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R C Murphy

Publications and source records attributed to R C Murphy.

At least 19 recordsLinked to original sources
PubMed1995

Metabolism of 6-trans-isomers of leukotriene B4 in cultured hepatoma cells and in human polymorphonuclear leukocytes. Identification of a delta 6-reductase metabolic pathway.

The intermediate metabolic events which degrade hydroxy polyunsaturated fatty acids is largely unknown. Such molecules are common products of lipid peroxidation and lipoxygenase catalyzed oxidation of arachidonic acid. Metabolism of two 5,12-dihydroxyeicosatetraenoic acids, 6-trans-LTB4 (leukotriene B4), and 6-trans-12-epi-LTB4 was studied in HepG2 cells (a human-derived hepatoma cell line). Extensive metabolism was observed with a major metabolite identified as 4-hydroxy-6-dodecenoic acid for both epimers. Incubation of 6-trans-LTB4 epimers at shorter times revealed the formation of intermediate metabolites, including 6-hydroxy-4,8-tetradecadienoic acid and 8-hydroxy-4,6,10-hexadecatrienoic acid suggesting beta-oxidation as the major pathway leading to the formation of the common terminal metabolite. Two additional metabolites were structurally elucidated as 5-oxo-6,7-dihydro-LTB4 and 6,7-dihydro-LTB4 which have not been previously described. Formation of 5-oxo-6,7-dihydro-LTB4 and 6,7-dihydro-LTB4 were also observed during metabolism of 6-trans-12-epi-LTB4 in human polymorphonuclear leukocytes. Of particular interest is the metabolism of these compounds by beta-oxidation from the carboxyl terminus, a process which is not observed with leukotriene B4 or leukotriene C4. Identification of these metabolites suggested the operation of the 5-hydroxyeicosanoid dehydrogenase pathway followed by a delta 6-reductase metabolic pathway which has not been previously described. This pathway of beta-oxidation may limit the activity of various 5,12-diHETEs including nonenzymatic hydrolysis products of LTA4 and also the recently described B4-isoleukotrienes.

Carcinoma, Hepatocellular↗
PubMed1995

Metabolism of leukotriene B4 in cultured hepatoma cells.

Incubation of leukotriene B4 (LTB4) with Hep G2 cells (a human-derived hepatoma cell line) resulted in the production of several metabolites indicative of alternative pathways of LTB4 metabolism not previously observed in normal hepatocytes. The major extracellular LTB4-derived metabolites were structurally identified using mass spectrometry and ancillary techniques including electrospray ionization. The major metabolite was 10-hydroxy-4,6,8,12-octadecatetraenoic acid (10-HOTE), an unexpected metabolite which lost the hydroxy group at carbon 5 from the parent LTB4. Two other major metabolites were 3(R)-hydroxy-LTB4 and 3(S)-hydroxy-LTB4. The formation of these three metabolites revealed that beta-oxidation from the carboxyl terminus can be a significant metabolic pathway for degradation of this hydroxy unsaturated fatty acid. The normal hepatocyte LTB4-derived metabolite, 20-carboxy-LTB4, was observed as only a minor product. The metabolic profile for Hep G2 cells suggests that the efficient cytochrome P-450 pathway involved in omega-oxidation in typical hepatocytes is absent in these cells. Several minor metabolites were also identified which included dihydro products resulting from metabolism by a 12-hydroxydehydrogenase/delta 10-reductase pathway. The formation of the major metabolite reveals the operation of steps in beta-oxidation of hydroxy, unsaturated fatty acids not anticipated by previously identified steps of fatty acid beta-oxidation.

Biotransformation↗
PubMed1995

Isoleukotrienes are biologically active free radical products of lipid peroxidation.

The free radical oxidation of arachidonic acid esterified to glycerophospholipids is known to generate complex metabolites, termed isoprostanes, that share structural features of prostaglandins derived from prostaglandin H2 synthase. Furthermore, certain isoprostanes have been found to exert biological activity through endogenous receptors on cell surfaces. Using mass spectrometry and ancillary techniques, the free radical oxidation of 1-hexadecanoyl-2-arachidonoyl-glycerophosphocholine was studied in the search for products of arachidonic acid isomeric to the leukotrienes that are derived from 5-lipoxygenase-catalyzed metabolism of arachidonic acid. Several conjugated triene metabolites were chromatographically separated from known 5-lipoxygenase products and structures characterized as 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid esterified to the glycerophosphocholine backbone. We have termed these products as B4-isoleukotrienes. Following saponification some, but not all, B4-isoleukotrienes were found to exert biological activity in elevating intracellular calcium in Indo-1-loaded human polymorphonuclear leukocytes. This activity could be blocked by a leukotriene B4 receptor antagonist. An EC50 of approximately 30 nM was determined for one unique B4-isoleukotriene with a relative retention index of 2.54. We have shown that free radical processes can lead to the formation of biologically active isoleukotrienes in glycerophosphocholine liposomes, and we propose that B4-isoleukotrienes may also be formed in membrane glycerophospholipids as a result of lipid peroxidation during tissue injury. Such B4-isoleukotrienes could then mediate events of tissue damage through activation of leukotriene B4 receptors on target cells.

Arachidonic Acid↗
PubMed1995

Identification of functional platelet-activating factor receptors on human keratinocytes.

Platelet-activating factor (PAF) is a potent inflammatory mediator that has been shown to be produced by human keratinocytes and is thought to play a role in cutaneous inflammation. Immunofluorescence and radioligand binding studies were used to characterize PAF receptors (PAF-R) on human keratinocytes and the human epidermoid cell lines A-431 and HaCaT. Indirect immunofluorescence studies demonstrated anti-PAF-R staining of primary cultures of human keratinocytes, A-431 cells, and HaCaT cells. Primary cultures of human fibroblasts and the melanoma cell line SK-30 failed to show immunostaining above that seen with control antiserum. With indirect immunofluorescence studies of sections of normal human skin, a granular anti-PAF-R staining pattern was noted on the keratinocyte cell membranes. A-431 cells readily metabolized PAF by deacetylation-reacylation at 37 degrees C, but not at 4 degrees C. Binding studies on crude membrane preparations of A-431 cells conducted at 4 degrees C demonstrated specific binding that reached saturation by 120 min. Scatchard analysis of PAF binding data revealed a single class of high-affinity (KD = 6.3 +/- 0.3 nM) PAF binding sites. The immunofluorescence and radioligand binding sites were shown to be functional PAF-Rs, as 10 pM to 1 microM PAF increased intracellular calcium in primary cultures of human keratinocytes, A-431 cells, and HaCaT cells, whereas PAF treatment of primary cultures of human fibroblasts or the melanoma cell line SK-30 did not result in changes in the intracellular calcium concentration. The structurally dissimilar PAF-R antagonists CV-6209, Ro19-3704, and alprazolam all inhibited the PAF-induced calcium changes in A-431 cells. The CV-6209 inhibition was seen at doses that competed with the PAF binding to these cells. These studies provide the first evidence for the presence of a functional PAF-R expressed on human keratinocytes, suggesting that this lipid mediator may play an important role in normal keratinocytes or in inflammatory dermatology.

Calcium↗
PubMed1992

Fast atom bombardment tandem mass spectrometric identification of diacyl, alkylacyl, and alk-1-enylacyl molecular species of glycerophosphoethanolamine in human polymorphonuclear leukocytes.

Fast atom bombardment ionization with tandem mass spectrometry of both positive and negative ions is a useful technique for the identification of intact glycerophosphoethanolamine (GPE) phospholipids, providing information as to polar head group and fatty acyl substituents. In the identification of GPE molecular species, positive ion neutral loss scanning for 141 units was attempted to confirm the presence of the phosphoethanolamine polar head group. This scan was found to discriminate against the abundant subclass of phospholipids having an 1-O-alk-1'-enyl linkage, termed plasmalogens, as well as 1-O-alkyl ether species. The neutral loss process is suggested to involve attack of a carbonyl oxygen from either sn-1 or sn-2 on the sn-3 methylene carbon with loss of neutral phosphoethanolamine. Using FAB/MS/MS alone, it is not possible to differentiate between plasmalogens and other 1-O-alkyl ether molecular species having the same molecular weight. The combination of mild acid hydrolysis, which selectively hydrolyzes the labile 1-O-alk-1'-enyl bond, with subsequent FAB/MS/MS distinguished species of these distinct subclasses. Using these techniques and precursor ion scans for the arachidonoyl carboxylate anion, m/z 303, the arachidonic acid containing glycerophosphoethanolamine molecular species were identified and the relative abundance of arachidonoyl plasmalogen, alkylacyl, and 1,2-diacyl GPE molecular species in the human polymorphonuclear leukocyte (neutrophil) was determined to be 75.4%, 12.1%, and 12.5%, respectively. These values were not significantly different from that reported in the literature using conventional methodology.

Arachidonic Acid↗
PubMed1992

Reversible membrane association of neutrophil 5-lipoxygenase is accompanied by retention of activity and a change in substrate specificity.

Ionophore activation of the human polymorphonuclear neutrophil results in eicosanoid synthesis and the accumulation of inactive 5-lipoxygenase in a membrane compartment. We report here that inhibition of self-inactivation of 5-lipoxygenase in ionophore-treated neutrophils with the reversible inhibitor zileuton, results in the accumulation of active 5-lipoxygenase in the membrane fraction. In zileuton plus ionophore-treated cells, 77% of the specific activity of the cytosolic enzyme from resting cells was diverted to the membrane fraction compared to 22% of the activity translocated when ionophore alone was used to activate the neutrophils. Accumulation of active membrane-associated 5-lipoxygenase was inhibited and reversed by the 5-lipoxygenase translocation inhibitor MK-886. The membrane-associated 5-lipoxygenase was two times more efficient in the production of leukotriene A4 from arachidonate-derived 5-hydroperoxyeicosatetraenoic acid than the cytosolic enzyme. Unlike the cytosolic enzyme, membrane-associated 5-lipoxygenase could metabolize 12(S)- and 15(S)-hydroxyeicosatetraenoic acid to 5(S),12(S)- and 5(S),15(S)-dihydroxyeicosatetraenoic acid, respectively. The ability to metabolize hydroxy fatty acids was dependent upon 5-lipoxygenase-activating protein association, but was lost if 5-lipoxygenase was eluted from the membrane by MK-886. These studies reveal for the first time that significant quantities of active 5-lipoxygenase can be detected in the membrane fraction of activated neutrophils and show that membrane association can alter the substrate specificity of 5-lipoxygenase which is further evidence for the role of the membrane-associated enzyme in the synthesis of 5-lipoxygenase metabolites.

Arachidonate 5-Lipoxygenase↗
PubMed1992

Time-dependent utilization of platelet arachidonic acid by the neutrophil in formation of 5-lipoxygenase products in platelet-neutrophil co-incubations.

The biosynthesis of leukotrienes is known to occur through a series of complex processes which, in part, can be influenced by cell-cell interactions. Several studies have suggested that arachidonic acid availability is a major limiting step for leukotriene biosynthesis and that its transfer between cells can represent a significant source of this precursor. Accordingly, effect of time and source of arachidonic acid on transcellular leukotriene synthesis was studied in mixed platelet/neutrophil populations challenged with the calcium ionophore A23187. A time-dependent contribution of platelet-derived as well as neutrophil-derived arachidonate was found in the selective formation of neutrophil 5-lipoxygenase metabolites. Utilization of platelet or neutrophil arachidonate was followed by incorporation of radiolabeled arachidonic acid into platelet or neutrophil phospholipids prior to stimulation. Specific activity of liberated arachidonic acid along with numerous 5-lipoxygenase products (including LTB4, 20-hydroxy-LTB4, 5-HETE and LTC4) was determined in order to follow mass and radiolabel. A large amount of platelet-derived arachidonic acid was released in the first 1.5 min, whereas 10 min platelet-derived arachidonate was much lower in amount but significantly higher in specific activity, suggesting different precursor pools. The platelet-derived arachidonate was heavily utilized by the neutrophils at the early time points for formation of 5-HETE and delta 6-trans-LTB4 isomers, but appeared to contribute only marginally to the constitutive metabolism of neutrophil arachidonate into LTB4. Results from these experiments suggest different pools of 5-lipoxygenase in the neutrophil and indicate a time and source dependent modulation of arachidonate metabolism in mixed cell interactions.

Arachidonate 5-Lipoxygenase↗
PubMed1992

Chemotactic LTB4 metabolites produced by hepatocytes in the presence of ethanol.

Ethanol in low concentrations significantly alters the hepatocyte metabolism of the neutrophil chemotactic lipid leukotriene B4 (LTB4). Two novel metabolites of LTB4 which are encountered only when ethanol is present, retained significant biological activity. One metabolite, 3-hydroxy-LTB4 increased intracellular free calcium in the human neutrophil at concentrations as low as 3 x 10(-10) M as well as induced shape change and adherence to albumin-coated latex beads at 10 nM. The 3-hydroxy-LTB4 and 3,20-hydroxy-LTB4 metabolites were also potent chemotactic agonists with an ED50 at 3.0 and 9.0 nM, respectively. These results suggest that the presence of ethanol can substantially alter inactivation of LTB4 by the liver and may mediate neutrophil accumulation into the liver, thereby contributing to the pathogenesis of alcoholic hepatitis even when LTB4 biosynthesis occurs at some site distant to the liver.

Animals↗
PubMed1992

Incorporation and distribution of epoxyeicosatrienoic acids into cellular phospholipids.

The different regioisomers of epoxyeicosatrienoic acids derived from cytochrome P-450 monooxygenase are readily esterified into phospholipids of mastocytoma cells. Incorporation of 14,15-epoxyeicosatrienoic acid was concentration-dependent, with Km = 1.1 microM and Vmax = 36 pmol/min/10(7) cells. Half-maximal incorporation occurred in 30 min, reaching a steady-state concentration of 470 pmol/10(6) cells. This was slightly lower than the values for arachidonic acid (665 pmol/10(6) cells) or 5-hydroxyeicosatetraenoic acid (554 pmol/10(6) cells). The distribution of 14,15-epoxyeicosatrienoic acid was preferential in the order phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylinositol greater than phosphatidyl serine much greater than neutral lipids plus fatty acids. This contrasted with 5(S)-hydroxyeicosatetraenoic acid, which was distributed primarily into phosphatidylcholine. Fast atom bombardment/tandem mass spectrometry facilitated identification of molecular species containing epoxyeicosatrienoic acids without relying on radioisotopes. Phosphatidylethanolamine plasmalogens with 16:1 or 18:2 at the sn-1 position, or an 18:0 acyl group, and phosphatidylcholine with 16:0 alkyl ether or an acyl group at the sn-1 position incorporated all possible epoxyeicosatrienoic acid regioisomers. Under basal conditions, cells eliminated 14,15-cis-epoxyeicosatrienoic acid slowly with a half-life of 34.9 +/- 7 h. Cells stimulated with calcium ionophore A23187 eliminated 14,15-epoxyeicosatrienoic acid rapidly. It was notable that its rate of release from phosphatidylcholine and phosphatidylinositol exceeded that for arachidonic acid. A coenzyme A-independent transacylase also catalyzed the transfer of epoxyeicosatrienoic acids from mastocytoma cell membranes into 1-palmitoyl-2-lysophosphatidylcholine. The cellular incorporation, release, and distribution of epoxyeicosatrienoic acids is distinctive and contrasts with most other eicosanoids, suggesting that these compounds may have both autocoid and nonautocoid functions.

8,11,14-Eicosatrienoic Acid↗
PubMed1992

Quantitation of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) produced by human polymorphonuclear leukocytes using electron capture ionization gas chromatography/mass spectrometry.

Arachidonic acid can be enzymatically oxidized at the terminal methyl group by the cytochrome P450 system found in several tissues and cells, including the human polymorphonuclear leukocyte. The omega-hydroxy metabolite, 20- hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) has recently been found to have interesting and diverse biological activities. Accurate measurement of quantities of this metabolite using physical chemical methods has not been previously described, but is necessary to assess biosynthesis of this eicosanoid from endogenous arachidonic acid. A procedure is described to quantitate 20-HETE produced by the human polymorphonuclear leukocyte following physiological stimulation using (18O2)carboxy-20-HETE as internal standard. Since the human neutrophil produces relatively small amounts of this eicosanoid, such a study required substantial sensitivity in the quantitative assay. Following stimulation of the neutrophil, cell extracts and supernatants were purified by reverse-phase high-performance liquid chromatography, catalytically reduced then derivatized to the pentafluorobenzyl ester, trimethylsilyl ethers before electron capture ionization gas chromatography/mass spectrometry. Using selected ion monitoring, the amount of 20-HETE present in a biological extract could be detected when as little as 60 pg per sample were available. Following stimulation of the human neutrophil with formyl-methionyl-leucyl-phenylalanine (0.1 microM), platelet activating factor (0.1 microM) as well as with the calcium ionophore A23187 (2 microM), 20-HETE was generated from endogenous arachidonate in concentrations of 1.2, 1.3 and 5.7 pg per 10(6) cells, respectively.

Gas Chromatography-Mass Spectrometry↗
PubMed1992

Leukotriene B4-induced human melanocyte pigmentation and leukotriene C4-induced human melanocyte growth are inhibited by different isoquinolinesulfonamides.

Leukotriene C4 (LTC4) is known to be a potent mitogen for cultured human neonatal melanocytes. We now demonstrate that leukotriene B4 (LTB4) can induce pigmentation in cultured human neonatal melanocytes in a dose-dependent fashion. The LTC4-induced mitogenesis is blocked by the cyclic nucleotide-dependent kinase inhibitor N-[2-(methyl-amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8). The LTB4-induced pigmentation is blocked by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7). We propose that LTB4-induced pigmentation and LTC4-induced mitogenesis are important in vivo signals. Their different effects in our culture system are blocked by different protein kinase inhibitors.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Biological activity and metabolism of 20-hydroxyeicosatetraenoic acid in the human platelet.

1. The cytochrome P-450 metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), was found to be a potent, dose-dependent inhibitor of platelet aggregation and inhibitor of thromboxane biosynthesis induced by arachidonic acid (IC50 5.2 +/- 1.5 microM), A23187 (IC50 16.2 +/- 5.4 microM), and U46619 (IC50 7.8 +/- 2.4 microM). 20-HETE did not inhibit thrombin-induced aggregation. 2. The human platelet metabolized 20-HETE to a series of novel metabolites formed by cyclo-oxygenase as well as lipoxygenase pathways. The structures of the metabolites were identified by mass spectrometry as 20-hydroxy-thromboxane B2, 12,17-dihydroxyheptadecatrienoic acid, 12,20-dihydroxyeicosatetraenoic acid, and 11,20-dihydroxyeicosatetraenoic acid. 3. The identification of the 11-hydroxy metabolite of 20-HETE suggests that 20-HETE is less efficiently cyclized to an endoperoxide intermediate by cyclo-oxygenase than is arachidonate. 4. Although some biological activity of 20-HETE may be related to competition with endogenous arachidonate for cyclo-oxygenase metabolism, the predominant mechanism of action of 20-HETE appears to be through antagonism of the prostaglandin H2/thromboxane A2 receptor.

Adult↗
PubMed1992

Cloning and expression of the cryIVD gene of Bacillus thuringiensis subsp. israelensis in the cyanobacterium Agmenellum quadruplicatum PR-6 and its resulting larvicidal activity.

A mosquitocidal cyanobacterium has been developed by introducing the mosquito-toxic cryIVD gene from Bacillus thuringiensis subsp. israelensis into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain PCC 7002). The cryIVD gene was introduced into the cyanobacterium on a derivative of the PR-6 expression vector pAQE19 delta Sal in which the cryIVD gene was translationally fused to the initial coding sequence of the highly expressed PR-6 cpcB gene. Coomassie blue staining and immunoblot analysis of gel-fractionated cell extract polypeptides indicate that the cpcB-cryIVD gene fusion is expressed at high levels in the cyanobacterial cells, with little or no apparent degradation of the cryIVD gene product. Larvicidal assays revealed that freshly hatched Culex pipiens mosquito larvae readily ingested the transformed cyanobacteria and that the cells proved to be toxic to the larvae.

Amino Acid Sequence↗
PubMed1992

Entry rate and metabolism of leukotriene C4 into vascular compartment in healthy subjects.

We measured the excretion of a major urinary metabolite of leukotriene (LT) C4, i.e., LTE4, during the infusion of exogenous LTC4 to enable estimation of the rate of entry of endogenous LTC4 into the bloodstream. Four healthy volunteers received 2-h intravenous infusions of vehicle alone and LTC4 at 0.063, 0.32, 1.6, and 2.9 pmol.kg-1.min-1 in random order. Urinary LTE4 was measured before, during, and up to 24 h after the infusions. The fractional elimination of LTE4 was independent of the rate of LTC4 infusion and averaged 4.3 +/- 0.9%. Calculation of the mean rate of entry of LTC4 into the circulation was found to be 0.06 pmol.kg-1.min-1. In addition, we characterized further metabolism of [14C]LTC4. The two major urinary metabolites were the omega- and beta-oxidation products (16-COOH-LTE4 and 14-COOH-LTE3), which accounted for 6-8% of the total infused amount of [14C]LTC4. We conclude that 1) LTC4 is produced at a low rate under physiological circumstances and is rapidly converted in the vasculature to LTE4, 2) changes in the urinary excretion of the latter may reliably reflect short-term changes in the rate of secretion of LTC4, and 3) measurement of the omega- and beta-oxidation products may reflect chronic changes in cysteinyl leukotriene biosynthesis.

Adult↗
PubMed1992

FMLP causes eicosanoid-dependent vasoconstriction and edema in lungs from endotoxin-primed rats.

Recruitment of inflammatory cells to the lung capillaries has been proposed as an important step in the sequence of events that lead to acute lung injury. Frequently, in the clinical setting, bacteremia and sepsis syndrome precede the acute lung failure and endotoxin priming may represent a comparable paradigm, useful for experimental pursuit. Following addition of the chemotactic tripeptide FMLP (10(-9) to 10(-6) M) to the cell-free, salt solution perfusate of isolated rat lungs, only a small degree of vasoconstriction was observed. However, in lungs isolated from rats that received 2 mg/kg intraperitoneal Salmonella enteritidis endotoxin 2 h before lung perfusion, FMLP dose dependently caused a large, transient pulmonary pressor response, edema formation, and release of large amounts of thromboxane and leukotriene B4. Since in vitro priming with endotoxin, direct vascular injury by neutrophil elastase, nor direct stimulation with FMLP of pulmonary artery rings from endotoxin-pretreated rats, mimicked the effects of in vivo endotoxin priming, we conclude that the presence of inflammatory cells in the lung capillaries accounted for the large amount of eicosanoids produced by the lungs after FMLP stimulation. In fact, by retrograde lavage of the lung circulation with a collagenase solution, previously adherent cell clumps were mobilized and identified. These cell clumps, composed of red blood cells, neutrophils, and platelets, were not seen in the vascular lavage sediment obtained from unprimed control lungs. Indomethacin, a thromboxane antagonist, AA861, a 5-lipoxygenase inhibitor, and WEB 2086, a platelet-activating factor (PAF) antagonist, reduced the thromboxane synthesis and release after FMLP (10(-7) M) in in vivo endotoxin-primed lungs. None of the inhibitors employed exclusively inhibited only one particular eicosanoid mediator but rather affected the release of several mediators, suggesting a close link between the different synthetic arachidonic acid pathways. An inhibitor of phospholipase C (2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate), NCDC, but not an inhibitor of phospholipase D (Wortmannin) or of protein kinase C (staurosporine) inhibited the FMLP-stimulated pulmonary pressure rise and eicosanoid release in endotoxin-primed lungs in vivo. Our data suggest that eicosanoids (in particular thromboxane) released from cells trapped in the lung circulation, but not from constitutive lung cells, contribute to vasoconstriction and edema formation caused by the chemoattractant FMLP in endotoxin-primed lungs.

Alkaloids↗