Thelephoric acid is an antioxidant produced by the hydrolysis of polyozellin, which is isolated from Polyozellus multiplex. In the present study, the inhibitory effects of polyozellin and thelephoric acid on 9 cytochrome P450 (CYP) family members (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were examined in pooled human liver microsomes (HLMs) using a cocktail probe assay. Polyozellin exhibited weak inhibitory effects on the activities of all 9 CYPs examined, whereas thelephoric acid exhibited dose- and time-dependent inhibition of all 9 CYP isoforms (IC50 values, 3.2-33.7 muM). Dixon plots of CYP inhibition indicated that thelephoric acid was a competitive inhibitor of CYP1A2 and CYP3A4. In contrast, thelephoric acid was a noncompetitive inhibitor of CYP2D6. Our findings indicate that thelephoric acid may be a novel, non-specific CYP inhibitor, suggesting that it could replace SKF-525A in inhibitory studies designed to investigate the effects of CYP enzymes on the metabolism of given compounds.
Cytochrome P-450 CYP1A2 ; Cytochrome P-450 CYP2D6 ; Cytochrome P-450 CYP2E1 ; Cytochrome P-450 Enzyme System* ; Humans ; Hydrolysis ; Metabolism ; Microsomes, Liver ; Proadifen* ; Protein Isoforms

We have reported that hypoxia stimulates EDRF(s) release from endothelial cells and the release may be augmented by previous hypoxia. As a mechanism, it was hypothesized that reoxygenation can stimulate EDRF(s) release from endothelial cells and we tested the hypothesis via bioassay experiment. In the bioassay experiment, rabbit aorta with endothelium was used as EDRF donor vessel and rabbit carotid artery without endothelium as a bioassay test ring. The test ring was contracted by prostaglandin F2a (3 X 10-6 M) which was added to the solution perfusing through the aorta. Hypoxia was evoked by switching the solution aerated with 95% O2/5% CO2 mixed gas to one aerated with 95% N2/5% CO2 mixed gas Hypoxia/reoxygenation were interexchanged at intervals of 2 minutes (intermittent hypoxia). In some experiments, endothelial cells were exposed to 10-minute hypoxia (continuous hypoxia) and then exposed to reoxygenation and intermittent hypoxia. In other experiments, the duration of re oxygenation was extended from 2 minutes to 5 minutes. When the donor aorta was exposed to intermittent hypoxia, hypoxia stimulated EDRF(s) release from endothelial cells and the hypoxia-induced EDRF(s) release was augmented by previous hypoxia/reoxygenation. When the donor aorta was exposed to continuous hypoxia, there was no increase of hypoxia-induced EDRF(s) release during hypoxia. But, after the donor aorta was exposed to reoxygenation, hypoxia-induced EDRF(s) release was markedly increased. When the donor aorta was pretreated with nitro-L-arginine (10-5 M for 30 minutes), the initial hypoxia-induced EDRF(s) release was almost completely abolished, but the mechanism for EDRF(s) release by the reoxygenation and subsequent hypoxia still remained to be clarified. TEA also blocked incompletely hypoxia-induced and hypoxia/reoxygenation-induced EDRF(s) release EDRF(s) release by repetitive hypoxia and reoxygenation was completely blocked by the combined treatment with nitro-L-arginine and TEA. Cytochrome P450 blocker, SKF-525A, inhibited the EDRF(s) release reversibly and endothelin antgonists, BQ 123 and BQ 788, had no effect on the release of endothelium-derived vasoactive factors. Superoxide dismutase (SOD) and catalase inhibited the EDRF(s) release from endothelial cells. From these data, it could be concluded that reoxygenation stimulates EDRF(s) release and hypoxia/reoxygenation can release not only NO but also another EDRF from endothelial cells by the production of oxygen free radicals.
Anoxia ; Aorta* ; Biological Assay ; Carotid Arteries ; Catalase ; Cytochrome P-450 Enzyme System ; Endothelial Cells* ; Endothelins ; Endothelium ; Free Radicals ; Humans ; Oxygen ; Proadifen ; Superoxide Dismutase ; Tea ; Tissue Donors

AIMTo investigate the role and mechanism of endothelium-derived hyperpolarizing factor (EDHF) in shear stress induced vasorelaxation of rat mesenteric artery.

METHODSThe changes in vessel diameter in response to variable flow (0-300 microL.min(-1)) were continuously examined. The contribution of prostacyclin (PGI2), NO and EDHF to shear stress induced relaxation were analyzed by inhibitory effects of indomethacin, N(G)-nitro-L-arginine (L-NA) and KCl. The nature and hyperpolarizing mechanism of EDHF were examined by the inhibitory effects of inhibitors of cytochrome P450 pathway and of various K+ channels.

RESULTSThe shear stress-induced relaxation were endothelium dependent and the contribution of NO was more prominent in large mesenteric arteries (400-500 microm) than that in resistance arteries (150-250 microm), whereas that of EDHF was noted in both-sized blood vessels. Tetrabutylammonium (a nonselective inhibitor of K channels) almost abolished, whereas the combination of charybdotoxin (an inhibitor of both large and intermediate-conductance Ca2+-activated K channels) and apamin (an inhibitor of small-conductance Ca2+-activated K channels) significantly inhibited the EDHF-mediated component of the shear stress-induced relaxations.

CONCLUSIONEDHF plays an important role in shear stress-induced endothelium-dependent relaxations, and K channels especially calcium-activated K channels appear to be involved.

Animals ; Apamin ; pharmacology ; Biological Factors ; physiology ; Charybdotoxin ; pharmacology ; Cytochrome P-450 Enzyme Inhibitors ; Endothelium, Vascular ; drug effects ; physiology ; In Vitro Techniques ; Large-Conductance Calcium-Activated Potassium Channels ; antagonists & inhibitors ; Male ; Mesenteric Arteries ; drug effects ; physiology ; Nitric Oxide ; physiology ; Potassium Channel Blockers ; pharmacology ; Proadifen ; pharmacology ; Quaternary Ammonium Compounds ; pharmacology ; Rats ; Rats, Wistar ; Small-Conductance Calcium-Activated Potassium Channels ; antagonists & inhibitors ; Vasodilation ; drug effects

OBJECTIVETo investigate the mechanism of enhanced large conductance calcium-activated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA).

METHODSCoronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16, 17-epoxydocosapentaenoic acid (16, 17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration.

RESULTSBK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2 ± 2.7)% of total potassium currents (n = 20). DHA could activate BK channels, and its 50% effective concentration (EC(50)) was (0.23 ± 0.03) µmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16, 17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC(50) was (19.7 ± 2.8) nmol/L.

CONCLUSIONDHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.

Animals ; Coronary Vessels ; cytology ; drug effects ; metabolism ; Cytochrome P-450 Enzyme Inhibitors ; Docosahexaenoic Acids ; pharmacology ; Fatty Acids, Unsaturated ; pharmacology ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Proadifen ; pharmacology ; Rats ; Rats, Sprague-Dawley