Degradation of Benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.     

Formation of the 11,12-diol as a metabolite of benzo(a)pyrene by rat skin in vivo

The dihydrodiols present as metabolites in rat skin after topical application of ³H-labelled benzo(a)pyrene included a significant amount of radioactivity that cochromatographed with synthetic $\text{trans}$-11,12-dihydro-11,12-dihydroxybenzo(a)pyrene. Treatment of the radioactive metabolite with hot mineral acid gave a product that had chromatographic properties identical to those of the phenol similarly formed from the synthetic dihydrodiol and acetylation of the metabolite yielded a product that cochromatographed with the diacetate of the synthetic dihydrodiol. These observations show that the 11,12-dihydrodiol is formed as a metabolite of BP in rat skin $\text{in vivo}$. The metabolite was not detected in mouse skin.     

Effects of tensile stress on the α1 nicotinic acetylcholine receptor expression in maxillofacial skeletal myocytes

The present study was to test the hypothesis that 11,12-epoxyeicosatrienoic acid (11,12-EET), a metabolic product of arachidonic acid by cytochrome P450 epoxygenase, regulates nitric oxide (NO) generation of the l-arginine/NO synthase (NOS) pathway in human platelets. Human platelets were incubated in the presence or absence of different concentrations of 11,12-EET for 2 h at 37°C, followed by measurements of activities of the l-arginine/NOS pathway. Incubation with 11,12-EET increased the platelet NOS activity, nitrite production, cGMP content, and the platelet uptake of l-[³H]arginine in a concentration-dependent manner. In addition, 11,12-EET attenuated intracellular free Ca²⁺ accumulation stimulated by collagen, which was at least partly mediated by EET-activated l-arginine/NOS pathway. It is suggested that 11,12-EET regulates platelet function through up-regulating the activity of the l-arginine/NOS/NO pathway.     

Eudesmane alcohols from jasonia glutinosa

Three new sesquiterpene alcohols have been isolated from Jasonia glutinosa. Their structures were elucidated by spectroscopic methods and chemical correlations as (?)-[11R]-4α,14-epoxyeudesm-11,12-diol, (?)-[11R]-eudesm-4(14)-en-5β,11,12-triol and (+)-[11R]-eudesm-4(14)-en-5α,11,12-triol and they are called α-epoxy kudtdiol, 5-epi-kudtriol and kudtriol respectively.     

The metabolism of 3-methylcholanthrene and some related compounds by rat-liver homogenates

1. A chromatographic investigation of the products of the metabolism of 3-methylcholanthrene by rat-liver homogenates showed the formation of compounds with the properties of 1- and 2-hydroxy-3-methylcholanthrene, cis- and trans-1,2-dihydroxy-3-methylcholanthrene and 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene. A glutathione conjugate that is probably S-(11,12-dihydro-12-hydroxy-3-methyl-11-cholanthrenyl)glutathione was also detected. 3-Methylcholanthrene-1- and -2-one and -1,2-quinone were also present, but these products may have arisen by the chemical oxidation of the corresponding hydroxy compounds. 2. Other metabolic products were tentatively identified as 9- and 10-hydroxy-3-methylcholanthrene, 4,5-dihydro-4,5-dihydroxy-3-methylcholanthrene and 3-hydroxymethylcholanthrene. 3. 1- and 2-Hydroxy-3-methylcholanthrene were converted by homogenates into the related ketones and into products with the properties of cis- and trans-1,2-dihydroxy-3-methylcholanthrene: 3-methylcholanthren-1- and -2-one were converted into their related hydroxy compounds and into the isomeric 1,2-dihydroxy compounds. The isomeric 1,2-dihydroxy compounds were each partly converted into the other isomer by these homogenates. All the above substrates also yielded products that appeared to be derivatives of 3-hydroxymethylcholanthrene. 4. 3-Methylcholanthrylene was converted by rat-liver homogenates into products with the properties of trans-1,2-dihydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene and 3-methylcholanthren-2-one. A small amount of the cis-1,2-dihydroxy compound was also formed, together with a glutathione conjugate that is possibly S-(2-hydroxy-3-methyl-1-cholanthrenyl)glutathione or its positional isomer. 5. An unidentified product was detected in the metabolism of 3-methylcholanthrene, the monohydroxy compounds, the ketones and the dihydroxy compounds, the formation of which appeared to involve metabolism at the 1,2-bond. 6. 11,12-Epoxy-11,12-dihydro-3-methylcholanthrene was converted by rat-liver homogenates into products with the properties of 11-hydroxy-3-methylcholanthrene (or, less likely, the 12-isomer), 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene and the glutathione conjugate described above. Products with the properties of these compounds were formed when the epoxide was allowed to react with glutathione in an aqueous medium. 7. Mouse-liver homogenate converted 3-methylcholanthrene into products with the chromatographic properties of 1- and 2-hydroxy-3-methylcholanthrene, cis- and trans-1,2-dihydroxy-3-methylcholanthrene, 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene, 3-methylcholanthrene-1- and -2-one and -1,2-quinone and the unidentified hydroxy-3-methylcholanthrenes. 8. The syntheses of cis- and trans-1,2-dihydroxy-3-methylcholanthrene, 3-methylcholanthren-2-one, 2-hydroxy-3-methylcholanthrene, 3-methylcholanthrylene, 11,12-epoxy-11,12-dihydro-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene are described.     

Novel bioconversion products of andrographolide by Aspergillus ochraceus and their cytotoxic activities against human tumor cell lines

Andrographolide (1), a major labdane diterpenoidal constituent of a famous traditional Chinese of Andrographis paniculata, exhibits a wide spectrum of biological activities including antibacterial, anti-inflammatory, and antitumor properties. Bioconversion of andrographolide (1) by Aspergillus ochraceus (ATCC 1008) was investigated. Five bioconversion products were isolated and identified. Their structures were identified to be 8β-hydroxy-8(17)-dihydroandrographolide (2), 8β-hydroxy-8(17)-dihydro-14-deoxy-11,12-didehydroandrographolide (3), 8β-hydroxy-8(17)-dihydro-14-deoxy-11,12-didehydroandrographolide 19-oic acid (4), 14-deoxy-11,12-didehydroandrographolide (5), and 14-deoxy-11,12-didehydroandrographolide 19-oic acid (6). Metabolites 2–4 were novel compounds. The proposed biosynthetic pathways of andrographolide by A. ochraceus were drawn. Most bioconversion products showed potential cytotoxic activities against human breast cancer (MCF-7), human colon cancer (HCT-116) and leukemia (HL-60) cell lines.     

The polar lipid composition of Mesorhizobium ciceri

The extractable lipid composition of Mesorhizobium ciceri strain HAMBI 1750 grown in a phosphate sufficient medium (79CA) is reported. Cardiolipin (CL—27% of total lipids), phosphatidylglycerol (PG—18%), phosphatidylethanolamine (PE—1%), phosphatidylcholine (PC—30%) and two methylated derivatives of PE, i.e. phosphatidyl-N, N-dimethylethanolamine (DMPE—1%) and phosphatidyl-N-monomethylethanolamine (MMPE—1%), were found to make up the phospholipids of the analysed bacteria. Nonphosphorus, ornithine-containing lipid (OL—10%) was also detected. Polar groups of phospholipids were predominantly acylated with cis-11,12-methyleneoctadecanoyl (lactobacillic) residues, whereas the ornithine lipid contained mainly 3-hexadecanoyloxy-11,12-methyleneoctadecanoic acid bound to the α-amino group.     

LC-MS/MS analysis of epoxyalcohols and epoxides of arachidonic acid and their oxygenation by recombinant CYP4F8 and CYP4F22

CYP4F22 and CYP4F8 are expressed in epidermis, and mutations of CYP4F22 are associated with lamellar ichthyosis. Epoxyalcohols (HEETs) and epoxides (EETs) of 20:4n−6 appear to be important for the water permeability barrier of skin. Our aim was to study the MS/MS spectra and fragmentation of these compounds and to determine whether they were oxidized by CYP4F22 or CYP4F8 expressed in yeast. HEETs were prepared from 15-hydroperoxyeicosatetraenoic acid (15-HPETE), 12-HPETE, and their [²H₈]labeled isotopomers, and separated by normal phase-HPLC with MS/MS analysis. CYP4F22 oxygenated 20:4n−6 at C-18, whereas metabolites of HEETs could not be identified. CYP4F8 formed ω3 hydroxy metabolites of HEETs derived from 12R-HPETE with 11,12-epoxy-10-hydroxy configuration, but not HEETs derived from 15S-HPETE. 8,9-EET and 11,12-EET were also subject to ω3 hydroxylation by CYP4F8. We conclude that CYP4F8 and CYP4F22 oxidize 20:4n−6 and that CYP4F8 selectively oxidizes 8,9-EET, 11,12-EET, and 10,11R,12R-HEET at the ω3 position.     

Synthesis and metabolism of 5beta-[11,12-3H]cholestane-3alpha, 7alpha-diol

5beta-[11,12-3H]Cholestane-3alpha,7alpha-diol was synthesized as follows. 5beta-Cholestane-3alpha,7alpha,12atriol 3,7-diacetate was treated with phosphorus oxychloride in pyridine solution and then the product, 5beta-cholest-11-ene-3alpha,7alpha-diol diacetate, was hydrogenated in acetic acid solution using platinum oxide as a catalyst under an atmosphere of tritium gas. 5beta-[11,12-3H]Cholestane-3alpha,7alpha-diol thus obtained was readily hydroxylated at C-26 by mitochondria in the presence of isocitric acid, magnesium chloride and potassium cyanide.     

A taxoid from the needles of the Japanese yew,Taxus cuspidata

5α-Hydroxy-2α,9α,10β-triacetoxy-11,12-epoxy-taxa-4(20)-en-13-one (taxinine A 11,12-epoxide) was isolated from the needles of the Japanese yew, Taxus cuspidata Sieb et Zucc, together with 10 other taxoids. Its structure was established on the basis of spectroscopic analysis.     

Cadinane-type sesquiterpenoids from Schisandra bicolor

The phytochemical investigation on the stems of Schisandra bicolor led to the isolation of seven cadinane-type sesquiterpenoids, 2-hydroxy-11,12-dehydrocalamenene (1), ent-T-muurolol (2), (+)-ent-epicubenol (3), (1S,4S)-7,8-dihydroxy-11,12-dehydrocalamenene (4), cadinane-T-cadinol (5), (−)-cadin-4,10(15)-dien-11-oic acid (6), and cadina-4,10(15)-dien-3-one (7). Their structures were determined by extensive analysis of their spectroscopic data. All the isolates were isolated from this species for the first time. The chemotaxonomic significance of these compounds was also summarized.     

Pathways of epoxyeicosatrienoic acid metabolism in endothelial cells. Implications for the vascular effects of soluble epoxide hydrolase inhibition

Epoxyeicosatrienoic acids (EETs) are products of cytochrome P-450 epoxygenase that possess important vasodilating and anti-inflammatory properties. EETs are converted to the corresponding dihydroxyeicosatrienoic acid (DHET) by soluble epoxide hydrolase (sEH) in mammalian tissues, and inhibition of sEH has been proposed as a novel approach for the treatment of hypertension. We observed that sEH is present in porcine coronary endothelial cells (PCEC), and we found that low concentrations of N,N'-dicyclohexylurea (DCU), a selective sEH inhibitor, have profound effects on EET metabolism in PCEC cultures. Treatment with 3 microM DCU reduced cellular conversion of 14,15-EET to 14,15-DHET by 3-fold after 4 h of incubation, with a concomitant increase in the formation of the novel beta-oxidation products 10,11-epoxy-16:2 and 8,9-epoxy-14:1. DCU also markedly enhanced the incorporation of 14,15-EET and its metabolites into PCEC lipids. The most abundant product in DCU-treated cells was 16,17-epoxy-22:3, the elongation product of 14,15-EET. Another novel metabolite, 14,15-epoxy-20:2, was present in DCU-treated cells. DCU also caused a 4-fold increase in release of 14,15-EET when the cells were stimulated with a calcium ionophore. Furthermore, DCU decreased the conversion of [3H]11,12-EET to 11,12-DHET, increased 11,12-EET retention in PCEC lipids, and produced an accumulation of the partial beta-oxidation product 7,8-epoxy-16:2 in the medium. These findings suggest that in addition to being metabolized by sEH, EETs are substrates for beta-oxidation and chain elongation in endothelial cells and that there is considerable interaction among the three pathways. The modulation of EET metabolism by DCU provides novel insight into the mechanisms by which pharmacological or molecular inhibition of sEH effectively treats hypertension.     

Identification of unusual cyclopropane monounsaturated fatty acids from the deep-water lake invertebrate Acanthogammarus grewingkii

In the deep-lake invertebrate, Acanthogammarus grewingkii a number of unusual fatty acids such as cis-11,12-methylene-5-eicosenoate, cis,cis-11,12–14,15-bis-methylene-5-eicosenoate, and their homologues, were identified. The structures were determined from spectra (¹HNMR, ¹³CNMR, mass, IR) after their isolation and quantification by means of Ag⁺-TLC, semipreparative RP-HPLC and capillary GC-MS. The possibility of biosynthesis and function of these unusually fatty acids is discussed, e.g. they may represent a specific feature of animals dwelling in a deep lake or may also be metabolites that are formed in cellular membranes or be of dietary origin.     

Effect of soluble epoxide hydrolase inhibition on epoxyeicosatrienoic acid metabolism in human blood vessels

We investigated the effects of soluble epoxide hydrolase (sEH) inhibition on epoxyeicosatrienoic acid (EET) metabolism in intact human blood vessels, including the human saphenous vein (HSV), coronary artery (HCA), and aorta (HA). When HSV segments were perfused with 2 micromol/l 14,15-[3H]EET for 4 h, >60% of radioactivity in the perfusion medium was converted to 14,15-dihydroxyeicosatrienoic acid (DHET). Similar results were obtained with endothelium-denuded vessels. 14,15-DHET was released from both the luminal and adventitial surfaces of the HSV. When HSVs were incubated with 14,15-[3H]EET under static (no flow) conditions, formation of 14,15-DHET was detected within 15 min and was inhibited by the selective sEH inhibitors N,N'-dicyclohexyl urea and N-cyclohexyl-N'-dodecanoic acid urea (CUDA). Similarly, CUDA inhibited the conversion of 11,12-[3H]EET to 11,12-DHET by the HSV. sEH inhibition enhanced the uptake of 14,15-[3H]EET and facilitated the formation of 10,11-epoxy-16:2, a beta-oxidation product. The HCA and HA converted 14,15-[3H]EET to DHET, and this also was inhibited by CUDA. These findings in intact human blood vessels indicate that conversion to DHET is the predominant pathway for 11,12- and 14,15-EET metabolism and that sEH inhibition can modulate EET metabolism in vascular tissue.     

Tumor formation in the neonatal mouse bioassay indicates that the potent carcinogen dibenzo[def,p]chrysene (dibenzo[a,l]pyrene) is activated in vivo via its trans-11,12-dihydrodiol

The hexacyclic aromatic hydrocarbon dibenzo[def,p]chrysene, better known as dibenzo[a,l]pyrene (DBP) in the field of chemical carcinogenesis, is present in the environment as a combustion product of organic matter. This compound is probably the strongest chemical carcinogen ever tested. As ultimate genotoxic metabolites of DBP two electrophilically reactive species are discussed: (i) radical cations generated by one-electron oxidation, and (ii) fjord region dihydrodiol epoxides formed via the trans-11,12-dihydroxy 11,12-dihydro derivative of DBP (11,12-dihydrodiol). In order to delineate the metabolic pathway(s) involved in tumor formation by DBP, newborn Crl:CD-1(ICR)BR mice were intraperitoneally treated with the parent compound, its 11,12-dihydrodiol, and the two diastereomeric fjord region dihydrodiol epoxides. Due to severe acute and chronic toxicity, the total dose of DBP and of the 11,12-dihydrodiol was limited to 40 nmol. For the same reason the dihydrodiol epoxides could only be applied in doses up to 0.4 nmol. The tumor incidence was determined 55 +/- 1 weeks after treatment. Under these conditions, DBP and its 11,12-dihydrodiol induced lung tumors (incidence: 86.5% versus 92.0%; yield: 2.88 versus 7.44 tumors per mouse), liver (incidence: 57.7% versus 60.0%; yield: 3.63 versus 5.28 tumors per mouse) and other organs (incidence: 36.5% versus 32.0%; yield: 0.56 versus 0.52 tumors per mouse). By contrast, only lung tumors at low incidence were detected in mice treated with solvent only (incidence: 28.8%; yield: 0.58 tumors per mouse). As with the parent hydrocarbon, mice treated with low doses of diastereomeric syn- and anti-dihydrodiol epoxides of DBP showed increased tumor incidences in liver (incidence: 19.0 and 46.7%; yield: 0.36 and 1.47 tumors per mouse, respectively), and in various other organs (incidence: 7.1 and 20.0%; yield: 0.07 and 0.20 tumors per mouse, respectively). In consideration of the 100-fold differences in the doses of compounds applied in this study, the tumor-inducing potency increases in the order DBP < 11,12-dihydrodiol < anti-dihydrodiol epoxide. This result provides strong evidence that the potent carcinogen DBP is activated in vivo in the mouse via its 11,12-dihydrodiol and not preferentially through alternative pathways.     

Abietane diterpenoids from the root of Salvia phlomoides

From the root of Salvia phlomoides three new abietane diterpenoids, demethylcryptojaponol, 14-deoxycoleon U and salviphlomone, have been isolated, besides the previously known diterpenes 8,13-abietadiene, 8,11,13-abietatriene, royleanone, 7α-acetoxyroyleanone, taxodione, taxodone, cryptojaponol and sugiol. The structures of demethylcryptojaponol (11,12-dihydroxy-8,11,13-abietatrien-7-one), 14-deoxycoleon U (6,11,12-trihydroxy-5,8,11,13-abietatetraen-7-one) and salviphlomone (6α,7β-dihydroxy-8,13-abietadiene-11,12-dione) were established by chemical and spectroscopic means.     

A C-formylated azulene from Lactarius deterrimus

1-Formyl-4-methyl-7-isopropyl azulene (11,12-dihydro-lactaroviolin) was characterized from Lactarius deterrimus together with other known compounds.     

Enzymic conversion of 11,12-leukotriene A4 to 11,12-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid. Purification of an epoxide hydrolase from the guinea pig liver cytosol

(11S,12S)-Epoxy-5,14-cis-7,9-trans-eicosatetraenoic acid (11,12-leukotriene A4) was nonenzymically converted to seven compounds: two diastereomers of (12S)-hydroxyeicosatetraeno-delta-lactones (major products), two diastereomers of (5,12S)-dihydroxyeicosatetraenoic acid and three stereoisomers of (11,12S)-dihydroxyeicosatetraenoic acid. Among these compounds, (11R,12S)-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid proved to be the only enzymic product. This hydrolysis activity was present in the cytosol fractions of various tissues of guinea pig such as liver, adrenal gland, small intestine, and brain. We purified the epoxide hydrolase to an apparent homogeneity from the guinea pig liver. The enzyme had a molecular weight of 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 7.3. The partial amino acid sequence was different from that of the microsomal enzyme. Km and Vmax values for 11,12-leukotriene A4 were 18 microM and 2.4 mumol/min/mg protein, respectively. These results indicate that 11,12-dihydroxyeicosatetraenoic acid is enzymically synthesized from 11,12-leukotriene A4 by the action of the cytosolic epoxide hydrolase in vitro.     

The formation of dihydrodiols by chemical or enzymic oxidation of 3-methylcholanthrene.

The chemical oxidation of 3-methylcholanthrene in an ascorbic acid-ferrous sulphate-EDTA reaction mixture gave all five possible dihydrodiols. The structures and stereochemistry of the dihydrodiols were shown by UV, mass and NMR spectral studies and by chemical examination to be cis-2a,3-dihydroxy-3-methylcholanthrene, trans-4,5-dihydro-4,5-dihydroxy-3-methylcholanthrene, trans-7,8-dihydro-7,8-dihydroxy-3-methylcholanthrene, trans-9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene, cis-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene. An examination by HPLC of the dihydrodiols formed in the metabolism of 3-methylcholanthrene by rat-liver microsomal preparations showed the presence of trans-4,5-dihydro-4,5-dihydoxy-3-methylcholanthrene, trans-7,8-dihydro-7,8-dihydroxy-3-methylcholanthrene, trans-9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene, identified by comparison of their UV and chromatographic characteristics with those of authentic standards. Tentative identification of cis- and trans-1,2-dihydroxy-3-methylcholanthrene, cis-2a,3-dihydroxy-3-methylcholanthrene and cis-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene as metabolites were made from their mobilities using HPLC. A quantitative comparison of the dihydrodiols formed from 3H-labelled 3-methylcholanthrene by microsomal preparations from the livers of normal and 3-methylcholanthrene-treated rats was carried out. trans-9,10-Dihydro-9,10-dihydroxy-3-methylcholanthrene and cis- and trans-1,2-dihydroxy-3-methylcholanthrene were formed when 3-methylcholanthrene was incubated with mouse skin in organ culture.     

Role of ADP-ribose in 11,12-EET-induced activation of K(Ca) channels in coronary arterial smooth muscle cells

We recently reported that cADP-ribose (cADPR) and ADP-ribose (ADPR) play an important role in the regulation of the Ca(2+)-activated K(+) (K(Ca)) channel activity in coronary arterial smooth muscle cells (CASMCs). The present study determined whether these novel signaling nucleotides participate in 11,12-epoxyeicosatrienoic acid (11,12-EET)-induced activation of the K(Ca) channels in CASMCs. HPLC analysis has shown that 11,12-EET increased the production of ADPR but not the formation of cADPR. The increase in ADPR production was due to activation of NAD glycohydrolase as measured by a conversion rate of NAD into ADPR. The maximal conversion rate of NAD into ADPR in coronary homogenate was increased from 2.5 +/- 0.2 to 3.4 +/- 0.3 nmol*(-1) *mg protein(-1) by 11,12-EET. The regioisomers of 8,9-EET, 11,12-EET, and 14,15-EET also significantly increased ADPR production from NAD. Western blot analysis and immunoprecipitation demonstrated the presence of NAD glycohydrolase, which mediated 11,12-EET-activated production of ADPR. In cell-attached patches, 11,12-EET (100 nM) increases K(Ca) channel activity by 5.6-fold. The NAD glycohydrolase inhibitor cibacron blue 3GA (3GA, 100 microM) significantly attenuated 11,12-EET-induced increase in the K(Ca) channel activity in CASMCs. However, 3GA had no effect on the K(Ca) channels activity in inside-out patches. 11,12-EET produced a concentration-dependent relaxation of precontracted coronary arteries. This 11,12-EET-induced vasodilation was substantially attenuated by 3GA (30 microM) with maximal inhibition of 57%. These results indicate that 11,12-EET stimulates the production of ADPR and that intracellular ADPR is an important signaling molecule mediating 11,12-EET-induced activation of the K(Ca) channels in CASMCs and consequently results in vasodilation of coronary artery.