A novel activity of microsomal epoxide hydrolase: metabolism of the endocannabinoid 2-arachidonoylglycerol.

Microsomal epoxide hydrolase (EPHX1, EC 3.3.2.9) is a highly abundant α/ß-hydrolase enzyme that is known for its catalytical epoxide hydrolase activity. A wide range of EPHX1 functions have been demonstrated including xenobiotic metabolism; however, characterization of its endogenous substrates is limited. In this study, we present evidence that EPHX1 metabolizes the abundant endocannabinoid 2-arachidonoylglycerol (2-AG) to free arachidonic acid (AA) and glycerol. The EPHX1 metabolism of 2-AG was demonstrated using commercially available EPHX1 microsomes as well as PC-3 cells overexpressing EPHX1. Conversely, EPHX1 siRNA markedly reduced the EPHX1 expression and 2-AG metabolism in HepG2 cells and LNCaP cells. A selective EPHX1 inhibitor, 10-hydroxystearamide, inhibited 2-AG metabolism and hydrolysis of a well-known EPHX1 substrate, cis-stilbene oxide. Among the inhibitors studied, a serine hydrolase inhibitor, methoxy-arachidonyl fluorophosphate, was the most potent inhibitor of 2-AG metabolism by EPHX1 microsomes. These results demonstrate that 2-AG is an endogenous substrate for EPHX1, a potential role of EPHX1 in the endocannabinoid signaling and a new AA biosynthetic pathway.

Roles of endothelial nitric oxide synthase (eNOS) and mitochondrial permeability transition pore (MPTP) in epoxyeicosatrienoic acid (EET)-induced cardioprotection against infarction in intact rat hearts.

We previously demonstrated that 11,12 and 14,15-epoxeicosatrienoic acids (EETs) produce cardioprotection against ischemia-reperfusion injury in dogs and rats. Several signaling mechanisms have been implicated in the cardioprotective actions of the EETs; however, their mechanisms remain largely elusive. Since nitric oxide (NO) plays a significant role in cardioprotection and EETs have been demonstrated to induce NO production in various tissues, we hypothesized that NO is involved in mediating the EET actions in cardioprotection. To test this hypothesis, we used an in vivo rat model of infarction in which intact rat hearts were subjected to 30-min occlusion of the left coronary artery and 2-hr reperfusion. 11,12-EET or 14,15-EET (2.5mg/kg) administered 10min prior to the occlusion reduced infarct size, expressed as a percentage of the AAR (IS/AAR), from 63.9±0.8% (control) to 45.3±1.2% and 45.5±1.7%, respectively. A nonselective nitric oxide synthase (NOS) inhibitor, L-NAME (1.0mg/kg) or a selective endothelial NOS inhibitor, L-NIO (0.30mg/kg) alone did not affect IS/AAR but they completely abolished the cardioprotective effects of the EETs. On the other hand, a selective neuronal NOS inhibitor, nNOS I (0.03mg/kg) and a selective inducible NOS inhibitor, 1400W (0.10mg/kg) did not affect IS/AAR or block the cardioprotective effects of the EETs. Administration of 11,12-EET (2.5mg/kg) to the rats also transiently increased the plasma NO concentration. 14,15-EET (10µM) induced the phosphorylation of eNOS (Ser(1177)) as well as a transient increase of NO production in rat cardiomyoblast cell line (H9c2 cells). When 11,12-EET or 14,15-EET was administered at 5min prior to reperfusion, infarct size was also reduced to 42.8±2.2% and 42.6±1.9%, respectively. Interestingly, L-NAME (1.0mg/kg) and a mitochondrial KATP channel blocker, 5-HD (10mg/kg) did not abolish while a sarcolemmal KATP channel blocker, HMR 1098 (6.0mg/kg) and a mitochondrial permeability transition pore (MPTP) opener, atractyloside (5.0mg/kg) completely abolished the cardioprotection produced by the EETs. 14,15-EET (1.5mg/kg) with an inhibitor of MPTP opening, cyclosporin A (CsA, 1.0mg/kg) produced a greater reduction of infarct size than their individual administration. Conversely, an EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 2.5mg/kg) completely abolished the cardioprotective effects of CsA, suggesting a role of MPTP in mediating the EET actions. Taken together, these results suggest that the cardioprotective effects of the EETs in an acute ischemia-reperfusion model are mediated by distinct mediators depending on the time of EET administration. The cardioprotective effects of EETs administered prior to ischemia were regulated by the activation of eNOS and increased NO production, while sarcKATP channels and MPTP were involved in the beneficial effects of the EETs when administered just prior to reperfusion.

Acute and chronic cardioprotection by the enkephalin analogue, Eribis peptide 94, is mediated via activation of nitric oxide synthase and adenosine triphosphate-regulated potassium channels.

BACKGROUND/AIMS: Eribis peptide 94 (EP 94) is a new enkephalin derivative which potently binds to the µ- and δ-opioid receptor. In this study, we determined the effects of EP 94 and potential mechanism(s) involved in cardioprotection of the rat heart. METHODS AND RESULTS: An acute (5 and10 min into ischemia) and a chronic (24 h prior to ischemia) EP 94 administration produced a similar 30-40% reduction in infarct size/area at risk and the effects were blocked by the K(ATP) channel antagonists, HMR 1098 and 5-HD. The cardioprotective effects were blocked by a nonselective nitric oxide synthase (NOS) inhibitor (L-NAME) following acute administration and by a selective iNOS inhibitor (1400W) following chronic administration. CONCLUSION: These results suggest that EP 94 may have potential for the treatment of ischemic heart disease via a nitric oxide (NO)-K(ATP)-mediated mechanism.

Cannabinoid receptor type 1 (CB1) activation inhibits small GTPase RhoA activity and regulates motility of prostate carcinoma cells.

The cannabinoid receptor type 1 (CB1) is a G protein-coupled receptor that is activated in an autocrine fashion by the endocannabinoids (EC), N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG). The CB1 and its endogenous and synthetic agonists are emerging as therapeutic targets in several cancers due to their ability to suppress carcinoma cell invasion and migration. However, the mechanisms that the CB1 regulates cell motility are not well understood. In this study, we examined the molecular mechanisms that diminish cell migration upon the CB1 activation in prostate carcinoma cells. The CB1 activation with the agonist WIN55212 significantly diminishes the small GTPase RhoA activity but modestly increases the Rac1 and Cdc42 activity. The diminished RhoA activity is accompanied by the loss of actin/myosin microfilaments, cell spreading, and cell migration. Interestingly, the CB1 inactivation with the selective CB1 antagonist AM251 significantly increases RhoA activity, enhances microfilament formation and cell spreading, and promotes cell migration. This finding suggests that endogenously produced EC activate the CB1, resulting in chronic repression of RhoA activity and cell migration. Consistent with this possibility, RhoA activity is significantly diminished by the exogenous application of AEA but not by 2-AG in PC-3 cells (cells with very low AEA hydrolysis). Pretreatment of cells with a monoacylglycerol lipase inhibitor, JZL184, which blocks 2-AG hydrolysis, decreases the RhoA activity. These results indicate the unique CB1 signaling and support the model that EC, through their autocrine activation of CB1 and subsequent repression of RhoA activity, suppress migration in prostate carcinoma cells.