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D Reynaud

Publications and source records attributed to D Reynaud.

At least 55 records · Page 3Linked to original sources
PubMed1995

Extractive derivatization of the 12-lipoxygenase products, hepoxilins, and related compounds into fluorescent anthryl esters for their complete high-performance liquid chromatography profiling in biological systems.

Facile methods for the detection of intact hepoxilins, monohydroxy-epoxide derivatives of arachidonic acid formed through the 12-lipoxygenase pathway, are unavailable because (i) an absence in these compounds of an appropriate chromophore for sensitive detection by uv exists, (ii) these compounds are sensitive to the acidic workup leading to varying degrees of decomposition, and (iii) they decompose to the derivatization procedures required for their analysis by gas chromatography mass spectrometry. Herein we apply a method which introduces a fluorescent ester chromophore to the carboxylic group of the hepoxilins under conditions which do not require acidification leading to stabilization of the derivative which is extracted into an organic solvent in situ. This procedure quantitatively derivatizes hepoxilins in a biological sample, permitting the detection of hepoxilins after a TLC purification with a limit of 50 pg/sample. This method permits the profiling of 12-HETE, hepoxilins A3 and B3, as well as the corresponding epoxide hydrolase products, trioxilins A3 and B3, in a biological sample by reverse-phase HPLC with fluorescent detection. We also report on the fluorescent and mass spectral properties of these derivatives using a liquid chromatography mass spectrometry LCMS interface with thermospray ionization.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1995

Hepoxilin binding in human neutrophils.

Hepoxilins have previously been shown to release intracellular calcium in human neutrophils. We show herein that tritium-labeled hepoxilin A3 of high specific radioactivity binds to human neutrophils, and this binding is reversed by the addition of unlabeled compound, demonstrating that specific binding for these compounds exists in these cells. Specific binding of both the methyl ester derivative as well as the free acid form of the hepoxilin takes place in broken membrane fragments. In contrast only the methyl ester derivative binds specifically to the intact cells. We also show that intact neutrophils form hepoxilin A3 when incubated in the presence of the hepoxilin precursor, 12(S)-HPETE. These data demonstrate that hepoxilin synthesis can occur in the neutrophil and that hepoxilin binding sites, which appear to be located intracellularly, exist in these cells.

8,11,14-Eicosatrienoic Acid↗
PubMed1994

Hepoxilin A3 formation in the rat pineal gland selectively utilizes (12S)-hydroperoxyeicosatetraenoic acid (HPETE), but not (12R)-HPETE.

Hepoxilins A3 and B3 have previously been shown to be formed from 12-hydroperoxyeicosatetraenoic acid (12-HPETE) through a heat-insensitive ferriheme catalysis as indicated by experiments with hemin or hemoglobin, typical of nonenzymatic rearrangement (Pace-Asciak, C. R. (1984) Biochim. Biophys. Acta 793, 485-488; Pace-Asciak, C. R. (1984) J. Biol. Chem. 259, 8332-8337). In this paper, we demonstrate through use of a mixture of (12S)- and (12R)-HPETE that an enzyme system exists in the rat pineal gland that selectively utilizes (12S)-HPETE for the transformation into hepoxilin A3, while the hemin-catalyzed transformation is not selective, with both (12S)- and (12R)-HPETE being utilized. A new procedure was established to rapidly extract (in the absence of acid) and directly derivatize from the incubation mixture the products of incubation using 9-anthryldiazomethane reagent to form the 9-anthryldiazomethane derivatives of the hydroxyeicosatetraenoic acids (HETEs), hepoxilins and trioxilins, which could be detected by high performance liquid chromatography using a flow-through fluorescence detector. The following observations were made: 1) the pineal experiments showed a high preference for the formation of hepoxilin A3 with minor amounts of hepoxilin B3, while experiments with heme catalysis showed formation of both hepoxilins B3 and A3; 2) chiral phase analysis of the HETEs recovered from the incubation mixture showed the predominance of (12R)-HETE from the pineal experiments, indicating that (12S)-HPETE was selectively used up, whereas both (12S)- and (12R)-HPETE were utilized in the hemin experiments; 3) chiral phase analysis of hepoxilin A3 indicated the presence in the pineal experiments of only hepoxilin with the 11S,12S-configuration formed from (12S)-HPETE, while the hemin experiments contained both the native 11S,12S-configuration as well as the unnative or "bis-epi"-11R,12R-configuration in the epoxide group of hepoxilin A3; 4) reverse phase analysis of the epoxide hydrolase product of hepoxilin A3, i.e. trioxilin A3, showed that the pineal experiments contained only the products of enzymatic hydrolysis derived from the native hepoxilin A3, whose formation was inhibited by the epoxide hydrolase inhibitor, trichloropropene oxide. The pineal system is heat-sensitive, with formation of hepoxilins being abolished by tissue boiling, while the hemin system is insensitive to boiling. These experiments demonstrate that hepoxilin A3 formation in the pineal gland utilizes (12S)-HPETE exclusively, being transformed by a "hepoxilin synthase" primarily into hepoxilin A3.

8,11,14-Eicosatrienoic Acid↗
PubMed1994

Formation and electrophysiological actions of the arachidonic acid metabolites, hepoxilins, at nanomolar concentrations in rat hippocampal slices.

Metabolites of arachidonic acid are known to be formed in the mammalian central nervous system. When intact hippocampal slices were incubated in artificial cerebrospinal fluid, 12-hydroxyeicosatetraenoic acid and two isomers of hepoxilin A3 (8R and 8S) were released as measured by gas chromatography-mass spectrometry. These compounds were released in greater amounts in the presence of noradrenaline or when arachidonic acid was added to the slices. The neuronal actions of chemically derived preparations of 8R and 8S hepoxilins and the glutathione conjugate, hepoxilin A3-C, were examined using intracellular and whole-cell electrophysiological recordings in hippocampal CA1 neurons in vitro. All compounds had the excitatory effects of lowering spike threshold and decreasing spike frequency adaptation, and the inhibitory actions of membrane hyperpolarization, enhanced postspike train afterhyperpolarizations and increased inhibitory postsynaptic potentials or currents. A synthetic analog of hepoxilin A3-C, in which the glutathione moiety is placed at carbon position 9 instead of carbon position 11 as in hepoxilin A3-C, was inactive. The actions of the hepoxilins showed a sharp dose-response relationship, with minimal threshold or no effect at 3 nM (n = 21) and maximal effects at 10 nM (n = 33). There were no significant differences between the responses to either the 8R or 8S isomers, or between hepoxilin A3 and hepoxilin A3-C. These data suggest that hepoxilins formed by the brain have significant neuromodulatory actions.

2-Amino-5-phosphonovalerate↗
PubMed1994

Formation, metabolism, and action of hepoxilin A3 in the rat pineal gland.

The present study was undertaken to investigate the possible formation of hepoxilin A3 in the rat pineal gland and to study the potential physiological role for this compound in this tissue. Incubation of homogenates of rat pineal glands with arachidonic acid (66 microM) led to the appearance of hepoxilin A3 (HxA3) analyzed as its stable trihydroxy derivative, trioxilin A3 by gas chromatography in both the electron impact and negative ion chemical ionization modes. Endogenous formation of HxA3 is estimated to be 1.43 +/- 0.66 ng/micrograms of protein. This amount is not modified when the tissue is boiled (2.07 +/- 0.66 ng/micrograms of protein). However, the formation of this compound was stimulated to 21.26 +/- 5.82 ng/micrograms of protein when exogenous arachidonic acid was added to the homogenate. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (10 micrograms) resulted in a partial blockade of hepoxilin formation. Using [1-14C]HxA3, we demonstrated that the pineal gland contained hepoxilin epoxide hydrolase, which hydrolyzed HxA3 into trioxilin A3. This hydrolysis was inhibited by 1 mumol/L of 3,3,3-trichloropropene-1,2-oxide. In a separate study, HxA3 in the presence of 3,3,3-trichloropropene-1,2-oxide to block the hydrolysis of HxA3 decreased the production of cyclic AMP in cultured organ rat pineals after stimulation with 5'-N-ethylcarboxamidoadenosine, an A1/A2 adenosine receptor agonist. This effect is stereospecific because the (8S)-enantiomer is more active in decreasing cyclic AMP production (-88.7%) than the (8R)-enantiomer. This is the first demonstration of the presence, metabolism, and action of HxA3 in the rat pineal gland.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1993

Enzymatic formation of hepoxilins A3 and B3.

Available data obtained so far has indicated that hepoxilin formation from 12-HPETE is catalyzed by hemin and hemoglobin and is not affected through heating of these ferri-heme compounds suggestive of nonenzymatic processes. The present paper demonstrates for the first time that 12-HPETE is transformed into the hepoxilins A3 and B3 by intact cells (skin subcutis layer) and slices of several tissues (brain hippocampus and pineal gland) and that this transformation is inhibited by tissue boiling, indicating an enzymatic catalysis. The tissues employed are pharmacologically responsive to hepoxilins and hence the present data offer biochemical support of a potential biological role for the hepoxilins in these tissues.

8,11,14-Eicosatrienoic Acid↗
PubMed1993

Hepoxilin A3 inhibits the rise in free intracellular calcium evoked by formyl-methionyl-leucyl-phenylalanine, platelet-activating factor and leukotriene B4.

We have previously shown that the hepoxilins are capable of increasing the intracellular free concentration of calcium ([Ca2+]i) in human neutrophils through a pertussis toxin-sensitive, extracellular calcium-independent pathway involving the mobilization of calcium from internal stores. A subsequent hepoxilin-induced and extracellular calcium-dependent influx of calcium is observed. In an effort to investigate further the role of these compounds in the human neutrophil, we investigated their potential effects on the action of known agonists such as formyl-methionine-leucine-phenylalanine (fMLP), platelet-activating factor (PAF) and leukotriene B4 (LTB4) on the mobilization of calcium. Hepoxilis dose-dependently inhibited the increases in [Ca2+]i induced by fMLP, PAF and LTB4. The hepoxilin concentration required for inhibition was around 100 ng/ml (3 x 10(-7) M). This concentration of hepoxilin did not cause any measurable change in [Ca2+]i. The extent of inhibition of the agonist-evoked rise in [Ca2+]i by hepoxilins was proportional to the increase in the calcium response evoked by hepoxilin beyond its threshold concentration. Additional experiments were carried out to investigate the mechanism for the hepoxilin effect. Using calcium-free medium and in the presence of sufficient amounts of thapsigargin (200 ng/ml) to maximally block the calcium pump (thereby achieving a constant rate of calcium leakage from stores), hepoxilin A3 increased further this rate of calcium leakage, indicating that hepoxilin acts by rapidly draining calcium from stores. Its potential (additional) thapsigargin-like action in blocking the pump, however, cannot be ruled out by these experiments. These observations suggest that the hepoxilins may serve an important negative regulatory function in the agonist-induced mobilization of calcium in these cells by depleting calcium stores.

8,11,14-Eicosatrienoic Acid↗
PubMed1993

Hepoxilin A3 is the endogenous lipid mediator opposing hypotonic swelling of intact human platelets.

When human blood platelets are exposed to hypotonic medium they swell first but, shortly thereafter, revert toward their original volume in a process termed regulatory volume decrease (RVD). RVD is the result of an enhanced efflux of K+ and Cl- ions and associated water. Platelet RVD is controlled by a short-lived lipoxygenase-derived product (LP). By using a combination of high-performance liquid chromatography, gas chromatography-mass spectrometry, and RVD reconstitution bioassay, we show that LP is identical with hepoxilin A3. In addition we demonstrate that authentic hepoxilin A3 possesses the same biological properties on RVD reconstitution as LP and that the activity of both compounds is amplified through epoxide hydrolase inhibition with 3,3,3-trichloropropene-1,2-oxide. Therefore, we report here that volume expansion causes the formation and release of hepoxilin A3 from intact human platelets and that this hepoxilin plays a major role in volume regulation.

8,11,14-Eicosatrienoic Acid↗
PubMed1993

Facile preparation and structural determination of monohydroxy derivatives of docosahexaenoic acid (HDoHE) by alpha-tocopherol-directed autoxidation.

Polyunsaturated fatty acids are oxidized through both enzymatic and nonenzymatic reactions into hydroxy derivatives. With increasing interest in dietary manipulations through ingestion of the highly unsaturated fish oil fatty acids, eicosapentaenoic acid and docosahexaenoic acid (DHA), methods to measure their metabolism are required. In this study we report the simple and expedient alpha-tocopherol-directed autoxidative preparation of a series of monohydroxy derivatives of DHA to provide a relatively homogeneous hydroxylation along each of the double bonds of the fatty substrate. Products were purified by high-performance liquid chromatography (HPLC) and their structures elucidated by the characteristic fragmentation pattern of the hydrogenated methyl ester trimethylsilyl ether derivatives by gas chromatography-mass spectrometry. Nine products were isolated in 20.2% yield overall, ranging from 1.55 to 4.14% yield of isolated compound. These were identified as 7, 8, 10, 11, 13, 14, 16, 17, and 20-HDoHEs (monohydroxydocosahexaenoic acids). Two of these products (14- and 17-HDoHE) could not be separated under the HPLC conditions used but were clearly distinguished using selected ion chromatography by their distinct mass spectral fragmentation. This method is highly suitable for the generation of standards to investigate the metabolism of DHA in tissues.

Chromatography, High Pressure Liquid↗
PubMed1990

Characterization of A-2 receptors in postmortem human pineal gland.

We have examined the binding of the adenosine agonist radioligands [3N]N6-cyclohexyladenosine ([3H]CHA) and [3H]5'-N-ethylcarboxamidoadenosine ([3H]NECA) to membranes prepared from postmortem human pineal glands. The results showed that the A-1-specific ligand CHA did not bind to membranes. By contrast, [3H]NECA, a nonselective A-1/A-2 ligand, gave 68% specific binding of the total binding. This specific binding was nearly insensitive to the N-ethyl-maleimide pretreatment method. To characterize this binding, we used cyclopentyladenosine (50 nM). Under those conditions [3H]NECA binding at 30 degrees C was rapid and reversible; the KD determined from the kinetic studies was 141 nM. In postmortem human pineal gland, the rank order of potency of adenosine analogues and drugs competing with [3H]NECA showed the specificity for an A-2 receptor: NECA greater than 2-chloroadenosine greater than L-N6(2-phenylisopropyl)adenosine greater than 8-phenyltheophylline greater than 3-isobutyl-1-methylxanthine greater than caffeine. Guanylylimidodiphosphate (100 microM) induced a decrease in the affinity of [3H]NECA, a result suggesting the involvement of a G protein mechanism in the coupling of the adenosine receptor to other components of the receptor complex. Scatchard analysis revealed one class of binding sites for [3H]NECA with KD and Bmax ranging from 175 to 268 nM and 11.0 to 14.1 pmol/mg protein, respectively. The binding of [3H]NECA was not affected by age, sex, or postmortem delay. [3H]NECA should be a useful tool to assess brain A-2 receptor density in a variety of neuropsychiatric disorders.

Adenosine↗
PubMed1989

Adenosine analogs elevate N-acetylserotonin and melatonin in rat pineal gland.

Adenosine, S-adenosyl-L-homocysteine, adenosine analogs such as 5'-N-ethylcarboxamidoadenosine and mioflazine, a nucleoside transport inhibitor, injected intraperitoneally at 09.00 h during the light period increased melatonin levels in the pineal gland of the rat. The largest increase occurred with 1 mg/kg 5'-N-ethylcarboxamidoadenosine. A representative time-response curve with 5'-N-ethylcarboxamidoadenosine (1 mg/kg) showed a maximal peak of N-acetylserotonin and melatonin 2 and 4 h after injection, respectively. These results are discussed in relation with the possible modulation through A2 receptors of melatonin synthesis in the pineal gland.

Adenosine↗