n-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide and Hypoxia

Transient receptor potential vanilloid 4 (TRPV4) is a multi-functional non-selective channel expressed in pulmonary vasculatures. TRPV4 contributes to serotonin- (5-HT-) induced pulmonary vasoconstriction and is responsible in part for the enhanced 5-HT response in pulmonary arteries (PAs) of chronic hypoxia mice. Epoxyeicosatrienoic acid (EET) is an endogenous agonist of TRPV4 and is known to regulate vasoreactivity. The levels of EETs, the expression of cytochrome P450 (CYP) epoxygenase for EET production, and epoxide hydrolase for EET degradation are altered by chronic hypoxia. Here, we examined the role of EET-dependent TRPV4 activation in the 5-HT-mediated PA contraction. In PAs of normoxic mice, inhibition of TRPV4 with a specific inhibitor HC-067047 caused a decrease in the sensitivity of 5-HT-induced PA contraction without affecting the maximal contractile response. Application of the cytochrome P450 epoxygenase inhibitor MS-PPOH had no effect on the vasoreactivity to 5-HT. In contrast, inhibition of CYP epoxygenase or TRPV4 both attenuated the 5-HT-elicited maximal contraction to a comparable level in PAs of chronic hypoxic mice. Moreover, the inhibitory effect of MS-PPOH on the 5-HT-induced contraction was obliterated in PAs of chronic hypoxic

Topics: Amides; Animals; Chronic Disease; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Eicosanoids; Hypertension, Pulmonary; Hypoxia; Ion Channel Gating; Male; Mice, Inbred C57BL; Morpholines; Pulmonary Artery; Pyrroles; Serotonin; TRPV Cation Channels; Vasoconstriction

Short, nonlethal ischemic episodes administered to hearts directly after ischemic events (ischemic postconditioning, IPost) have an advantage over ischemic preconditioning (IPC). The endogenous cytochrome P450 2J3/11,12-epoxyeicosatrienoic acid (CYP2J3/11,12-EET) is upregulated by IPost, but not IPC, in the rat heart. The CYP epoxygenase inhibitor N-methylsulphonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH) reduces the cardioprotective effects of IPost, but not IPC. We proposed that upregulation of CYP2J3/11,12-EET during IPost induces cardioprotection by inhibiting cardiomyocyte apoptosis and that multiple apoptotic signals, including changes in mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) opening, mitochondrial cytochrome c leakage, caspase-3 levels, and levels of protective kinases such as Bcl-2 and Bax, are involved in the process. Neonatal rat cardiomyocytes underwent 3-h hypoxia followed by 2-, 5-, or 6-h reoxygenation (H/R) or three cycles of 5-min reoxygenation followed by 5-min hypoxia before 90-min reoxygenation (HPost); or were transfected with pcDNA3.1-CYP2J3 for 48 h before H/R; or were treated with MS-PPOH for 10 min before HPost. For HPost alone, pcDNA3.1-CYP2J3 transfection attenuated cardiomyocyte apoptosis to 68.4% (p<0.05) of that with H/R. pcDNA3.1-CYP2J3 transfection significantly decreased MMP and inhibited mPTP opening induced by H/R, reduced mitochondrial cytochrome c leakage, cleaved caspase-3 protein expression, and increased the ratio of Bcl-2 to Bax expression. MS-PPOH abolished this effect. Therefore, upregulation of CYP2J3/11,12-EET during HPost is involved in cardioprotection by inhibiting apoptosis via a caspase-dependent pathway, and the apoptosis-suppressive effect may have important clinical implications during HPost.

Topics: 8,11,14-Eicosatrienoic Acid; Amides; Animals; Animals, Newborn; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Survival; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Cytochromes c; Hypoxia; Membrane Potential, Mitochondrial; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocytes, Cardiac; Oxygen; Rats; Rats, Wistar; Up-Regulation

We assessed pulmonary cytochrome P450 (CYP) epoxygenase expression and activity during hypoxia and explored the effects of modulating epoxygenase activity on pulmonary hypertension. The acute hypoxic vasoconstrictor response was studied in Swiss Webster mice, who express CYP2C29 in their lungs. Animals were pretreated with vehicle, the epoxygenase inhibitor (N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide) or an inhibitor of the soluble epoxide hydrolase. Whereas the epoxygenase inhibitor attenuated hypoxic pulmonary constriction (by 52%), the soluble epoxide hydrolase inhibitor enhanced the response (by 39%), indicating that CYP epoxygenase-derived epoxyeicosatrienoic acids elicit pulmonary vasoconstriction. Aerosol gene transfer of recombinant adenovirus containing the human CYP2C9 significantly elevated mean pulmonary artery pressure and total pulmonary resistance indices, both of which were sensitive to the inhibitor sulfaphenazole. The prolonged exposure of mice to hypoxia increased CYP2C29 expression, and transcript levels increased 5-fold after exposure to normobaric hypoxia (FIO2 0.07) for 2 hours. This was followed by a 2-fold increase in protein expression and by a significant increase in epoxyeicosatrienoic acid production after 24 hours. Chronic hypoxia (7 days) elicited pulmonary hypertension and pulmonary vascular remodeling, effects that were significantly attenuated in animals continually treated with N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide (-46% and -55%, respectively). Our results indicate that endogenously generated epoxygenase products are associated with hypoxic pulmonary hypertension in mice and that selective epoxygenase inhibition significantly reduces acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary vascular remodeling. These observations indicate potential novel targets for the treatment of pulmonary hypertension and highlight a pivotal role for CYP epoxygenases in pulmonary responses to hypoxia.

Topics: Adenoviridae; Amides; Animals; Aryl Hydrocarbon Hydroxylases; Blood Vessels; Chronic Disease; Cytochrome P-450 CYP2C9; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 2; Gene Transfer Techniques; Genetic Vectors; Hemodynamics; Humans; Hypertension, Pulmonary; Hypoxia; Lung; Mice; Oxygenases; Vasoconstriction