Biomedical subjects

David F Grant

Publications and source records attributed to David F Grant.

13 recordsLinked to original sources

PubMed2006

Distribution of soluble epoxide hydrolase, cytochrome P450 2C8, 2C9 and 2J2 in human malignant neoplasms.

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme with a C-terminal epoxide hydrolase activity and an N-terminal phosphatase activity. Arachidonic acid epoxides, previously suggested to be involved in apoptosis, oncogenesis and cell proliferation, are generated by cytochrome P450 epoxygenases and are good substrates of the sEH C-terminal domain. In addition, the N-terminal phosphatase domain hydrolyzes isoprenoid mono- and pyrophosphates, which are involved in cell signaling and apoptosis. Here we provide a comprehensive analysis of the distribution of sEH, CYP2C8, 2C9 and 2J2 in human neoplastic tissues using tissue micro-arrays. The human neoplastic tissue micro-arrays provide a well-controlled side by side analysis of a wide array of neoplastic tissues and their surrounding normal tissue controls. Many of the neoplastic tissues showed altered expression of these enzymes as compared to normal tissues. Altered expression was not limited to the neoplastic tissues but also found in the surrounding non-neoplastic tissues. For example, sEH expression in renal and hepatic malignant neoplasms and surrounding non-neoplastic tissues was found to be significantly decreased, whereas expression was found to be increased in seminoma as compared to normal tissues. Our study warrants further investigation of the role of altered expression of these enzymes in neoplastic tissues.

Aryl Hydrocarbon Hydroxylases↗

PubMed2006

NMR and HPLC-MS/MS analysis of synthetically prepared linoleic acid diol glucuronides.

Hydroxylated fatty acids are important mediators of many physiological and pathophysiological processes in a variety of human tissues. Recent evidence shows that in humans many of these are ultimately excreted in the urine as the glucuronide conjugates. In this paper we describe a general approach for the chemical synthesis of glucuronide conjugate derivatives of fatty acids. The synthesis strategy employs three steps (epoxidation, hydrolysis and glucuronidation) using methyl linoleate as a model non-hydroxylated starting compound. Hydroxylated starting compounds would require only the glucuronidation step. NMR and HPLC-MS/MS experiments were used to help determine the structure of the synthesized glucuronide conjugates and to identify fragmentation product ions useful for discriminating positional isomers in biological samples. This synthetic strategy should prove useful for generating analytical standards in order to identify and quantify glucuronide metabolites of hydroxylated fatty acids in humans.

Chemical Phenomena↗

PubMed2006

Effects of human soluble epoxide hydrolase polymorphisms on isoprenoid phosphate hydrolysis.

Soluble epoxide hydrolase (sEH) is highly expressed in human liver and contains a C-terminal epoxide hydrolase activity and an N-terminal phosphatase activity. Endogenous C-terminal hydrolase substrates include arachidonic acid epoxides, however, data are limited regarding possible endogenous substrates for the N-terminal phosphatase. Possible sEH N-terminal substrates include isoprenoid phosphate precursors of cholesterol biosynthesis and protein isoprenylation. Here, we report the kinetic analysis for a range of sEH isoprenoid substrates. We also provide an analysis of the effects of human sEH polymorphisms on isoprenoid hydrolysis. Interestingly, the Arg287Gln polymorphism recently suggested to be involved in hypercholesterolemia was found to possess a higher isoprenoid phosphatase activity than the wild type sEH. Consistent with the finding of isoprenoid phosphates as substrates for sEH, we identified isoprenoid-derived N-terminal inhibitors with IC50 values ranging from 0.84 (+/-0.9) to 55.1 (+/-30.7) microM. Finally, we evaluated the effects of the different isoprenoid compounds on the C-terminal hydrolase activity.

Enzyme Activation↗

PubMed2005

Cell-specific subcellular localization of soluble epoxide hydrolase in human tissues.

Soluble epoxide hydrolase (sEH) is a phase-I xenobiotic metabolizing enzyme having both an N-terminal phosphatase activity and a C-terminal epoxide hydrolase activity. Endogenous hydrolase substrates include arachidonic acid epoxides, which have been involved in regulating blood pressure and inflammation. The subcellular localization of sEH has been controversial. Earlier studies using mouse and rat liver suggested that sEH may be cytosolic and/or peroxisomal. In this study we applied immunofluorescence and confocal microscopy using markers for different subcellular compartments to evaluate sEH colocalization in an array of human tissues. Results showed that sEH is both cytosolic and peroxisomal in human hepatocytes and renal proximal tubules and exclusively cytosolic in other sEH-containing tissues such as pancreatic islet cells, intestinal epithelium, anterior pituitary cells, adrenal gland, endometrium, lymphoid follicles, prostate ductal epithelium, alveolar wall, and blood vessels. sEH was not exclusively peroxisomal in any of the tissues evaluated. Our data suggest that human sEH subcellular localization is tissue dependent, and that sEH may have tissue- or cell-type-specific functionality. To our knowledge, this is the first report showing the subcellular localization of sEH in a wide array of human tissues.

Amino Acid Sequence↗

PubMed2004

Polymorphisms in human soluble epoxide hydrolase: effects on enzyme activity, enzyme stability, and quaternary structure.

Human soluble epoxide hydrolase (hsEH) has been shown to play a role in regulating blood pressure and inflammation. HsEH consists of an N-terminal phosphatase and a C-terminal epoxide hydrolase domain. In the present study, we examined the effects of polymorphisms in the hsEH gene on phosphatase activity, enzyme stability, and protein quaternary structure. The results showed that mutants Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the Arg103Cys/Arg287Gln (double mutant) have significantly lower phosphatase activity compared to the most frequent allele (MFA) of hsEH. In addition, the Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the double mutant have significantly lower kcat/Km values. The stabilities at 37 degrees C of purified Arg287Gln and Arg103Cys/Arg287Gln mutants were also significantly reduced compared to the MFA. HPLC size-exclusion studies showed that the MFA exists predominantly as a dimer. However, the Arg287Gln and Arg103Cys/Arg287Gln mutants show increased concentration of the monomer. We conclude that the Arg287Gln polymorphism disrupts putative intra- and inter-monomeric salt-bridges responsible for dimerization.

Amino Acid Substitution↗

PubMed2004

Linoleic acid epoxide promotes the maintenance of mitochondrial function and active Na+ transport following hypoxia.

Low concentrations of arachidonic acid monoepoxides protect against ischemia/reperfusion injury. This study examined whether low concentrations of the linoleic acid monoepoxide, cis-12,13-epoxy-9-octadecenoic acid (12,13-EOA), protect renal cells against decreases in mitochondrial and transport functions induced by hypoxia/reoxygenation. Primary cultures of rabbit renal proximal tubular cells (RPTC) were pretreated with diluent or 1, 5, or 10 microM 12,13-EOA for 1 h and exposed to 2 h hypoxia/0.5 h reoxygenation in the absence of 12,13-EOA. Basal respiration, oligomycin-sensitive oxygen consumption (QO2), and ATP content decreased 31, 35 and 65%, respectively, following hypoxia/reoxygenation. Hypoxia/reoxygenation also increased mitochondrial membrane potential (DeltaPsi(m)). Pretreatment with 12,13-EOA prevented decreases in basal and oligomycin-sensitive QO2s and increases in DeltaPsi(m). Despite the protection against decreases in mitochondrial function, 12,13-EOA pretreatment did not prevent the initial decrease in intracellular ATP content following hypoxia. However, pretreatment did accelerate the recovery of intracellular ATP levels during reoxygenation. Pretreatment with 12,13-EOA also prevented hypoxia-induced decreases in active Na+ transport. Ouabain-sensitive QO2 (a marker of active Na+ transport) decreased 38% following hypoxia/reoxygenation but was maintained in RPTC pretreated with 1, 5 or 10 microM 12,13-EOA prior to hypoxia. Pretreatment of RPTC with the hydrolyzed product of 12,13-EOA, 12,13-dihydroxyoctadecenoic acid, did not have any protective effects against mitochondrial dysfunction and decreases in active Na+ transport. Thus, this is the first report demonstrating that preconditioning of RPTC with low concentrations of 12,13-EOA, but not its hydrolyzed product, maintains mitochondrial respiration, accelerates restoration of ATP levels, and prevents decreases in active Na+ transport following hypoxia/reoxygenation.

Adenosine Triphosphate↗

PubMed2004

Distribution of soluble epoxide hydrolase and of cytochrome P450 2C8, 2C9, and 2J2 in human tissues.

Soluble epoxide hydrolase (sEH) hydrolyzes a wide variety of endogenous and exogenous epoxides. Many of these epoxides are believed to be formed by cytochrome P450 epoxygenases. Here we report the distribution of sEH and cytochrome P450 epoxygenases 2C8, 2C9, and 2J2 by immunohistochemistry. A large number of different tissues from different organs were evaluated using high-throughput tissue microarrays. sEH was found in the liver, kidney, and in many other organs, including adrenals, pancreatic islets, pituitary gland, lymphoid tissues, muscles, certain vascular smooth muscles, and epithelial cells in the skin, prostatic ducts, and the gastrointestinal tract. Immunolabeling for sEH was highly specific for particular tissues and individual cell types. CYP2C9 was also found in almost all of these organs and tissues, suggesting that 2C9 and sEH are very similar in their tissue-specific patterns of expression. CYP2C8 and 2J2 were also widely distributed in human tissues but were less frequently associated with sEH. The results suggest potentially distinct pathways of endogenous fatty acid epoxide production and hydrolysis in a variety of human tissues.

Aryl Hydrocarbon Hydroxylases↗

PubMed2003

Polymorphisms in human soluble epoxide hydrolase.

Human soluble epoxide hydrolase (hsEH) metabolizes a variety of epoxides to the corresponding vicinal diols. Arachidonic and linoleic acid epoxides are thought to be endogenous substrates for hsEH. Enzyme activity in humans shows high interindividual variation (e.g., 500-fold in liver) suggesting the existence of regulatory and/or structural gene polymorphisms. We resequenced each of the 19 exons of the hsEH gene (EPHX2) from 72 persons representing black, Asian, and white populations. A variety of polymorphisms was found, six of which result in amino acid substitutions. Amino acid variants were localized on the crystal structure of the mouse sEH, resulting in the prediction that at least two of these (Arg287Gln and Arg103Cys) might significantly affect enzyme function. The six variants of the hsEH cDNA corresponding to each single polymorphism and one corresponding to a double polymorphism were then constructed by site-directed mutagenesis and expressed in insect cells. As predicted, Arg287Gln and the double mutant Arg287Gln/Arg103Cys showed decreased enzyme activity using trans-stilbene oxide, trans-diphenylpropene oxide, and 14,15-epoxyeicosatrienoic acid as substrates. Lys55Arg and Cys154Tyr mutants had elevated activity for all three substrates. Detailed kinetic studies revealed that the double mutant Arg287Gln/Arg103Cys showed significant differences in Km and Vmax. In addition, stability studies showed that the double mutant was less stable than wild-type protein when incubated at 37 degrees C. These results suggest that at least six hsEH variants exist in the human population and that at least four of these may influence hsEH-mediated metabolism of exogenous and endogenous epoxide substrates in vivo.

Animals↗

PubMed2002

Effect of linoleic acid metabolites on Na(+)/K(+) pump current in N20.1 oligodendrocytes: role of membrane fluidity.

Metabolic derivatives of linoleic acid, both monoepoxides and diols, have been reported to be toxic in humans and multiple animal tissue preparations. A previous electrophysiological study has shown these compounds produce multiple effects on the electrical activity of rat ventricular myocytes. The hydrophobic nature of these compounds suggests the possibility that these effects may be due to nonspecific lipid interactions, i.e., changes in membrane fluidity. This study investigates membrane fluidity as a possible mechanism by which linoleic acid metabolites inhibit Na(+)/K(+) pump current (I(p)). This study showed that positional isomers 9,10- and 12,13-epoxy-octadecenoic acid (EOA) and 9,10- and 12,13-dihydroxy-OA (DHOA) inhibit I(p) in a dose-dependent manner in N20.1 mouse oligodendrocytes, with greater inhibition produced by EOAs. These compounds, at 10 microM, inhibited I(p) by 4.7 +/- 1.6, 18.2 +/- 0.5, 11.7 +/- 0.5, and 25.1 +/- 0.9% for 12,13-DHOA, 9,10-DHOA, 12,13-EOA, and 9,10-EOA, respectively, in oligodendrocytes. Fluorescence recovery after photobleaching measurements showed that both DHOA isomers produced a 7-8% increase in diffusion coefficient of the probe at 10 microM, whereas the diffusion coefficient was decreased by 5 and 13% by 9,10-EOA and 12,13-EOA, respectively. There was no apparent correlation between membrane fluidity and inhibition of I(p) by these four linoleic acid metabolites. These results indicate that membrane fluidity alone cannot explain the effects of these compounds on I(p) and suggest that they have a specific interaction with the Na(+)/K(+) pump.

Animals↗

PubMed2002

Linoleic acid, cis-epoxyoctadecenoic acids, and dihydroxyoctadecadienoic acids are toxic to Sf-21 cells in the absence of albumin.

Conversion of 12,13-cis-epoxyoctadecenoic acid (12,13-EOA) to 12,13-dihydroxyoctadecenoic acid (12,13-DHOA) by soluble epoxide hydrolase has been suggested to be a critical step in mediating the toxicity of epoxidized linoleic acid. The current study tests the hypothesis that low levels of albumin in the normal culturing media of Sf-21 cells can protect these cells from exposures to 12,13-EOA, but not 12,13-DHOA. In albumin-free media, Sf-21 cells exposed to 100 microM 12,13-EOA, and 12,13-DHOA for 1 min showed significant signs of mitochondrial dysfunction which led to cytotoxicity. The addition of bovine serum albumin (BSA) at a concentration (3 microM) found in normal serum-supplemented media protected Sf-21 cells exposed to 12,13-EOA, but not 12,13-DHOA while BSA (500 microM) fully protected Sf-21 cells exposed to these fatty acids. These data resolve previous discrepancies observed among in vitro models and help clarify our understanding of how these metabolites affect human health.

Animals↗

PubMed2002

Linoleic acid metabolites act to increase contractility in isolated rat heart.

Previous in vivo studies in dogs suggest that the 9,10-monoepoxide of linoleic acid (9,10-cis-epoxyoctadecenoic acid [9,10-EOA]) has toxic cardiovascular effects that result in death at higher doses. More recent work with rabbit renal proximal tubule cells suggests that the 12,13-metabolites of linoleic acid are more toxic than the 9,10-isomers. Thus, in the current study, we tested the hypothesis that 12,13-EOA and 12,13-dihydroxyoctadecadienoic acid (12,13-DHOA) have direct adverse effects on the heart. Langendorff-perfused rat hearts were exposed to 30 microM linoleic acid, 30 microM 12,13-EOA, or 30 microM 12,13-DHOA for 60 min followed by a 30-min recovery period. As indicated by peak left intraventricular pressure and/or +dP/dt(max), all three of the agents elicited moderate increases in contractile function that peaked within 10 20 min. The effects of linoleic acid and 12,13-EOA returned to control values during the remainder of the 60-min exposure, whereas the positive inotropic response to 12,13-DHOA was maintained until washout. Sustained arrhythmias and negative inotropic actions were not observed with any of the three compounds. Subsequently, the monoepoxides were infused into conscious rats (35 mg/kg/h) while blood pressure, heart rate, and EKG were monitored for 24 h using biotelemetry techniques. The only effect observed was a slight decline in blood pressure. Thus, current data suggest that linoleic acid and its oxidative metabolites do not have direct cardiotoxic effects during acute exposure.

Animals↗

PubMed2002

Divergent effects of selective peroxisome proliferator-activated receptor-gamma 2 ligands on adipocyte versus osteoblast differentiation.

PPAR gamma is activated by diverse ligands and regulates the differentiation of many cell types. Based on evidence that activation of PPAR gamma 2 by rosiglitazone stimulates adipogenesis and inhibits osteoblastogenesis in U-33/gamma 2 cells, a model mesenchymal progenitor of adipocytes and osteoblasts, we postulated that the increase in marrow fat and the decrease in osteoblast number that occur during aging are due to increased PPAR gamma 2 activation. Here, we show that the naturally occurring PPAR gamma ligands 9,10-dihydroxyoctadecenoic acid, and 15-deoxy-Delta(12,14)-PGJ(2), also stimulate adipocytes and inhibit osteoblast differentiation of U-33/gamma 2 cells. Strikingly, 9,10-epoxyoctadecenoic acid and the thiazolidine acetamide ligand GW0072 [(+/-)-(2S,5S)-4-(4-(4-carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thaizolidineN,N-dibenzyl-acetamide] prevent osteoblast differentiation, but do not stimulate adipogenesis, whereas 9-hydroxyoctadecadienoic acid stimulates adipogenesis but does not affect osteoblast differentiation. The divergent effects of PPAR gamma 2 ligands on osteoblast and adipocyte differentiation were confirmed in primary murine bone marrow cultures using rosiglitazone and GW0072. These findings indicate that the proadipogenic and antiosteoblastogenic effects of PPAR gamma 2 are mediated by distinct regulatory pathways that can be differentially modulated depending on the nature of the ligand, and they support the idea that increased fatty acid oxidation during aging may inhibit osteoblast differentiation. Moreover, there may be selective PPAR gamma 2 modulators that block the adverse effects of fatty acid oxidation products while retaining beneficial activities such as insulin sensitization.

Adipocytes↗

PubMed2001

Identification of the gamma-aminobutyric acid receptor beta(2) and beta(3) subunits in rat, rabbit, and human kidneys.

The properties and functions of gamma-aminobutyric acid (GABA(A)) receptors in the mammalian central nervous system are well studied. However, the presence and significance of GABA(A) receptors in nonneural tissue is less clear. The goal of this study was to examine the expression and localization of the GABA(A) receptor beta(2) and beta(3) subunits in the kidney. Reverse transcriptase products from RNA isolated from rat and rabbit kidney cortex and cerebellum and rabbit S(2) segments were amplified by use of PCR and GABA(A) beta(2) and beta(3) subunit-specific primers. Sequencing of the kidney PCR products revealed that the rat kidney cortex and rat neuronal GABA(A) receptor beta(2) subunit were identical in nucleotide composition. The rabbit kidney and rabbit neuronal GABA(A) receptor beta(2) subunit were 99% identical in nucleotide composition. Sequencing of the kidney PCR products revealed that the rat kidney cortex and rat neuronal GABA(A) receptor beta(3) subunits were 93% and 95% identical in nucleotide and amino acid composition, and rabbit kidney cortex and rabbit neuronal GABA(A) receptor beta(3) subunits were 95% and 98% identical in nucleotide and amino acid composition, respectively. PCR screening of a human kidney cDNA library and sequencing revealed that the human kidney cortex and neuronal beta(3) subunits were identical in nucleotide composition. Immunoblot analysis of rat kidney cortex and brain identified immunoreactive proteins in the 55 to 57 kD region, corresponding to the GABA(A) receptor beta(2) and beta(3) subunits. Immunohistochemistry revealed cytosolic and basolateral staining of the proximal convoluted and straight tubule. These results provide compelling evidence for the expression of the GABA(A) receptor beta(2) and beta(3) subunits in the kidney of multiple species and the localization of the beta(2)/beta(3) subunits to the renal proximal tubule.

Amino Acid Sequence↗