance and selectivity of the chlorine (or other halogen) displacement, density functional theory calculations were performed to determine the charge on the principal cation involved in the chlorine loss (in the ortho, meta, and para positions), the charge for the neutral radical (noncation), the excess alpha-electron density on the relevant atom and the energy to form the cation from the neutral atom (ionization energy). Results showed that the selectivity and extent of halogen displacement correlated highly to the electrophilicity of the radical cation as well as the neutral radical. These data further support the proposed fragmentation mechanism involving intramolecular radical elimination.

Metabolic/inflammatory/vascular comorbidity in psychiatric disorders; soluble epoxide hydrolase (sEH) as a possible new target.

The common and severe psychiatric disorders, including major depressive disorder (MDD) and bipolar disorder (BD), are associated with inflammation, oxidative stress and changes in peripheral and brain lipid metabolism. Those pathways are implicated in the premature development of vascular and metabolic comorbidities, which account for considerable morbidity and mortality, including increased dementia risk. During endoplasmic reticulum stress, the soluble epoxide hydrolase (sEH) enzyme converts anti-inflammatory fatty acid epoxides generated by cytochrome p450 enzymes into their corresponding and generally less anti-inflammatory, or even pro-inflammatory, diols, slowing the resolution of inflammation. The sEH enzyme and its oxylipin products are elevated post-mortem in MDD, BD and schizophrenia. Preliminary clinical data suggest that oxylipins increase with symptoms in seasonal MDD and anorexia nervosa, requiring confirmation in larger studies and other cohorts. In rats, a soluble sEH inhibitor mitigated the development of depressive-like behaviors. We discuss sEH inhibitors under development for cardiovascular diseases, post-ischemic brain injury, neuropathic pain and diabetes, suggesting new possibilities to address the mood and cognitive symptoms of psychiatric disorders, and their most common comorbidities.

Effect of angiotensin-converting enzyme blockade, alone or combined with blockade of soluble epoxide hydrolase, on the course of congestive heart failure and occurrence of renal dysfunction in Ren-2 transgenic hypertensive rats with aorto-caval fistula.

We showed recently that increasing kidney epoxyeicosatrienoic acids (EETs) by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for EETs degradation, retarded the development of renal dysfunction and progression of aorto-caval fistula(ACF)-induced congestive heart failure (CHF) in Ren-2 transgenic hypertensive rats (TGR). In that study the final survival rate of untreated ACF TGR was only 14 % but increased to 41 % after sEH blockade. Here we examined if sEH inhibition added to renin-angiotensin system (RAS) blockade would further enhance protection against ACF-induced CHF in TGR. The treatment regimens were started one week after ACF creation and the follow-up period was 50 weeks. RAS was blocked using angiotensin-converting enzyme inhibitor (ACEi, trandolapril, 6 mg/l) and sEH with an sEH inhibitor (sEHi, c-AUCB, 3 mg/l). Renal hemodynamics and excretory function were determined two weeks post-ACF, just before the onset of decompensated phase of CHF. 29 weeks post-ACF no untreated animal survived. ACEi treatment greatly improved the survival rate, to 84 % at the end of study. Surprisingly, combined treatment with ACEi and sEHi worsened the rate (53 %). Untreated ACF TGR exhibited marked impairment of renal function and the treatment with ACEi alone or combined with sEH inhibition did not prevent it. In conclusion, addition of sEHi to ACEi treatment does not provide better protection against CHF progression and does not increase the survival rate in ACF TGR: indeed, the rate decreases significantly. Thus, combined treatment with sEHi and ACEi is not a promising approach to further attenuate renal dysfunction and retard progression of CHF.

Inhibition of 2-arachidonoylglycerol catabolism modulates vasoconstriction of rat middle cerebral artery by the thromboxane mimetic, U-46619.

BACKGROUND AND PURPOSE: Cerebrovascular smooth muscle cells express the CB1 cannabinoid receptor and CB1 agonists produce vasodilatation of the middle cerebral artery (MCA). The thromboxane A2 mimetic, U-46619, increased the content of the endocannabinoid, 2-arachidonoylglycerol (2-AG) in the MCA and 2-AG moderated the vasoconstriction produced by U46619 in this tissue. The purposes of this study were to examine the extent to which 2-AG is catabolized by cerebral arteries and to determine whether blockade of 2-AG inactivation potentiates its feedback inhibition of U-44619-mediated vasoconstriction. EXPERIMENTAL APPROACH: The diameters of isolated, perfused MCA from male rats were measured using videomicroscopy. KEY RESULTS: Exogenous 2-AG produces a CB1 receptor-dependent and concentration-related increase in the diameter of MCA constricted with 5-HT. The E (max) for 2-AG dilation is increased 4-fold in the presence of the metabolic inhibitors 3-(decylthio)-1,1,1-trifluropropan-2-one (DETFP), URB754 and URB597. To examine the role of catabolism in the effects of endogenous 2-AG, vasoconstriction induced by U-46619 was studied. DETFP and URB754, but not the fatty acid amide hydrolase inhibitor, URB597, significantly increased the EC(50) for U-46619. These data support a physiological role for endocannabinoid feedback inhibition in the effects of U-46619 and indicate that endogenously produced 2-AG is also efficiently catabolized within the MCA. CONCLUSIONS AND IMPLICATIONS: MCA express mechanisms for the efficient inactivation of 2-AG, providing further support for an endocannabinoid feedback mechanism that opposes thromboxane-mediated vasoconstriction. These data suggest that potentiation of endogenously produced 2-AG could be a novel therapeutic approach to the treatment of thrombotic stroke.