Development of a Primary Human Co-Culture Model of Inflamed Airway Mucosa.

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface.

Epoxyeicosatrienoic acids protect cardiac cells during starvation by modulating an autophagic response.

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid involved in regulating pathways promoting cellular protection. We have previously shown that EETs trigger a protective response limiting mitochondrial dysfunction and reducing cellular death. Considering it is unknown how EETs regulate cell death processes, the major focus of the current study was to investigate their role in the autophagic response of HL-1 cells and neonatal cardiomyocytes (NCMs) during starvation. We employed a dual-acting synthetic analog UA-8 (13-(3-propylureido)tridec-8-enoic acid), possessing both EET-mimetic and soluble epoxide hydrolase (sEH) inhibitory properties, or 14,15-EET as model EET molecules. We demonstrated that EETs significantly improved viability and recovery of starved cardiac cells, whereas they lowered cellular stress responses such as caspase-3 and proteasome activities. Furthermore, treatment with EETs resulted in preservation of mitochondrial functional activity in starved cells. The protective effects of EETs were abolished by autophagy-related gene 7 (Atg7) short hairpin RNA (shRNA) or pharmacological inhibition of autophagy. Mechanistic evidence demonstrated that sarcolemmal ATP-sensitive potassium channels (pmKATP) and enhanced activation of AMP-activated protein kinase (AMPK) played a crucial role in the EET-mediated effect. Our data suggest that the protective effects of EETs involve regulating the autophagic response, which results in a healthier pool of mitochondria in the starved cardiac cells, thereby representing a novel mechanism of promoting survival of cardiac cells. Thus, we provide new evidence highlighting a central role of the autophagic response in linking EETs with promoting cell survival during deep metabolic stress such as starvation.

Distribution of soluble and microsomal epoxide hydrolase in the mouse brain and its contribution to cerebral epoxyeicosatrienoic acid metabolism.

Epoxide hydrolases comprise a family of enzymes important in detoxification and conversion of lipid signaling molecules, namely epoxyeicosatrienoic acids (EETs), to their supposedly less active form, dihydroxyeicosatrienoic acids (DHETs). EETs control cerebral blood flow, exert analgesic, anti-inflammatory and angiogenic effects and protect against ischemia. Although the role of soluble epoxide hydrolase (sEH) in EET metabolism is well established, knowledge on its detailed distribution in rodent brain is rather limited. Here, we analyzed the expression pattern of sEH and of another important member of the EH family, microsomal epoxide hydrolase (mEH), in mouse brain by immunohistochemistry. To investigate the functional relevance of these enzymes in brain, we explored their individual contribution to EET metabolism in acutely isolated brain cells from respective EH -/- mice and wild type littermates by mass spectrometry. We find sEH immunoreactivity almost exclusively in astrocytes throughout the brain, except in the central amygdala, where neurons are also positive for sEH. mEH immunoreactivity is abundant in brain vascular cells (endothelial and smooth muscle cells) and in choroid plexus epithelial cells. In addition, mEH immunoreactivity is present in specific neuronal populations of the hippocampus, striatum, amygdala, and cerebellum, as well as in a fraction of astrocytes. In freshly isolated cells from hippocampus, where both enzymes are expressed, sEH mediates the bulk of EET metabolism. Yet we observe a significant contribution of mEH, pointing to a novel role of this enzyme in the regulation of physiological processes. Furthermore, our findings indicate the presence of additional, hitherto unknown cerebral epoxide hydrolases. Taken together, cerebral EET metabolism is driven by several epoxide hydrolases, a fact important in view of the present targeting of sEH as a potential therapeutic target. Our findings suggest that these different enzymes have individual, possibly quite distinct roles in brain function and cerebral EET metabolism.

Epoxyeicosatrienoic acid prevents postischemic electrocardiogram abnormalities in an isolated heart model.

Cytochrome P450 epoxygenases metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) which are in turn converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). The main objective of this study was to investigate the protective effects of EETs following ischemic injury using an ex vivo electrocardiogram (EKG) model. Hearts from C57Bl/6, transgenic mice with cardiomyocyte-specific overexpression of CYP2J2 (Tr) and wildtype (WT) littermates were excised and perfused with constant pressure in a Langendorff apparatus. Electrodes were placed superficially at the right atrium and left ventricle to assess EKG waveforms. In ischemic reperfusion experiments hearts were subjected to 20 min of global no-flow ischemia followed by 20 min of reperfusion (R20). The EKG from C57Bl/6 hearts perfused with 1 microM 14,15-EET showed less QT prolongation (QTc) and ST elevation (STE) (QTc=41+/-3, STE=2.3+/-0.3; R20: QTc=42+/-2 ms, STE=1.2+/-0.2mv) than control hearts (QTc=36+/-2, STE=2.3+/-0.2; R20: QTc=53+/-3 ms; STE=3.6+/-0.4mv). Similar results of reduced QT prolongation and ST elevation were observed in EKG recording from CYP2J2 Tr mice (QTc=35+/-1, STE=1.9+/-0.1; R20: QTc=38+/-4 ms, STE=1.3+/-0.2mv) compared to WT hearts. The putative epoxygenase inhibitor MS-PPOH (50 microM) and EET antagonist 14,15-EEZE (10 microM) both abolished the cardioprotective response, implicating EETs in this process. In addition, separate exposure to the K(ATP) channel blockers glibenclamide (1 microM) and HMR1098 (10 microM), or the PKA protein inhibitor H89 (50 nM) during reperfusion abolished the improved repolarization in both the models. Consistent with a role of PKA, CYP2J2 Tr mice had an enhanced activation of the PKAalpha regulatory II subunit in plasma membrane following IR injury. The present data demonstrate that EETs can enhance the recovery of ventricular repolarization following ischemia, potentially by facilitating activation of K(+) channels and PKA-dependent signaling.

Stimulation of rat erythrocyte P2X7 receptor induces the release of epoxyeicosatrienoic acids.

BACKGROUND AND PURPOSE: Red blood cells (RBCs) are reservoirs of vasodilatory, antiaggregatory, and antiinflammatory lipid mediators-epoxyeicosatrienoic acids (EETs). This study addresses the formation and release of erythrocyte-derived EETs in response to ATP receptor stimulation that may represent an important mechanism regarding circulatory regulation. EXPERIMENTAL APPROACH: Erythrocyte EET formation and release were investigated by incubating rat RBCs in physiological salt solution with agents that effected ATP release via P2 receptor stimulation of phospholipase A2 and epoxygenase-like activities with activation of the ATP secretory mechanism. EETs were analyzed by gas and liquid chromatography-mass spectrometry. KEY RESULTS: EETs were released from rat RBCs: 14,15-, 11,12-, 8,9- and 5,6-EETs in a ratio of 1.2:1.0:0.9:0.8. EETs were produced by epoxidation of arachidonic acid catalyzed by hemoglobin. Spontaneous release of EETs, 0.66+/-0.14 ng per 10(9) RBCs, was dose-dependently increased by an ATP analog, BzATP, and inhibited by P2X(7) receptor antagonists. 5 microM ATP increased release of EETs over 20% to 0.83+/-0.15 ng per 10(9) RBCs; 10 microM BzATP tripled the amount of EET release to 1.87+/-0.20 ng per 10(9) RBCs. EET release by ATP or BzATP was not associated with hemolysis. Carbenoxolone, a gap junction inhibitor that inhibits ATP release, and glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), which is required for ATP release, inhibited the spontaneous and stimulated EET release from RBCs. CONCLUSIONS AND IMPLICATIONS: EETs are produced and released from RBCs via a mechanism that is mediated by ATP stimulation of P2X(7) receptors coupled to ATP transporters, pannexin-1 and CFTR.

Characterization of 14,15-epoxyeicosatrienoyl-sulfonamides as 14,15-epoxyeicosatrienoic acid agonists: use for studies of metabolism and ligand binding.

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid. EETs mediate numerous biological functions. In coronary arteries, they regulate vascular tone by the activation of smooth muscle large-conductance, calcium-activated potassium (BK(Ca)) channels to cause hyperpolarization and relaxation. We developed a series of 14,15-EET agonists, 14,15-EET-phenyliodosulfonamide (14,15-EET-PISA), 14,15-EET-biotinsulfonamide (14,15-EET-BSA), and 14,15-EET-benzoyldihydrocinnamide-sulfonamide (14,15-EET-BZDC-SA) as tools to characterize 14,15-EET metabolism and binding. Agonist activities of these analogs were characterized in precontraced bovine coronary arterial rings. All three analogs induced concentration-dependent relaxation and were equipotent with 14,15-EET. Relaxations to these analogs were inhibited by the BK(Ca) channel blocker iberiotoxin (100 nM), the 14,15-EET antagonist 14,15-epoxyeicosa-5(Z)-enoylmethylsulfonamide (10 muM), and abolished by 20 mM extracellular K(+). 14,15-EET-PISA is metabolized to 14,15-dihydroxyeicosatrienoyl-PISA by soluble epoxide hydrolase in bovine coronary arteries and U937 cells but not U937 cell membrane fractions. 14,15-EET-P(125)ISA binding to human U937 cell membranes was time-dependent, concentration-dependent, and saturable. The specific binding reached equilibrium by 15 min at 4 degrees C and remained unchanged up to 30 min. The estimated K(d) and B(max) were 148.3 +/- 36.4 nM and 3.3 +/- 0.5 pmol/mg protein, respectively. These data suggest that 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA are full 14,15-EET agonists. 14,15-EET-P(125)ISA is a new radiolabeled tool to study EET metabolism and binding. Our results also provide preliminary evidence that EETs exert their biological effect through a membrane binding site/receptor.

Preparation of N-tBoc L-glutathione dimethyl and di-tert-butyl esters: versatile synthetic building blocks.

The title l-glutathione derivatives, containing acid- and base-labile esters, respectively, were obtained in good overall yields. N-(t)Boc l-glutathione dimethyl ester was prepared via Fischer esterification of l-glutathione disulfide (GSSG) using HCl in dry methanol, protection of the amine with (t)Boc(2)O, and tributylphosphine cleavage of the disulfide in wet isopropanol. Alternatively, Fischer esterification and (t)Boc-protection of l-glutathione (GSH) also furnished N-(t)Boc glutathione dimethyl ester accompanied by a small amount of S-(t)Boc that was removed chromatographically. The di-tert-butyl ester was obtained by S-palmitoylation of GSH in TFA as solvent, N-(t)Boc-protection, esterification using (t)BuOH mediated by diisopropylcarbodiimide/copper(I) chloride, and saponification of the thioester. These l-glutathione derivatives are versatile synthetic building blocks for the preparation of S-glutathione adducts.

Adenosine2A receptor vasodilation of rat preglomerular microvessels is mediated by EETs that activate the cAMP/PKA pathway.

Dilation of rat preglomerular microvessels (PGMV) by activation of adenosine A2A receptors (A2AR) is coupled to epoxyeicosatrienoic acid (EET) release. We have investigated the commonality of this signal transduction pathway, i.e., sequential inhibition of G(salpha), adenylyl cyclase, PKA, and Ca2+-activated K+ (KCa) channel activity, to the vasoactive responses to A2AR activation by a selective A2A agonist, CGS-21680, compared with those of 11,12-EET. Male Sprague-Dawley rats were anesthetized, and microdissected arcuate arteries (110-130 microm) were cannulated and pressurized to 80 mmHg. Vessels were superfused with Krebs solution containing NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin and preconstricted with phenylephrine. We assessed the effect of 3-aminobenzamide (10 microM), an inhibitor of mono-ADP-ribosyltranferases, on responses to 11,12-EET (3 nM) and CGS-21680 (10 microM) and found that both were inhibited by approximately 70% (P<0.05), whereas the response to SNP (10 microM) was unaffected. Furthermore, 11,12-EET (100 nM), like cholera toxin (100 ng/ml), stimulated ADP-ribose formation in homogenates of arcuate arteries compared with control. SQ-22536 (10 microM), an inhibitor of adenylyl cyclase activity, and myristolated PKI (14-22) amide (5 microM), an inhibitor of PKA, decreased activity of 11,12-EET and CGS-21680. Incubation of 11,12-EET (3 nM-3 microM) with PGMV resulted in an increase in cAMP levels (P<0.05). The responses to both 11,12-EET and CGS-21680 were significantly reduced by superfusion of iberiotoxin (100 nM), an inhibitor of KCa channel activity. Thus in rat PGMV activation of A2AR is coupled to EET release upstream of adenylyl cyclase activation and EETs stimulate mono-ADP-ribosyltransferase, resulting in Gsalpha protein activation.

14,15-Dihydroxyeicosatrienoic acid activates peroxisome proliferator-activated receptor-alpha.

Epoxyeicosatrienoic acids (EETs), lipid mediators synthesized from arachidonic acid by cytochrome P-450 epoxygenases, are converted by soluble epoxide hydrolase (SEH) to the corresponding dihydroxyeicosatrienoic acids (DHETs). Originally considered as inactive degradation products of EETs, DHETs have biological activity in some systems. Here we examined the capacity of EETs and DHETs to activate peroxisome proliferator-activated receptor-alpha (PPARalpha). We find that among the EET and DHET regioisomers, 14,15-DHET is the most potent PPARalpha activator in a COS-7 cell expression system. Incubation with 10 microM 14,15-DHET produced a 12-fold increase in PPARalpha-mediated luciferase activity, an increase similar to that produced by the PPARalpha agonist Wy-14643 (20 microM). Although 10 microM 14,15-EET produced a threefold increase in luciferase activity, this was abrogated by the SEH inhibitor dicyclohexylurea. 14-Hexyloxytetradec-5(Z)-enoic acid, a 14,15-EET analog that cannot be converted to a DHET, did not activate PPARalpha. However, PPARalpha was activated by 2-(14,15-epoxyeicosatrienoyl)glycerol, which was hydrolyzed and the released 14,15-EET converted to 14,15-DHET. COS-7 cells incorporated 14,15-[3H]DHET from the medium, and the cells also retained a small amount of the DHET formed during incubation with 14,15-[3H]EET. Binding studies indicated that 14,15-[3H]DHET binds to the ligand binding domain of PPARalpha with a Kd of 1.4 microM. Furthermore, 14,15-DHET increased the expression of carnitine palmitoyltransferase 1A, a PPARalpha-responsive gene, in transfected HepG2 cells. These findings suggest that 14,15-DHET, produced from 14,15-EET by the action of SEH, may function as an endogenous activator of PPARalpha.

An Economic and Practical Synthesis of the 2-Tetrahydrofuranyl Ether Protective Group.

Primary, secondary, and tertiary alcohols as well as phenols and carbohydrates are efficiently transformed into the corresponding 2-tetrahydrofuranyl ethers by a combination of Mn(0) powder and CCl(4) in tetrahydrofuran.

5E, 8Z, 11Z, 14Z-eicosatetraenoic acid, a novel trans isomer of arachidonic acid, causes G1 phase arrest and induces apoptosis of HL-60 cells.

Trans arachidonic acid isomers (trans-AA) constitute a new group of trans fatty acids (trans-FA) generated in vivo via endogenous cis-trans isomerization stimulated by the NO2 radical. Because both NO2 and trans-FA have been implicated as causative factors in cancer, we studied the effect of the trans-AA isomers on proliferation and viability of human promyelocytic (HL-60) cells. The four trans arachidonic (trans-AA) acid isomers synthesized by us have been presently tested with respect to their competence to affect the proliferation and viability of human promyeolocytic HL-60 cells in culture. The data demonstrate that one of the isomers, 5,6-trans-AA, showed distinct activity by targeting cell progression through the cell cycle and inducing apoptosis. The effects were time- and concentration-dependent: the cytostatic effect of 5E-AA was observed at 10 microM following 72 h of treatment. This effect was manifested as a perturbation of cell progression through G1 phase, indicating the 'on' activation of the G1 checkpoint as evidenced by the flow- and laser scanning-cytometry techniques. Apoptotic cells were identified by comparison of their morphology, DNA fragmentation, caspase activation and collapse of mitochondrial potential with control cells. These observations suggested that 5E-AA induced a mitochondrial pathway of apoptosis. There was no evidence of cell-cycle phase specificity in induction of apoptosis by 5E-AA, as the cells showing highly fragmented DNA or caspase-3 activation were distributed in all phases of the cycle. The data suggest that 5E-AA may have at least two targets: one that is cell-cycle specific and associated with the observed arrest in the G1 phase and another, unrelated to the cell cycle, which is responsible for triggering apoptosis indiscriminately, regardless of cycle phase I.

Epoxyeicosatrienoic acids mediate adenosine-induced vasodilation in rat preglomerular microvessels (PGMV) via A2A receptors.

Activation of rat adenosine2A receptors (A2A R) dilates preglomerular microvessels (PGMV), an effect mediated by epoxyeicosatrienoic acids (EETs). Incubation of PGMV with a selective A2A R agonist, 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 100 microM), increased isolated PGMV EET levels to 7.57+/-1.53 ng mg-1 protein from 1.06+/-0.22 ng mg-1 protein in controls (P<0.05), without affecting hydroxyeicosatetraenoic acid (HETE) levels (10.8+/-0.69 vs 11.02+/-0.74 ng mg-1 protein). CGS 21680-stimulated EETs was abolished by preincubation with an A2A R antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385) (100 microM). A selective epoxygenase inhibitor, methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH; 12 microM) prevented CGS 21680-induced increase in EETs, indicating inhibition of de novo synthesis of EETs. In pressurized (80 mmHg) renal arcuate arteries (110-130 microm) preconstricted with phenylephrine (20 nM), superfusion with CGS 21680 (0.01-10 microM) increased the internal diameter (i.d.) concentration-dependently; vasodilation was independent of nitric oxide and cyclooxygenase activity. CGS 21680 (10 microM) increased i.d. by 32+/-6 microm; vasodilation was prevented by inhibition of EET synthesis with MS-PPOH. Addition of 3 nM 5,6-EET, 8,9-EET and 11,12-EET increased i.d. by 53+/-9, 17+/-4 and 53+/-5 microm, respectively, whereas 14,15-EET was inactive. The responses to 5,6-EET were, however, significantly inhibited by indomethacin. We conclude that 11,12-EET is the likely mediator of A2A R-induced dilation of rat PGMV. Activation of A2A R coupled to de novo EET stimulation may represent an important mechanism in regulating preglomerular microvascular tone. British Journal of Pharmacology (2004) 141, 441-448. doi:10.1038/sj.bjp.0705640

11,12-epoxyeicosatrienoic acid (11,12-EET): structural determinants for inhibition of TNF-alpha-induced VCAM-1 expression.

A series of 11,12-EET analogues were synthesized and compared using a human endothelial cell based TNF-alpha-induced VCAM-1 expression assay. The resulting data were used to map a putative recognition/binding domain for 11,12-EET.

Evidence for a membrane site of action for 14,15-EET on expression of aromatase in vascular smooth muscle.

Epoxyeicosatrienoic acids (EETs) are synthesized in the endothelial cells of vascular tissues. They are released from the endothelial cells and produce relaxation of the smooth muscle cells by hyperpolarization. The present findings demonstrate that EETs also regulate aromatase activity in vascular smooth muscle cells. Exposure of cultured rat aortic smooth muscle cells to either 1 microM 14,15-EET or 1 microM 11,12-EET inhibits dibutyryl cAMP-induced aromatase activity by 80-100%. 11,12-Dihydroxyeicosatrienoic acid, the hydration product of 11,12-EET, has no effect on dibutyryl cAMP-induced vascular smooth muscle aromatase activity. In contrast to 14,15-EET, the N-methylsulfanilamide derivative of 14,15-EET (14,15-EET-SA) was neither metabolized nor incorporated into cell lipids, but it retained the ability to inhibit cAMP-induced aromatase activity. Furthermore, the 14,15-EET-SA inhibition of cAMP-induced aromatase activity persisted when the sulfanilamide derivative of 14,15-EET was covalently tethered to silica beads (average diameter, 0.5 microm), which restricted 14,15-EET-SA from entering the cell. These data are consistent with the presence of a receptor for EETs in the plasma membrane and support the hypothesis that the inhibition of aromatase by EETs is initiated by the interaction of EET with the putative plasma membrane receptor.

An adjacent pair of human NUDT genes on chromosome X are preferentially expressed in testis and encode two new isoforms of diphosphoinositol polyphosphate phosphohydrolase.

Combinatorial expression of the various isoforms of diphosphoinositol synthases and phosphohydrolases determines the rates of phosphorylation/dephosphorylation cycles that have been functionally linked to vesicle trafficking, stress responses, DNA repair, and apoptosis. We now describe two new 19-kDa diphosphoinositol polyphosphate phosphohydrolases (DIPPs), named types 3alpha and 3beta, which possess the canonical Nudix-type catalytic motif flanked on either side by short Gly-rich sequences. The two enzymes differ only in that Pro-89 in the alpha form is replaced by Arg-89 in the beta form, making the latter approximately 2-fold more active in vitro. Another Nudix substrate, diadenosine hexaphosphate, was hydrolyzed less efficiently (k(cat)/K(m) = 0.2 x 10(5) m(-1) s(-1)) compared with diphosphoinositol polyphosphates (k(cat)/K(m) = 2-40 x 10(5) m(-1) s(-1)). Catalytic activity in vivo was established by individual overexpression of the human (h) DIPP3 isoforms in HEK293 cells, which reduced cellular levels of diphosphoinositol polyphosphates by 40-50%. The hDIPP3 mRNA is preferentially expressed in testis, accompanied by relatively weak expression in the brain, contrasting with hDIPP1 and hDIPP2 which are widely expressed. The hDIPP3 genes (NUDT10 encodes hDIPP3alpha; NUDT11 encodes hDIPP3beta) are only 152 kbp apart at p11.22 on chromosome X and probably arose by duplication. Transcription of both genes is inactivated on one of the X chromosomes of human females to maintain appropriate gene dosage. The hDIPP3 pair add tissue-specific diversity to the molecular mechanisms regulating diphosphoinositol polyphosphate turnover.

14,15-Dihydroxyeicosatrienoic acid relaxes bovine coronary arteries by activation of K(Ca) channels.

Epoxyeicosatrienoic acids (EETs) cause vascular relaxation by activating smooth muscle large conductance Ca(2+)-activated K(+) (K(Ca)) channels. EETs are metabolized to dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolase. We examined the contribution of 14,15-DHET to 14,15-EET-induced relaxations and characterized its mechanism of action. 14,15-DHET relaxed U-46619-precontracted bovine coronary artery rings but was approximately fivefold less potent than 14,15-EET. The relaxations were inhibited by charybdotoxin, iberiotoxin, and increasing extracellular K(+) to 20 mM. In isolated smooth muscle cells, 14,15-DHET increased an iberiotoxin-sensitive, outward K(+) current and increased K(Ca) channel activity in cell-attached patches and inside-out patches only when GTP was present. 14,15-[(14)C]EET methyl ester (Me) was converted to 14,15-[(14)C]DHET-Me, 14,15-[(14)C]DHET, and 14,15-[(14)C]EET by coronary arterial rings and endothelial cells but not by smooth muscle cells. The metabolism to 14,15-DHET was inhibited by the epoxide hydrolase inhibitors 4-phenylchalcone oxide (4-PCO) and BIRD-0826. Neither inhibitor altered relaxations to acetylcholine, whereas relaxations to 14,15-EET-Me were increased slightly by BIRD-0826 but not by 4-PCO. 14,15-DHET relaxes coronary arteries through activation of K(Ca) channels. Endothelial cells, but not smooth muscle cells, convert EETs to DHETs, and this conversion results in a loss of vasodilator activity.

Asymmetric total synthesis of (+)-fostriecin.

[structure: see text] The title compound, a potent protein phosphatase inhibitor and anticancer agent, was prepared by an efficient, multiconvergent asymmetric synthesis. Key transformations include a ring forming olefin metathesis leading to the alpha,beta-unsaturated lactone and creation of the triene moiety via Suzuki cross-coupling.

The CYP450 hydroxylase pathway contributes to P2X receptor-mediated afferent arteriolar vasoconstriction.

This study was conducted to test the hypothesis that the cytochrome P-450 (CYP450) metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the afferent arteriolar response to P2 receptor activation. Afferent arteriolar responses to ATP, the P2X agonist, alpha,beta-methylene ATP and the P2Y agonist UTP were determined before and after treatment with the selective CYP450 hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE). Stimulation with 1.0 and 10 microM ATP elicited an initial preglomerular vasoconstriction of 12 +/- 1% and 45 +/- 4% and a sustained vasoconstriction of 11 +/- 1% and 11 +/- 2%, respectively. DDMS or 20-HEDE significantly attenuated the sustained afferent arteriolar constrictor response to ATP. alpha,beta-Methylene ATP (1 microM) induced a rapid initial afferent vasoconstriction of 64 +/- 3%, which partially recovered to a stable diameter 10 +/- 1% smaller than control. Both DDMS and 20-HEDE significantly attenuated the initial vasoconstriction and abolished the sustained vasoconstrictor response to alpha,beta-methylene ATP. UTP decreased afferent diameter by 50 +/- 5% and 20-HEDE did not change this response. In addition, the ATP-induced increase in the intracellular Ca2+ concentration in preglomerular microvascular smooth muscle cells was significantly attenuated by 20-HEDE. Taken together, these results are consistent with the hypothesis that the CYP450 metabolite 20-HETE participates in the afferent arteriolar response to activation of P2X receptors.

Cytochrome P450 CYP2J9, a new mouse arachidonic acid omega-1 hydroxylase predominantly expressed in brain.

A cDNA encoding a new cytochrome P450 was isolated from a mouse brain library. Sequence analysis reveals that this 1,958-base pair cDNA encodes a 57-58-kDa 502-amino acid polypeptide that is 70-91% identical to CYP2J subfamily P450s and is designated CYP2J9. Recombinant CYP2J9 was co-expressed with NADPH-cytochrome P450 oxidoreductase (CYPOR) in Sf9 cells using a baculovirus system. Microsomes of CYP2J9/CYPOR-transfected cells metabolize arachidonic acid to 19-hydroxyeicosatetraenoic acid (HETE) thus CYP2J9 is enzymologically distinct from other P450s. Northern analysis reveals that CYP2J9 transcripts are present at high levels in mouse brain. Mouse brain microsomes biosynthesize 19-HETE. RNA polymerase chain reaction analysis demonstrates that CYP2J9 mRNAs are widely distributed in brain and most abundant in the cerebellum. Immunoblotting using an antibody raised against human CYP2J2 that cross-reacts with CYP2J9 detects a 56-kDa protein band that is expressed in cerebellum and other brain segments and is regulated during postnatal development. In situ hybridization of mouse brain sections with a CYP2J9-specific riboprobe and immunohistochemical staining with the anti-human CYP2J2 IgG reveals abundant CYP2J9 mRNA and protein in cerebellar Purkinje cells. Importantly, 19-HETE inhibits the activity of recombinant P/Q-type Ca(2+) channels that are known to be expressed preferentially in cerebellar Purkinje cells and are involved in triggering neurotransmitter release. Based on these data, we conclude that CYP2J9 is a developmentally regulated P450 that is abundant in brain, localized to cerebellar Purkinje cells, and active in the biosynthesis of 19-HETE, an eicosanoid that inhibits activity of P/Q-type Ca(2+) channels. We postulate that CYP2J9 arachidonic acid products play important functional roles in the brain.

Mechanism and signal transduction of 14 (R), 15 (S)-epoxyeicosatrienoic acid (14,15-EET) binding in guinea pig monocytes.

14(R), 15(S)-epoxyeicosatrienoic acid (14,15-EET) is a cytochrome P-450 monooxygenase (epoxygenase) metabolite of arachidonic acid (AA). In this study, we have identified a population of specific high affinity binding sites for 14,15-EET in the guinea pig mononuclear (GPM) cells. The results of competition studies showed that 14(R), 15(S)-EET was an effective competing ligand with a Ki of 226.3 nM followed by 11(R), 12(S)-EET, 14(S), 15(R)-EET, 14,15 thia(S)-ET, and 14,15-aza(N)-ET. The binding was sensitive to various protease treatments suggesting that the binding site is protein in nature. Cholera toxin (CT) and dibutyryl cAMP attenuated 14,15-EET binding in GPM cells. Mean binding site density (Bmax), decreased 32.0% and 19.1% by the pretreatment with cholera toxin (200 micrograms/ml) and dibutyryl cAMP (100 nM), respectively, without changing the dissociation constant. A specific protein kinase A (PKA) inhibitor, H-89, but not the PKC inhibitor K252a reversed the down regulation of 14,15-EET receptor binding caused by dibutyryl cAMP in GPM cells. Thus, the results sug-gest that the specific binding site of 14,15-EET in GPM cells be associated with a receptor that could be down regulated through an increase in intracellular cAMP and activation of a PKA signal trans-duction. We propose that the signal transduction mechanism begins with the binding of 14,15-EET to its receptor that leads to increase intracellular cAMP levels and the activation of PKA, and finally, with the down regulation of 14,15-EET receptor binding.