Effects of Soluble Epoxide Hydrolase Deficiency on Acute Pancreatitis in Mice

Background

Acute pancreatitis (AP) is a frequent gastrointestinal disorder that causes significant morbidity, and its incidence has been progressively increasing. AP starts as a local inflammation in the pancreas that often leads to systemic inflammatory response and complications. Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition in murine models has beneficial effects in inflammatory diseases, but its significance in AP remains unexplored.

Methodology/Principal Findings

To investigate whether sEH may have a causal role in AP we utilized Ephx2 knockout (KO) mice to determine the effects of sEH deficiency on cerulein- and arginine-induced AP. sEH expression increased at the protein and messenger RNA levels, as well as enzymatic activity in the early phase of cerulein- and arginine-induced AP in mice. In addition, amylase and lipase levels were lower in cerulein-treated Ephx2 KO mice compared with controls. Moreover, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B and IL-6 were lower in cerulein-treated Ephx2 KO mice compared with controls. Further, Ephx2 KO mice exhibited decreased cerulein- and arginine-induced NF-κB inflammatory response, MAPKs activation and decreased cell death. Conclusions -These findings demonstrate a novel role for sEH in the progression of cerulein- and arginine-induced AP.     

Inhibition of the Soluble Epoxide Hydrolase by Tyrosine Nitration

Inhibition of the soluble epoxide hydrolase (sEH) has beneficial effects on vascular inflammation and hypertension indicating that the enzyme may be a promising target for drug development. As the enzymatic core of the hydrolase domain of the human sEH contains two tyrosine residues (Tyr³⁸³ and Tyr⁴⁶⁶) that are theoretically crucial for enzymatic activity, we addressed the hypothesis that the activity of the sEH may be affected by nitrosative stress. Epoxide hydrolase activity was detected in human and murine endothelial cells as well in HEK293 cells and could be inhibited by either authentic peroxynitrite (ONOO⁻) or the ONOO⁻ generator 3-morpholino-sydnonimine (SIN-1). Protection of the enzymatic core with 1-adamantyl-3-cyclohexylurea in vitro decreased sensitivity to SIN-1. Both ONOO⁻ and SIN-1 elicited the tyrosine nitration of the sEH protein and mass spectrometry analysis of tryptic fragments revealed nitration on several tyrosine residues including Tyr³⁸³ and Tyr⁴⁶⁶. Mutation of the latter residues to phenylalanine was sufficient to abrogate epoxide hydrolase activity. In vivo, streptozotocin-induced diabetes resulted in the tyrosine nitration of the sEH in murine lungs and a significant decrease in its activity. Taken together, these data indicate that the activity of the sEH can be regulated by the tyrosine nitration of the protein. Moreover, nitrosative stress would be expected to potentiate the physiological actions of arachidonic acid epoxides by preventing their metabolism to the corresponding diols.Over the last decade, a great deal has been discovered about the physiological role of cytochrome P450-derived epoxides, such as those generated from arachidonic and linoleic acid, in the regulation of vascular homeostasis (1). For example, CYP2C- and CYP2J-derived epoxyeicosatrienoic acids (EETs)³ can acutely regulate vascular tone by inducing endothelial and smooth muscle cell hyperpolarization in the systemic circulation while promoting constriction in pulmonary circulation. EETs also stimulate a number of endothelial signaling cascades to promote angiogenesis (2).The arachidonic acid epoxides (apart from 5,6-EET) are chemically stable, and their intracellular level is tightly regulated by a number of different mechanisms including β-oxidation (3), chain elongation (4), and hydration. However, of these regulatory mechanisms, it appears that the physiologically most important enzyme for the intracellular regulation of EET levels is the soluble epoxide hydrolase (sEH) (5). The dihydroxyeicosatrienoic acids (DHETs) generated from the EETs by sEH are biologically active, although generally less so than the parent epoxides (for review, see Ref. 6). Indeed, when the EETs are converted to the more polar DHETs, they are not as readily incorporated into membrane lipids (7, 8) and rapidly leave cells as diols or as still more polar conjugates.Surprisingly little is known about the mechanisms that regulate sEH activity, and although there have been a number of studies linking changes in sEH expression with inflammatory or hormonal stimuli (9, 10), nothing is known about the regulation of sEH by post-translational modification. Given that two tyrosine residues (Tyr³⁸³ and Tyr⁴⁶⁶) in the active site of the hydrolase are reportedly essential for enzyme activity (11), we determined whether or not the sEH could be regulated by tyrosine nitration.     

Mechanism of Protection by Soluble Epoxide Hydrolase Inhibition in Type 2 Diabetic Stroke

Inhibition of soluble epoxide hydrolase (sEH) is a potential target of therapy for ischemic injury. sEH metabolizes neuroprotective epoxyeicosatrienoic acids (EETs). We recently demonstrated that sEH inhibition reduces infarct size after middle cerebral artery occlusion (MCAO) in type 1 diabetic mice. We hypothesized that inhibition of sEH would protect against ischemic injury in type 2 diabetic mice. Type 2 diabetes was produced by combined high-fat diet, nicotinamide and streptozotocin in male mice. Diabetic and control mice were treated with vehicle or the sEH inhibitor t-AUCB then subjected to 60-min MCAO. Compared to chow-fed mice, high fat diet-fed mice exhibited an upregulation of sEH mRNA and protein in brain, but no differences in brain EETs levels were observed between groups. Type 2 diabetic mice had increased blood glucose levels at baseline and throughout ischemia, decreased laser-Doppler perfusion of the MCA territory after reperfusion, and sustained larger cortical infarcts compared to control mice. t-AUCB decreased fasting glucose levels at baseline and throughout ischemia, improved cortical perfusion after MCAO and significantly reduced infarct size in diabetic mice. We conclude that sEH inhibition, as a preventative treatment, improves glycemic status, post-ischemic reperfusion in the ischemic territory, and stroke outcome in type 2 diabetic mice.     

Role of Endothelial Soluble Epoxide Hydrolase in Cerebrovascular Function and Ischemic Injury

Soluble Epoxide Hydrolase (sEH) is a key enzyme in the metabolism and termination of action of epoxyeicosatrienoic acids, derivatives of arachidonic acid, which are protective against ischemic stroke. Mice lacking sEH globally are protected from injury following stroke; however, little is known about the role of endothelial sEH in brain ischemia. We generated transgenic mice with endothelial-specific expression of human sEH (Tie2-hsEH), and assessed the effect of transgenic overexpression of endothelial sEH on endothelium-dependent vascular reactivity and ischemic injury following middle cerebral artery occlusion (MCAO). Compared to wild-type, male Tie2-hsEH mice exhibited impaired vasodilation in response to stimulation with 1 µM acetylcholine as assessed by laser-Doppler perfusion monitoring in an in-vivo cranial window preparation. No difference in infarct size was observed between wild-type and Tie2-hsEH male mice. In females, however, Tie2-hsEH mice sustained larger infarcts in striatum, but not cortex, compared to wild-type mice. Sex difference in ischemic injury was maintained in the cortex of Tie2-hsEH mice. In the striatum, expression of Tie2-hsEH resulted in a sex difference, with larger infarct in females than males. These findings demonstrate that transgenic expression of sEH in endothelium results in impaired endothelium-dependent vasodilation in the cerebral circulation, and that females are more susceptible to enhanced ischemic damage as a result of increased endothelial sEH than males, especially in end-arteriolar striatal region.     

Epoxy Fatty Acids and Inhibition of the Soluble Epoxide Hydrolase Selectively Modulate GABA Mediated Neurotransmission to Delay Onset of Seizures

In the brain, seizures lead to release of large amounts of polyunsaturated fatty acids including arachidonic acid (ARA). ARA is a substrate for three major enzymatic routes of metabolism by cyclooxygenase, lipoxygenase and cytochrome P450 enzymes. These enzymes convert ARA to potent lipid mediators including prostanoids, leukotrienes and epoxyeicosatrienoic acids (EETs). The prostanoids and leukotrienes are largely pro-inflammatory molecules that sensitize neurons whereas EETs are anti-inflammatory and reduce the excitability of neurons. Recent evidence suggests a GABA-related mode of action potentially mediated by neurosteroids. Here we tested this hypothesis using models of chemically induced seizures. The level of EETs in the brain was modulated by inhibiting the soluble epoxide hydrolase (sEH), the major enzyme that metabolizes EETs to inactive molecules, by genetic deletion of sEH and by direct administration of EETs into the brain. All three approaches delayed onset of seizures instigated by GABA antagonists but not seizures through other mechanisms. Inhibition of neurosteroid synthesis by finasteride partially blocked the anticonvulsant effects of sEH inhibitors while the efficacy of an inactive dose of neurosteroid allopregnanolone was enhanced by sEH inhibition. Consistent with earlier findings, levels of prostanoids in the brain were elevated. In contrast, levels of bioactive EpFAs were decreased following seizures. Overall these results demonstrate that EETs are natural molecules which suppress the tonic component of seizure related excitability through modulating the GABA activity and that exploration of the EET mediated signaling in the brain could yield alternative approaches to treat convulsive disorders.     

Effects of Direct Renin Inhibition on Myocardial Fibrosis and Cardiac Fibroblast Function

Myocardial fibrosis, a major pathophysiologic substrate of heart failure with preserved ejection fraction (HFPEF), is modulated by multiple pathways including the renin-angiotensin system. Direct renin inhibition is a promising anti-fibrotic therapy since it attenuates the pro-fibrotic effects of renin in addition to that of other effectors of the renin-angiotensin cascade. Here we show that the oral renin inhibitor aliskiren has direct effects on collagen metabolism in cardiac fibroblasts and prevented myocardial collagen deposition in a non-hypertrophic mouse model of myocardial fibrosis. Adult mice were fed hyperhomocysteinemia-inducing diet to induce myocardial fibrosis and treated concomitantly with either vehicle or aliskiren for 12 weeks. Blood pressure and plasma angiotensin II levels were normal in control and hyperhomocysteinemic mice and reduced to levels lower than observed in the control group in the groups treated with aliskiren. Homocysteine-induced myocardial matrix gene expression and fibrosis were also prevented by aliskiren. In vitro studies using adult rat cardiac fibroblasts also showed that aliskiren attenuated the pro-fibrotic pattern of matrix gene and protein expression induced by D,L, homocysteine. Both in vivo and in vitro studies demonstrated that the Akt pathway was activated by homocysteine, and that treatment with aliskiren attenuated Akt activation. In conclusion, aliskiren as mono-therapy has potent and direct effects on myocardial matrix turnover and beneficial effects on diastolic function.     

Soluble Epoxide Hydrolase as an Anti-inflammatory Target of the Thrombolytic Stroke Drug SMTP-7

Although ischemic stroke is a major cause of death and disability worldwide, only a small fraction of patients benefit from the current thrombolytic therapy due to a risk of cerebral hemorrhage caused by inflammation. Thus, the development of a new strategy to combat inflammation during thrombolysis is an urgent demand. The small molecule thrombolytic SMTP-7 effectively treats ischemic stroke in several animal models with reducing cerebral hemorrhage. Here we revealed that SMTP-7 targeted soluble epoxide hydrolase (sEH) to suppress inflammation. SMTP-7 inhibited both of the two sEH enzyme activities: epoxide hydrolase (which inactivates anti-inflammatory epoxy-fatty acids) and lipid phosphate phosphatase. SMTP-7 suppressed epoxy-fatty acid hydrolysis in HepG2 cells in culture, implicating the sEH inhibition in the anti-inflammatory mechanism. The sEH inhibition by SMTP-7 was independent of its thrombolytic activity. The simultaneous targeting of thrombolysis and sEH by a single molecule is a promising strategy to revolutionize the current stroke therapy.     

Development of a High Throughput Cell-Based Assay for Soluble Epoxide Hydrolase Using BacMam Technology

Epoxyeicosatrienoic acids (EETs) play important protective functions in cardiovascular and renal systems. Under physiological conditions, EETs are quickly converted by the soluble epoxide hydrolase (sEH) to diols which do not have the beneficiary roles. Inhibition of sEH with small molecules to increase the concentration of EETs therefore provides an attractive therapeutic strategy for cardiovascular diseases. We describe here the development of a high throughput cell-based assay to measure sEH activity and screen small molecular compounds as sEH inhibitors. This assay is based on the technology of fluorescence polarization (FP), utilizing a Cy3B labeled 14,15-DHET ligand and a rabbit anti-14,15-DHET antibody. With the optimized assay, we measured the cellular sEH activity of several cell lines expressing endogenous sEH as well as sEH BacMam transduced HEK-293 cells. The inhibitory effect of several known sEH inhibitors was evaluated in sEH BacMam transduced HEK-293 cells. Our data show that there is good agreement of pIC₅₀ values obtained between the FP format and a commercially available ELISA kit. To our knowledge, this is the first report of a high throughput cell-based assay for screening sEH inhibitors.     

Pharmacological Inhibition of AMP-Deaminase in Rat Cardiac Myocytes

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC ₅₀ = 0.5 μ M. AMPDI was much less effective with isolated cardiomyocytes (IC ₅₀ = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies.     

环氧化物水解酶1基因（EPHX1）多态性与新生儿出生体重间关系分析

为研究母亲环氧化物水解酶１基因（ＥＰＨＸ１）多态性与新生儿出生体重间关系。收集了某纺织厂３４２名女工的资料,用ＰＣＲ－限制性片段长度多态性法分析母亲ＥＰＨＸ１基因型,采用多元线性逐步回归模型分析母亲ＥＰＨＸ１多态性与新生儿出生体重关系。结果发现调整主要混杂因素后ＥＰＨＸ１的Ｈｉｓ１３９Ａｒｇ／Ａｒｇ１３９Ａｒｇ基因型与新生儿出生体重有显著性相关（β±ＳＥ＝－１４９ｇ ± ５６,Ｐ＝— ０．００８３）。经被动吸烟分层分析,结果显示：仅在有被动吸烟史人群中ＥＰＨＸ１的Ｈｉｓ１３９Ａｒｇ／Ａｒｇ１３９Ａｒｇ基因型与新生儿出生体重有显著性相关,（β±ＳＥ＝－ ２３４ｇ ± ８８,Ｐ＝ ０．００８８）;而根据工作紧张程度分层分析后发现,仅在有工作紧张史人群中发现ＥＰＨＸ１的Ｈｉｓ１３９Ａｒｇ／Ａｒｇ１３９Ａｒｇ基因型与新生儿出生体重有显著性相关（β±ＳＥ＝－ １５７ｇ ±ｔ ５９, Ｐ＝ ０．００７９）。我们的结果显示母亲 ＥＰＨＸ１多态性与新生儿出生体重有显著性相关,并存在基因－环境的交互作用。     

环氧化物水解酶1基因(EPHXl)多态性与新生儿出生体重间关系分析

为研究母亲环氧化物水解酶1基因(EPHXl)多态性与新生儿出生体重间关系.收集了某纺织厂342名女工的资料,用PCR-限制性片段长度多态性法分析母亲EPHXl基因型,采用多元线性逐步回归模型分析母亲EPHXl多态性与新生儿出生体重关系.结果发现调整主要混杂因素后EPHXl的His139Arg/AG139Arg基因型与新生儿出生体重有显著性相关(β±SE＝-149g±56,P＝0.0083).经被动吸烟分层分析,结果显示仅在有被动吸烟史人群中EPHXl的Hus139Arg/Arg139Arg基因型与新生儿出生体重有显著性相关,(β±SE=-234g±88,P＝0.0088);而根据工作紧张程度分层分析后发现,仅在有工作紧张史人群中发现EPHXI的His139Arg/Arg139Arg基因型与新生儿出生体重有显著性相关(β±SE＝-157g±59,P＝0.0079).我们的结果显示母亲EPHXl多态性与新生儿出生体重有显著性相关,并存在基因-环境的交互作用.     

Colorimetric Assays for Quantitative Analysis and Screening of Epoxide Hydrolase Activity

Focusing on directed evolution to tailor enzymes as usable biocatalysts for fine chemistry, we have studied in detail several colorimetric assays for quantitative analysis of epoxide hydrolase (EH) activity. In particular, two assays have been optimized to characterize variants issued from the directed evolution of the EH from Aspergillus niger. Assays described in this paper are sufficiently reliable for quantitative screening of EH activity in microtiter plates and are low cost alternatives to GC or MS analysis. Moreover, they are usable for various epoxides and not restricted to a type of substrate, such as those amenable to assay by UV absorbancy. They can be used to assay EH activity on any epoxide and to directly assay enantioselectivity when both (R) and (S) substrates are available. The advantages and drawbacks of these two methods to assay EH activity of a large number of natural samples are summarized.     

Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor

Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other α/β hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-Å resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of α/β hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif''s effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.Pseudomonas aeruginosa is a Gram-negative bacterium that acts as an opportunistic pathogen. In colonizing the urinary tract, eye, and lung, as well as the surfaces of implanted medical devices, it forms antibiotic-resistant biofilms (12). In nosocomial infections, such as ventilator-associated pneumonia, P. aeruginosa is the second most common bacterial agent, and it represents the leading cause of death due to hospital-acquired infection (1). Among patients with compromised pulmonary function, P. aeruginosa frequently establishes persistent lung infections, exacerbating outcomes in chronic obstructive pulmonary disease (42) and cystic fibrosis (30). Overall, nearly 80% of patients with cystic fibrosis have a chronic P. aeruginosa infection in the lung by age 18 (22). Preventing infection by limiting exposure to the pathogen is difficult due to its ubiquitous distribution in the environment (47). On the other hand, the treatment of chronic lung infections is similarly challenging due to the formation of antibiotic-resistant biofilms. As a result, there currently is no effective treatment to eradicate a chronic P. aeruginosa infection from the lung once established (15, 54).P. aeruginosa secretes a multitude of virulence factors that assist the bacterium during the initial process of airway colonization and biofilm formation (32), in some instances acting directly on host cells. In particular, it was shown recently in a coculture model that the presence of P. aeruginosa causes a decrease in the quantity of cystic fibrosis transmembrane conductance regulator (CFTR) at the apical membrane of human airway epithelial cells (49). CFTR is the ion channel responsible for chloride secretion into the airway surface liquid (ASL) in the lung. The removal of CFTR from the cell surface leads to reduced chloride efflux, ASL dehydration, and decreased mucociliary clearance, thus facilitating the establishment of a bridgehead for bacterial infection.The downregulation of plasma membrane CFTR is mediated by a single secreted protein, the CFTR inhibitory factor (Cif), which is encoded at the PA14_26090 or cif locus (37) and is delivered into the host cell by outer membrane vesicles (5). The CFTR inhibitory effect also can be replicated by the application of purified, recombinant Cif protein directly to the apical surface of human airway epithelial cells. Within an hour after treatment with Cif, the levels of CFTR in the apical membrane are significantly reduced (37). While the mechanism of Cif action is incompletely understood, Cif has been shown to inhibit the recycling of CFTR to the apical membrane following endocytic uptake (49). Additional work has shown that Cif treatment causes a similar effect on some ABC transporters while having no effect on others (56). The mechanism by which Cif is able to generate this selectivity currently is unknown.Based on sequence comparisons, Cif was predicted to belong to the α/β hydrolase family (37), which contains several different classes of enzymes with closely related sequences. Specifically, Cif showed the greatest degree of sequence similarity to the class of epoxide hydrolases (EHs), which catalyze the conversion of epoxide moieties to vicinal diols (Fig. ​(Fig.1).1). The EHs are conserved between bacteria and mammals and are used to detoxify products of oxidative metabolism as well as xenobiotic compounds. In mammals, they metabolize potent chemical signal mediators (9). As a family, they also are of potential biocatalytic interest (46). EHs have not previously been reported as bacterial virulence factors, but Pseudomonas is adept at exploiting a wide variety of biochemical strategies to subvert host cell functions.Open in a separate windowFIG. 1.Epoxide hydrolase activity. The EH class of enzymes is responsible for the catalytic addition of a water molecule to an epoxide ring, creating a vicinal diol.Preliminary mutagenesis experiments targeting Cif suggested a link between EH activity and host cell effects, but these studies relied on low-identity sequence alignments and activity assays performed with an artificial EH substrate (37) that also is susceptible to esterase activity (18). Sequence alignment of Cif with known EHs revealed substitutions in several conserved EH motifs thought to be required for the formation of the enzyme active site. Furthermore, sequence relationships suggest that several haloacetate dehalogenases (HADs) previously had been misclassified as EHs, all of which cluster in the EH subgroup that includes Cif (52). As a basis for a detailed structure-function analysis of Cif, we have determined its crystal structure and assayed its activity against a variety of candidate substrates for EHs and related α/β hydrolases both for wild-type protein and for a mutation that abrogates Cif''s host cell activity.     

Potent Natural Soluble Epoxide Hydrolase Inhibitors from Pentadiplandra brazzeana Baillon: Synthesis,Quantification, and Measurement of Biological Activities In Vitro and In Vivo

We describe here three urea-based soluble epoxide hydrolase (sEH) inhibitors from the root of the plant Pentadiplandra brazzeana. The concentration of these ureas in the root was quantified by LC-MS/MS, showing that 1, 3-bis (4-methoxybenzyl) urea (MMU) is the most abundant (42.3 μg/g dry root weight). All of the ureas were chemically synthesized, and their inhibitory activity toward recombinant human and recombinant rat sEH was measured. The most potent compound, MMU, showed an IC₅₀ of 92 nM via fluorescent assay and a Ki of 54 nM via radioactivity-based assay on human sEH. MMU effectively reduced inflammatory pain in a rat nociceptive pain assay. These compounds are among the most potent sEH inhibitors derived from natural sources. Moreover, inhibition of sEH by these compounds may mechanistically explain some of the therapeutic effects of P. brazzeana.     

Pharmacological Characterization of Endocannabinoid Transport and Fatty Acid Amide Hydrolase Inhibitors

  1. The mechanism of anandamide uptake and disposal has been an issue of considerable debate in the cannabinoid field. Several compounds have been reported to inhibit anandamide uptake or fatty acid amide hydrolase (FAAH; the primary catabolic enzyme of anandamide) activity with varying degrees of potency and selectivity. We recently reported the first evidence of a binding site involved in the uptake of endocannabinoids that is independent from FAAH. There are no direct comparisons of purported selective inhibitory compounds in common assay conditions measuring anandamide uptake, FAAH activity and binding activity.2. A subset of compounds reported in the literature were tested in our laboratory under common assay conditions to measure their ability to (a) inhibit [¹⁴C]-anandamide uptake in cells containing (RBL-2H3) or cells lacking (HeLa) FAAH, (b) inhibit purified FAAH hydrolytic activity, and (c) inhibit binding to a putative binding site involved in endocannabinoid transport in both RBL and HeLa cell membranes.3. Under these conditions, nearly all compounds tested inhibited (a) uptake of [¹⁴C]-anandamide, (b) enzyme activity in purified FAAH preparations, and (c) radioligand binding of [³H]-LY2183240 in RBL and HeLa plasma membrane preparations. General rank order potency was preserved within the three assays. However, concentration response curves were right-shifted for functional [¹⁴C]-anandamide uptake in HeLa (FAAH^−/−) cells.4. A more direct comparison of multiple inhibitors could be made in these three assay systems performed in the same laboratory, revealing more information about the selectivity of these compounds and the relationship between the putative endocannabinoid transport protein and FAAH. At least two separate proteins appear to be involved in uptake and degradation of anandamide. The most potent inhibitory compounds were right-shifted when transport was measured in HeLa (FAAH^−/−) cells suggesting a requirement for a direct interaction with the FAAH protein to maintain high affinity binding of anandamide or inhibitors to the putative anandamide transport protein.     

Deficiency of Ataxia Telangiectasia Mutated Kinase Modulates Cardiac Remodeling Following Myocardial Infarction: Involvement in Fibrosis and Apoptosis

Ataxia telangiectasia mutated kinase (ATM) is a cell cycle checkpoint protein activated in response to DNA damage. We recently reported that ATM plays a protective role in myocardial remodeling following β-adrenergic receptor stimulation. Here we investigated the role of ATM in cardiac remodeling using myocardial infarction (MI) as a model. Methods and Results: Left ventricular (LV) structure, function, apoptosis, fibrosis, and protein levels of apoptosis- and fibrosis-related proteins were examined in wild-type (WT) and ATM heterozygous knockout (hKO) mice 7 days post-MI. Infarct sizes were similar in both MI groups. However, infarct thickness was higher in hKO-MI group. Two dimensional M-mode echocardiography revealed decreased percent fractional shortening (%FS) and ejection fraction (EF) in both MI groups when compared to their respective sham groups. However, the decrease in %FS and EF was significantly greater in WT-MI vs hKO-MI. LV end systolic and diastolic diameters were greater in WT-MI vs hKO-MI. Fibrosis, apoptosis, and α-smooth muscle actin staining was significantly higher in hKO-MI vs WT-MI. MMP-2 protein levels and activity were increased to a similar extent in the infarct regions of both groups. MMP-9 protein levels were increased in the non-infarct region of WT-MI vs WT-sham. MMP-9 protein levels and activity were significantly lower in the infarct region of WT vs hKO. TIMP-2 protein levels similarly increased in both MI groups, whereas TIMP-4 protein levels were significantly lower in the infarct region of hKO group. Phosphorylation of p53 protein was higher, while protein levels of manganese superoxide dismutase were significantly lower in the infarct region of hKO vs WT. In vitro, inhibition of ATM using KU-55933 increased oxidative stress and apoptosis in cardiac myocytes.     

Effect of Interleukin 6 Deficiency on Renal Interstitial Fibrosis

Our recent studies have shown that bone marrow-derived fibroblast precursors contribute significantly to the pathogenesis of renal fibrosis. However, the molecular mechanisms underlying the recruitment and activation of bone marrow-derived fibroblast precursors are incompletely understood. We found that interleukin 6 was induced in the kidney in a murine model of renal fibrosis induced by unilateral ureteral obstruction. Therefore, we investigated if interleukin 6 play a role in the recruitment and maturation of bone marrow-derived fibroblast precursors in the kidney during the development of renal fibrosis. Wild-type and interleukin 6 knockout mice were subjected to unilateral obstructive injury for up to two weeks. Interleukin 6 knockout mice accumulated similar number of bone marrow-derived fibroblast precursors and myofibroblasts in the kidney in response to obstructive injury compared to wild-type mice. Furthermore, IL-6 knockout mice expressed comparable α-SMA in the obstructed kidney compared to wild-type mice. Moreover, targeted disruption of Interleukin 6 did not affect gene expression of profibrotic chemokine and cytokines in the obstructed kidney. Finally, there were no significant differences in renal interstitial fibrosis or expression of extracellular matrix proteins between wild-type and interleukin 6 knockout mice following obstructive injury. Our results indicate that interleukin 6 does not play a significant role in the recruitment of bone marrow-derived fibroblast precursors and the development of renal fibrosis.