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R C Murphy

Publications and source records attributed to R C Murphy.

At least 163 records · Page 9Linked to original sources
PubMed1985

Preparation of oxygen-18-labeled lipoxygenase metabolites of arachidonic acid.

Plasma pseudocholinesterase and porcine liver esterase were used to catalyse the incorporation of the stable isotope oxygen-18 into the carboxyl moiety of lipoxygenase metabolites of arachidonic acid. This simple method produces eicosanoid products containing two oxygen-18 atoms; but the enzymes studied were found to display large substrate specificity in the efficiencies at which oxygen-18 could be incorporated into the lipoxygenase metabolites. Furthermore, [18O2]LTB4 was found not to back exchange during in vitro incubation with human neutrophils. The methods involved for stable isotope incorporation are simple, efficient and produce highly enriched species in a short time. By varying the type of esterase, the amount of esterase or the length of incubation highly enriched species of all eicosanoids tested could be prepared.

Animals↗
PubMed1985

Synthesis of prostaglandins and leukotrienes. Effects of ethanol.

Prostaglandins, thromboxane, and leukotrienes are metabolites of arachidonic acid that have a variety of physiological effects at low concentrations. Effects include smooth muscle contraction, platelet aggregation, platelet disaggregation, bronchoconstriction, increased capillary permeability, cardiac dysfunction, and polymorphonuclear leukocyte accumulation. Although their formation does not appear to be essential for life, these eicosanoid activities are wide ranging and have important roles in normal physiology as well as pathophysiology. At the center of eicosanoid biosynthesis lies the plasma cell membrane which serves as the arachidonic acid reservoir. It has been widely appreciated that ethanol exerts effects on the lipid bilayer, and it is not surprising that a growing body of evidence supports the concept that important interactions between ethanol and eicosanoid biosynthesis can occur. Furthermore, at various steps leading to ultimate prostaglandin, thromboxane and leukotriene production, reactive intermediates such as radicals are involved whose lifetime in the biological milieu can be profoundly modulated by ethanol.

Animals↗
PubMed1985

Novel glutathione conjugates formed from epoxyeicosatrienoic acids (EETs).

The catalysis of glutathione (GSH) conjugation to epoxyeicosatrienoic acids (EETs) by various purified isozymes of glutathione S-transferase was studied. A GSH conjugate of 14,15-EET was isolated by HPLC and TLC; this metabolite contained one molecule of EET and one molecule of GSH. Fast atom bombardment mass spectrometry of the isolated metabolite confirmed the structure as a GSH conjugate of 14,15-EET. Studies designed to determine the isozyme specificity of this reaction demonstrated that two isozymes, 3-3, and 5-5, efficiently catalyzed this conjugation reaction. The Km values for 14,15-EET were approximately 10 microM and the Vmax values ranged from 25 to 60 nmol conjugate formed min-1 mg-1 purified transferase 3-3 and 5-5. The 5,6-, 8,9-, and 11,12-EETs were also substrates for the reaction, albeit at lower rates. These results demonstrate that the EETs can serve as substrates for the cytosolic glutathione S-transferases.

8,11,14-Eicosatrienoic Acid↗
PubMed1985

Surgical delay and arachidonic acid metabolites: evidence for an inflammatory mechanism: an experimental study in rats.

In a rat skin flap model, surgical delay produced an increase in the production of arachidonic acid metabolites, with a derangement of the normal equilibrium between PGE2 and PGF2 alpha and a marked increase in the vasoconstrictive substance, thromboxane. During the delay period, there is a gradual decrease in tissue levels toward normal. Subsequent elevation of the delayed flap produces a blunted response in thromboxane production, an increase in PGE2 levels and increased flap survival. Acute elevation of an undelayed flap produced more marked elevation of all metabolites, with prolonged elevation of the vasoconstrictive PGF2 alpha and thromboxane, progressive ischemia and decreased flap survival. A key role in inflammation, mediated by these inflammatory mediators, is postulated in the mechanism of delay.

Animals↗
PubMed1985

Identification and functional characterization of leukotriene B4 20-hydroxylase of human polymorphonuclear leukocytes.

A single reaction product was formed during the incubation of 1.5 microM (5S,12R)-dihydroxy-6,14-cis-8,10-trans-[3H]icosatetraenoic acid (leukotriene B4, LTB4) for 30 min at 37 degrees C in 10 mM potassium phosphate buffer (pH 7.5) with 100 microM NADPH and the 150,000 X g supernatant of sonicated human polymorphonuclear leukocytes (PMN). The reaction product exhibited the same mobility on reversed-phase HPLC (RP-HPLC) and TLC as standard 20-hydroxy-LTB4 (20-OH-LTB4). When the omega-oxidation product of [3H]LTB4 was eluted from a Sep-Pak, resolved by RP-HPLC, and analyzed by GC/MS, its structure was determined to be solely 20-OH-LTB4. The Km of the 20-hydroxylase for [3H]LTB4 at its optimal pH of 7.5 was 0.22 +/- 0.08 microM (mean +/- SD, n = 4) and the Vmax was 48 +/- 11 pmol/min X mg of protein (mean +/- SD, n = 4). When the concentration of [3H]LTB4 was fixed at 1.5 microM, the Km for NADPH was 1.01 +/- 0.59 microM (mean +/- SD, n = 3). The location in the 150,000 X g supernatant of the LTB4 20-hydroxylase distinguishes it from the cytochrome P-450 system of liver, lung, and kidney microsomes and from the NADPH oxidase-cytochrome b-245 system of the human PMN. The LTB4 20-hydroxylase is either a unique cytochrome P-450 or other monooxygenase.

Cytochrome P-450 Enzyme System↗
PubMed1985

Alveolar inflammation and arachidonate metabolism in monocrotaline-induced pulmonary hypertension.

We tested the hypothesis that monocrotaline would activate arachidonic acid metabolism in rats. If activation occurred before the pulmonary hypertension developed, arachidonate metabolites could play a role in the hypertensive monocrotaline injury. We found that 1 wk after monocrotaline administration 6-ketoprostaglandin F1 alpha and leukotriene C4 were increased in lung lavages. At 3 wk when pulmonary hypertension was well developed, lung lavage contained increased 6-ketoprostaglandin F1 alpha and thromboxane B2. In addition, the number and activity of white blood cells in the lavages was increased, and abnormal alveolar macrophages were present. The lung extract contained slow-reacting substances including leukotriene D4. Indomethacin administration inhibited the formation of cyclooxygenase metabolites but did not prevent pulmonary hypertension. Diethylcarbamazine administration reduced the numbers and activity of inflammatory cells, increased pulmonary hypertension, prevented right ventricular hypertrophy, and inhibited the formation of slow-reacting substances. We concluded that arachidonate metabolism was activated before pulmonary hypertension developed, that the inflammatory cells in the alveolus accompanied the hypertensive process, and that diethylcarbamazine attenuated both the monocrotaline-induced inflammatory response and the pulmonary hypertension.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

Effect of sustained water diuresis on prostaglandin E2 excretion in humans.

The relationship of the renal excretion of prostaglandin E2 (PGE2) to urine flow during a water diuresis was examined using radioimmunoassay (RIA) and gas chromatography-mass spectrometry (GC-MS). Seven normal women fasted overnight were water loaded with 20 ml/kg orally, and each hour for 3 h they drank water to replace the urine volume plus 20 ml. The osmolality of the collected urines ranged from 49 to 1,073 mosmol/kg. Assay of urinary PGE2 concentrations by both RIA and GC-MS gave a correlation coefficient of 0.94. Eight normal women were then studied with a water diuresis sustained for 6 h. The excretion of PGE2 (measured with the validated RIA) increased for the 1st 2-3 h (from 1.8 +/- 0.5 to 25.8 +/- 16.6 pg X kg-1 X min-1), but then fell to base-line level by the 5th h (to 2.9 +/- 0.8 pg X kg-1 X min-1) even though the water diuresis was sustained. The urinary concentration vs. time curves for PGE2 and for the freely diffusible solute urea were compared. PGE2 concentration remained elevated for 3 h before falling (from the 100 pg/ml range to 15 pg/ml) while urea concentration decreased steadily from the 1st h. This finding suggests that the early urinary PGE2 excretion was not a washout phenomenon and is consistent with a transient increase in PGE2 synthesis. We conclude that urinary excretion of PGE2 is not a simple function of urine flow after a water load. There is a transient initial increase in urinary PGE2 excretion at the start of a water load that probably reflects an increase in renal PGE2 synthesis. However, with a sustained water diuresis, PGE2 excretion falls, indicating that an enhanced PGE2 synthesis rate is not required to sustain a water diuresis.

Adult↗
PubMed1985

Lipoxygenase products of arachidonic acid stimulate LHRH release from rat median eminence.

Exogenous arachidonic acid (AA) incubated in presence of male rat hypothalamus, shows a low rate of conversion (less than 1%) of the substrate with a major product, identified as 12-hydroxyeicosatetraenoic acid (12-HETE) by reverse phase-high performance liquid chromatography (rpHPLC) and gas chromatography-mass spectrometry (GC-MS). Furthermore, immunoreactive 12-HETE estimated after purification on rpHPLC is produced by hypothalamus slices or median eminences (MEs) incubated in absence of any exogenous precursor. The effect of 12-HETE was tested on the release of LHRH from rat MEs after a 30-min incubation and was compared to the effect of another lipoxygenase product, 5-HETE, and to the well-known stimulatory effect of prostaglandin E2 (PGE2). The three AA metabolites stimulate LHRH release. A significant stimulatory effect on LHRH release is obtained with 10(-9) M of 12-HETE and only with 10(-8) M of 5-HETE or PGE2. Furthermore, the effect of higher concentrations is different according to the eicosanoid tested. The maximal response (176% of the control) is reached with 12-HETE at 10(-8) M. No significant change is observed at 10(-7) and 10(-6) M. The response with 5-HETE is also maximal (162% of the control) at 10(-8) M but decreases significantly (only 117% of the control) at 10(-6) M. The amplitude of the response to PGE2 is larger and higher, reaching a plateau (300% of the control) at 10(-6) M. 12-HETE has no effect on somatostatin (SRIF), release, as already known for PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1985

Diethylcarbamazine inhibits acute and chronic hypoxic pulmonary hypertension in awake rats.

Leukotriene inhibitors preferentially inhibit the acute pressor response to hypoxia in isolated rat lungs. If leukotrienes are important in hypoxic pulmonary vasoconstriction, then inhibition of their synthesis could prevent the development of chronic hypoxic pulmonary hypertension. We found that diethylcarbamazine (DEC), a leukotriene synthesis blocker, reversibly inhibited acute hypoxic pulmonary vasoconstriction in the awake rat. Rats exposed to chronic hypobaric hypoxia showed pulmonary hypertension and the production of a slow-reacting substance compared with low altitude control rats. The higher of 2 doses of DEC blocked the pulmonary hypertension and the production of slow-reacting substance in all of the hypoxic rats treated. The lower dose of DEC attenuated the pulmonary hypertension in only some of the rats. Changes in weight gain, hematocrit, or generation of prostacyclin did not explain the prevention of the pulmonary hypertension by DEC. We conclude that diethylcarbamazine inhibits acute and chronic pulmonary hypertension in the intact rat.

Acute Disease↗
PubMed1985

Leukotriene inhibitors attenuate rat lung injury induced by hydrogen peroxide.

It is known that reactive oxygen species cause lung injury in association with activation of arachidonate metabolism. Because metabolites of the cyclooxygenase pathway do not appear to mediate the injury, we considered that the 5-lipoxygenase pathway might be activated and that inhibition of the pathway could interfere with the development of the injury. Thus, we sought to induce an oxidant lung injury and to prevent such injury by inhibiting lipoxygenase pathway or by blocking leukotriene action. In isolated rat lungs, glucose oxidase added to a glucose-containing, cell-free perfusate was used to produce the injurious oxygen species. Lung edema occurred and increased with increasing oxygen tension in the inspired air. Light microscopy of the lung showed perivascular fluid cuffs, and electron microscopy showed endothelial cell damage. Measurements in the lung effluent showed that concentrations of 5-hydroxyeicosatetraenoic acid (5-HETE) and of cyclooxygenase metabolites increased after glucose oxidase administration; BW 755C, U60,257, and FPL 55712 inhibited the glucose-oxidase-induced lung edema. And U60,257 also inhibited the glucose-oxidase-induced increase in 5-HETE without concomitant inhibition of cyclooxygenase metabolites. Thus, glucose oxidase via generation of active oxygen species stimulated the lung 5-lipoxygenase pathway, and inhibitors of 5-lipoxygenase protected against the oxidant lung injury. Further, in these experiments, the injury occurred in the absence of circulating blood cells and was augmented by increasing the inspired oxygen concentration.

Animals↗
PubMed1984

Metabolism of leukotrienes B4 and C4 in the isolated perfused rat lung.

Isolated rat lungs perfused with physiological buffer containing leukotriene C4 were found to rapidly metabolize leukotriene C4 to leukotriene C4 sulfoxide, leukotriene D4, and leukotriene E4. Addition of leukotriene C4 to the recirculating perfusion buffer was observed to cause a persistent increase in the pulmonary arterial pressure. Leukotriene C4 instilled into the airway of the perfused lung was also rapidly metabolized to these same products with retention of the products within the lung. In contrast, leukotriene B4 injected into the perfusion fluid was recovered unchanged in the lung effluent. Leukotriene B4 instilled into the airway of the perfused lung was observed to rapidly traverse the alveolar membranes and was recovered intact in the lung effluent. No evidence for formation of the 20-hydroxy or the 20-carboxy metabolites of leukotriene B4 by the isolated perfused rat lung was observed.

Animals↗
PubMed1984

N-acylkansosamine. A novel N-acylamino sugar from the trehalose-containing lipooligosaccharide antigens of Mycobacterium kansasii.

A novel N-acylamino sugar was isolated from the antigenic trehalose-containing lipooligosaccharides IV-VII of Mycobacterium kansasii. The native reducing sugar, its O-acetyl derivative, the methylglycoside, the O-acetylated alditol, and the de-N-acylated N-, O-acetylated alditol were all examined by high resolution 1H NMR, 13C NMR, direct probe and gas-liquid chromatography-mass spectrometry in both the chemical ionization and electron impact modes, and by high resolution mass spectrometry. The dideoxy sugar had a formula weight of 277, an empirical formula of C12H23NO6, C- and O-methyl substituents, and a N-methoxypropionyl branch. Upon alkaline hydrolysis, methoxypropionic acid was released and shown to correspond to the synthetic compound by gas chromatography and chemical ionization and electron impact mass spectrometry. The structure 4,6-dideoxy-2-O-methyl-3-C-methyl-4-(2'-methoxypropionamido)-alpha and beta-L-manno-hexopyranose, with the trivial name N-acylkansosamine, is proposed. The sugar is present in the more polar, highly antigenic lipooligosaccharides and is regarded as exclusive to M. kansasii and as its primary cell wall immunodeterminant.

Amino Sugars↗
PubMed1984

Structure elucidation of platelet activating factor derived from human neutrophils.

Platelet activating factor (PAF) synthesized by human neutrophils challenged by opsonized zymosan or calcium ionophore was isolated from cells and buffer using Bligh and Dyer extraction following the addition of tracer amounts of tritiated-PAF. The extract was subjected to TLC separation of phospholipid classes, followed by reverse phase HPLC for molecular species separation. All fractions were measured for radioactivity, biological activity and fast atom bombardment mass spectrometry. While the radioactive tracer PAF could be separated into three molecular species, PAF biological activity eluted as a single component which was characterized as 1-O-hexadecyl-2-acetyl-glycero-3-phosphocholine. The lack of molecular species heterogeneity of PAF produced in response to stimuli implies a higher degree of control of biosynthesis than previously suspected.

Adult↗
PubMed1984

Oxidation of docosahexaenoic acid by rat liver microsomes.

[1-14C]Docosahexaenoic acid (n-3) was incubated at 37 degrees C for 30 min in the presence of rat liver microsomes and 1 mM NADPH. The products were isolated using organic solvent extractions, reverse phase, and normal phase high performance liquid chromatography. Isolates were identified using ultraviolet spectroscopy, capillary gas-liquid chromatography, and gas chromatography-mass spectrometry. The major metabolites were: 19,20-, 16,17-, 13,14-, 10,11-, and 7,8-dihydroxydocosapentaenoic acids, 22-hydroxydocosahexaenoic acid, and 21-hydroxydocosahexaenoic acid. The minor metabolites were 17-hydroxy-4,7,10,13,15,19-, 16-hydroxy-4,7,10,17,19-, 14-hydroxy-4,7,10,12,-16,19-, 13-hydroxy-4,7,10,14,16,19-, 11-hydroxy-4,7,9,13,16,19-, 10-hydroxy-4,7, 11,13,16,19-, 8-hydroxy-4,6,10,13,16,19-, and 7-hydroxy-4,8,10,13,16,19 -docosahexaenoic acids. These metabolites of docosahexaenoic acid resulted from four distinct classes of oxidation, omega-hydroxylations, (omega-1)-hydroxylations, epoxidations, and lipoxygenase-like hydroxylations. The similarity of these product profiles to those reported for comparable microsomal incubations with other essential fatty acids suggest that microsome cytochrome P-450 monooxygenases were involved.

Animals↗
PubMed1984

The detrimental effect of cigarette smoking on flap survival: an experimental study in the rat.

Cigarette smoking is commonly prohibited after surgery involving flaps. This admonition against smoking has been justified on the theoretical grounds that flap survival could be decreased by the cutaneous vasoconstriction and increased platelet aggregation produced by smoking cigarettes. A smoke chamber was constructed to evaluate directly the effect of intermittent cigarette smoke exposure on the survival of a McFarlane flap on the dorsum of a rat. The O2, CO2 and temperature were monitored in the chamber during smoke exposures and consistently approximated values seen in room air. Average flap survival at 7 days in control rats not exposed to cigarette smoke was 75.2% while flap survival decreased to 40.1% in rats intermittently exposed to cigarette smoke. The difference was statistically significant (p less than 0.01). Changes in carboxyhaemoglobin levels similar to those observed in human smokers were also found in the smoke-exposed rats. These findings support the concept that post-operative cigarette smoking is detrimental to flap survival.

Animals↗
PubMed1984

Prostanoid derivatives in experimental flap delay with formic acid.

The physiological changes that allow increased flap survival after flap delay have never been clearly defined. Inflammation has been postulated as a critical factor in flap delay and prostaglandins are known to be mediators of the inflammatory response. To evaluate the role of prostanoid derivatives in experimental flap delay, 0.2% formic acid was injected under the outline of modified McFarlane flaps in 20 rats and saline was injected under the flap outline in 20 control rats. Two weeks later the flaps were raised on all the rats. Average flap survival one week after flap elevation was 65.5% after formic acid delay and 42.9% after saline delay. Biopsies were taken from the flaps both during the delay period and after flap elevation. The specimens were stained for PGE2, PGF2 alpha and thromboxane. Changes noted in the levels of these prostanoid derivatives suggest that they may indeed mediate flap delay.

Animals↗
PubMed1984

Leukotriene C4 production during hypoxic pulmonary vasoconstriction in isolated rat lungs.

Leukotriene inhibitors preferentially inhibit hypoxic pulmonary vasoconstriction in isolated rat lungs. If lipoxygenase products are involved in the hypoxic pressor response they might be produced during acute alveolar hypoxia and a leukotriene inhibitor should block both the vasoconstriction and leukotriene production that occurs in response to hypoxia. We investigated in isolated blood perfused rat lungs whether leukotriene C4 (LTC4) could be recovered from whole lung lavage fluid during ongoing hypoxic vasoconstriction. Lung lavage from individual rats had slow reacting substance (SRS)-like myotropic activity by guinea pig ileum bioassay. Pooled lavage (10 lungs) as analyzed by reverse phase high performance liquid chromatography had an ultraviolet absorbing component at the retention time for LTC4. At radioimmunoassay, and SRS myotropic activity by bioassay. LTC4 was not found during normoxic ventilation, during normoxic ventilation after a hypoxic pressor response, or during vasoconstriction elicited by KCl. Diethylcarbamazine citrate, a leukotriene synthesis blocker, concomitantly inhibited the hypoxic vasoconstriction and LTC4 production. Thus 5-lipoxygenase products may play a role in the sequence of events leading to hypoxic pulmonary vasoconstriction.

Animals↗