Biomedical subjects

F Dray

Publications and source records attributed to F Dray.

At least 19 recordsLinked to original sources

PubMed1992

Active metabolism of arachidonic acid by Kaposi sarcoma cells cultured from lung biopsies (KS-3); identification by HPLC and MS/MS of the predominant metabolite secreted as the 11,12-epoxy-eicosatrienoic acid.

The development of long-term culture of AIDS-KS cells has allowed us to investigate further a possible vascular origin of Kaposi sarcoma. Taking into account the relative specificity of arachidonic acid (AA) metabolism according to cell type, the AA 'cascade' was analyzed in cultured KS-3 cells established from lung biopsies and compared to human umbilical venous endothelial (H-UVE) cells and human myometrial smooth muscle (H-MSM) cells, under basal conditions and after stimulation with vasoactive agents such as histamine or thrombin. Considering strictly the 'prostaglandin' profile given by RIAs, the metabolism of AA was closer, whilst not identical, to H-UVE than to H-MSM cells. However, evaluation of all the eicosanoids released from [3H]AA labeled KS-3 cells revealed that the predominant metabolite was not prostacyclin (PGI2), as suggested from PG RIAs, but an epoxy-eicosatrienoic acid (EET), identified as the 11, 12 isomer by HPLC and MS/MS. The synthesis of this EET is probably cytochrome P-450 monooxygenase dependent. Its potential role in the development of the KS tumor cells is under investigation.

8,11,14-Eicosatrienoic Acid↗

PubMed1991

Prostaglandin E2 and leukotriene C4-induced luteinizing hormone-releasing hormone release from immature and adult male rat median eminences in vitro: eicosanoid formation and binding parameters.

The amounts of prostaglandin E2 formed in vitro by the median eminences of adult male rats were greater than those produced by the median eminences of immature, 22 day-old rats. However, the amount of leukotriene C4 produced by the adult rat median eminences was lower than that produced by the immature rat median eminences. Analysis of the prostaglandin E2 binding parameters of hypothalamic P2 membrane fractions indicates that there are two binding components, one high affinity (RH) and one low affinity (RL) in both adult and immature rats. The maximal binding capacity of RH from adult rat membranes was significantly lower than that of immature rat membranes, correlating with greater prostaglandin E2 production by the adult rat median eminence. Only one leukotriene C4 binding site was detected in both adult and immature rat membranes. Exogenous prostaglandin E2 and leukotriene C4 both stimulated, the release of luteinizing hormone-releasing hormone to the same extent from both the adult and immature median eminences.

Aging↗

PubMed1991

Comparative study of biosynthetic human growth hormone immunogenicity in growth hormone deficient children.

The immunogenicities of six recombinant human growth hormone (rhGH) preparations, from KABI (A rhGH191 and B rhGH192), Eli Lilly (C), Nordisk (D), Sanofi (E) and Serono (F), used to treat 260 GH-deficient children, have been compared using a common specific and sensitive procedure for antibody determination. For this purpose we developed two immunoassays: a competitive liquid radioimmunoassay using 125I-rhGH, and an immunometric solid enzymoimmunoassay in which the rhGHs were immobilized. Blood samples were collected from the GH-deficient children before treatment and after 3, 6, 9, 12, 18 and 24 months of therapy. Human GH antibodies were detected in children treated with 3 of the 6 rhGH preparations. Seven percent of the patients treated with hormone A, 14% with hormone B and 22% with hormone C formed antibodies against the respective rhGH. Differences in capacity and affinity of the hGH antibodies were observed between these anti-GH-positive groups. They could be divided into 2 groups according to their immunopotency. One group (7, 14 and 6% of the patients treated with hormones A, B and C, respectively) developed anti-hGH antibodies with very low binding capacities (30-100 fmol/ml). The other group (16% of the patients treated with hormone C) developed IgG-type antibodies to hGH with higher binding capacities (200-1,200 fmol/ml) and a measurable binding affinity (Ka = 10(8) M-1). These hGH antibodies partially inhibited the binding of labeled GH to its specific liver membrane receptor. However, because of their low titer, they did not inhibit growth in the treated children.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1991

Practical method for optimizing radioimmunoassay detection and precision limits.

A model of the competitive radioimmunoassay standard curve, based on the Law of Mass Action, has been developed and used in conjunction with experimental and counting errors to predict the assay detection limit and precision profiles. We verified the model with hapten radioimmunoassays performed in our laboratory. The resulting computer program can be used to determine the optimum antiserum concentration--depending on its affinity--and labeled-antigen concentration--according to its specific activity and nonspecific binding. The graphical representation of this model provides radioimmunologists with a practical tool for assay optimization.

Antigen-Antibody Reactions↗

PubMed1991

Elimination of undesired cross-reactants by using mixtures of antibodies: experimental and theoretical evaluations of hapten radioimmunoassays.

This study shows that the specificity of radioimmunoassays can be improved by including a second antibody raised against an undesired cross-reactant. In a radioimmunoassay of prostaglandin E2 (PGE2) involving a monoclonal antibody, the cross-reactivity with 6-keto-prostaglandin E1 (6kPGE1) was decreased from 20% to 2% by including a high concentration of a polyclonal anti-6kPGE1. A similar increase in specificity was obtained in the assay of a larger hapten, luliberin (luteinizing hormone releasing hormone); the cross-reactivity of a luliberin analog was decreased 20-fold. Equations derived from the Law of Mass Action were used for the mathematical analysis and for the computer simulation of changes in assay affinity and specificity according to the quantity and quality of the mixed antibodies. The model gave values that agreed well with experimental data; it promises to be quite useful in designing specific radioimmunoassays.

Antibodies, Monoclonal↗

PubMed1991

[Prostaglandins and reproduction. I. Physiological aspects].

1) Eicosanoids are a family of polyunsaturated 20-carbon fatty acids and their metabolites. The metabolites are produced by three enzymatic pathways: the cyclooxygenase pathway, giving prostaglandins (PGs), the lipoxygenases and the epoxygenases pathways. Arachidonic acid (C20:4) is the most common fatty acid precursor in mammalian cells, where it is incorporated, as an ester, into the membrane lipid complex. 2) The eicosanoids have a variety of effects on several cell activities, including secretion, muscle contraction, cell growth and differentiation. The type of effect--stimulation or inhibition--depends on the metabolite, its concentration, the metabolic activity of the cell and the involvement of other humoral factors. 3) The message may be transmitted via a specific membrane receptor to a specific transduction system: the adenyl or guanyl cyclase system and mobilization of free cytosolic Ca2+, or via the participation of membrane ion channels. Depending on which is involved, the eicosanoid message applies to the cell in which it was synthesized or to neighboring cells (autocrine or paracrine action). 4) The eicosanoids, especially the PGs, take part in many reproductive processes; in the hypothalamic-pituitary axis, particularly through the synaptic modulation by PGE2 (stimulation of LHRH secretion and inhibition of noradrenaline secretion); in the ovary: follicle maturation and luteolysis; in the oviducts: gamete migration; in the uterus: ovum implantation and parturition. 5) PGs seem to have a variety of species-dependent effects on the normal onset of labor. In sheep there is an increase in fetal cortisol, a drop in the progesterone/estradiol ratio and increased PG synthesis. In women, there is an increase of phospholipase A2 activity in amnios and uterus with an increase of PGE2 in the first tissue and of PGF2 alpha in the second one. 6) The PGs from the seminal fluid have several actions. They effect fertility by acting on the female genital tract or on the spermatozoa. PGE1 and PGE2 influence the fertilization capacity. PGs also effect the process of ejaculation (inhibition of the stimulatory effect of noradrenaline). Finally, they effect the immune responses: PGEs and 19 hydroxy PGEs immuno-suppressive characteristics.

Animals↗

PubMed1990

Localization of arachidonate 12-lipoxygenase in parenchymal cells of porcine anterior pituitary.

12-Lipoxygenases oxygenate arachidonic acid producing its 12S-hydroperoxy derivative and are well known as platelet and leukocyte enzymes. When a peroxidase-linked immunoassay of the enzyme according to the avidin-biotin method was applied to the cytosol fractions from various parts of porcine brain, a considerable amount of the enzyme was found in the anterior pituitary. The enzyme level (about 200 ng/mg cytosol protein) corresponded to about 6% of the enzyme content in porcine peripheral leukocytes. Posterior and intermediate lobes showed about one-tenth of the enzyme level of anterior pituitary. Other parts of porcine brain contained the 12-lipoxygenase in amounts below 7 ng/mg cytosol protein. The cytosol fraction (0.7 mg of protein) of anterior pituitary produced 12S-hydroxy-5,8,10,14-eicosatetraenoic acid from 25 microM arachidonic acid in about 34% conversion at 24 degrees C for 5 min, giving a specific enzyme activity about 3 nmol/min/mg protein. Furthermore, various octadecapolyenoic acids were oxygenated almost as fast as the arachidonate 12-oxygenation. When anterior pituitary was investigated immunohistochemically with anti-12-lipoxygenase antibody, most of the immunostained cells were certain parenchymal cells with granules, which were not blood cells. These biochemical and immunohistochemical results provide a good reason for considering that 12-lipoxygenase does play an important role in pituitary function.

Animals↗

PubMed1990

Leukotriene C4-induced release of LHRH into the hypophyseal portal blood and of LH into the peripheral blood.

Intracerebroventricular (i.c.v.) administration of leukotriene (LT) C4 at doses of 2, 0.5 and 0.2 micrograms/rat significantly stimulated (3-12 fold) the release of LH into the peripheral blood of male rats. Injection of anti-LHRH serum had no effect on LTC4-stimulated LH release, but did block PGE2- stimulated LH release. I.c.v.- infused LTC4 also stimulated the release of LHRH into the hypophyseal portal blood. This is the first report of an in vivo action of LTC4 on the release of a hypothalamic releasing factor (LHRH) and a pituitary hormone (LH). These observations, plus in vitro results, clearly show that LTC4 stimulates LH release by acting on both the hypothalamus, causing LHRH release, and on the pituitary. Then the action of LTC4 on LH release in vivo is quite different from the indirect action of PGE2.

Animals↗

PubMed1990

Contribution of arachidonate metabolites to basal and thyrotropin releasing-hormone-stimulated release of prolactin from purified lactotrophs in primary culture.

Among the different biochemical pathways which have been suggested to play a role in the control of prolactin (Prl) release from anterior pituitaries, arachidonate and its metabolites have been proposed to be involved in the process of Prl release. In this study we investigated the contribution of arachidonate metabolites to both basal and TRH-stimulated Prl release from perifused lactotrophs in culture (derived from pituitary glands of lactating female rats), which exhibit a high sustained release of Prl in absence of inhibitory input. Inhibition of the general oxidative metabolism of arachidonate by 10(-5) M ETYA or of the arachidonate lipoxygenase metabolism by 10(-5) M NDGA decreased basal Prl release to 45 +/- 10% (n = 3) and 36 +/- 4% (n = 6) of the control release, respectively. Indomethacin, an inhibitor of the cyclooxygenase pathway, was without effect. Of the lipoxygenase metabolites tested at 10(-6) M only 15-HPETE and 15-HETE induced Prl release. 15-HETE elicited prolactin release in a concentration dependent manner with a maximal effect at 10(-6) M (10.72 +/- 3 ng/ml vs control 5.1 +/- 0.8 ng/ml, n = 3). The quantity of Prl release induced by TRH was markedly decreased in the presence of NDGA. However, the fraction of Prl release elicited by TRH, calculated as a percentage of the amount of Prl released prior to TRH application, was similar under control conditions, and in the presence of NDGA. In contrast, inhibition of the protein kinases A and G by H8 (10(-5) M) failed to alter basal Prl release but inhibited the effect of TRH by 58 +/- 6% (n = 3). These data suggest that in absence of inhibitory inputs the high sustained release of Prl observed in cultures of lactotrophs derived from lactating female rats depends on the availability of lipoxygenase metabolites, and that the blockade of lipoxygenase reduces the absolute amount of Prl released by TRH without suppressing the ability of TRH to stimulate Prl release.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed1990

Effect of castration on eicosanoid and catecholamine-induced luteinizing hormone-releasing hormone release in vitro.

The castration of adult rats four weeks before experimentation did not alter either the basal production of prostaglandin (PG) E2 (intact rats 1157 +/- 160 pg/mg protein, castrated rats 1093 +/- 90 pg/mg protein) or the A 23187-induced production of PGE2 (1591 +/- 209 or 1701 +/- 286 pg/mg protein respectively) or that of leucotriene (LT) C4 (474 +/- 33, 389 +/- 39 pg/mg protein). Castration significantly reduced the absolute amounts of luteinizing hormone-releasing hormone (LHRH) released under basal conditions (594 +/- 56 pg/mg protein to 34% of intact controls) and after A 23187 stimulation (to 28% of controls). The LHRH released by the median eminence (ME) of castrated rats in the presence of PGE2 (10(-6) M), LTC4 (10(-8) M), norepinephrine (NE) (10(-5) M) or dopamine (DA) (10(-5) M and 10(-4) M) was significantly lower than the LHRH released by intact animals in the presence of these factors. However the relative values for LHRH release (e.g. as % of controls) indicated that the degree of stimulation was identical in intact and castrated rats (2-3 times). These data suggest that the absence of gonadal steroids resulting from long-term castration does not block the stimulatory action of eicosanoids or catecholamines on the release of LHRH by the ME of adult male rats in vitro. Furthermore, castration does not interfere with the biosynthesis of PGE2 and LTC4.

Animals↗

PubMed1990

Validation in rat plasma of a direct radioimmunoassay for a luteinizing hormone-releasing hormone antagonist (BIM 21009)

Rabbits were immunized with [Ac-D-beta-Nal1-, D-p-Cl-Phe2, D-Phe3, D-Arg6, Phe7, D-Ala10]LHRH (BIM 21009) coupled to bovine serum albumin using bis-diazotized benzidine. The best antiserum had an affinity of 5. 10(-10) M and a specificity directed against the C-terminal part of the molecule. The antiserum was not affected by native LHRH but reacted to some extent with detergents. Assay of free-peptide plasma after gel filtration on Ultrogel AcA 34 showed apparent immunoreactivity associated with albumin and lipoproteins. The sensitivity of direct assay was 0.4 ng/ml. Measurements of BIM 21009 after s.c. injection in rats showed the resistance of the peptide to elimination. The specificity of the determinations in plasma were checked by High Performance Liquid Chromatography.

Amino Acid Sequence↗

PubMed1990

Effects of N-acetyl-aspartyl glutamic acid and sodium cromoglycate on leukotriene B4 secretion by human leukocytes.

Peripheral leukocytes from allergic subjects were treated for 30 min with sodium cromoglycate (SCG) or with N-acetyl-aspartyl glutamic acid (NAAGA) and challenged for leukotriene B4 (LTB4) production with calcium ionophore A 23187. NAAGA significantly inhibits LTB4 release at concentrations of 10(-2) M (-86%), 5 x 10(-3) M (-49%) and 10(-3) M (-34%), while SCG was not able to block LTB4 production within the range of 10(-2)-10(-4) M. In spite of the fact that SCG and NAAGA are chemically unrelated and that both show antiallergic properties, only NAAGA is able in this model to block production of LTB4, a chemical mediator strongly involved in inflammatory and hypersensitivity reactions.

Bronchodilator Agents↗

PubMed1990

Detection of synenkephalin, the amino-terminal portion of proenkephalin, by antisera directed against its carboxyl terminus.

Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C-terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.

Adrenal Gland Neoplasms↗

PubMed1990

Intracerebroventricular injection of platelet-activating factor induces secretion of adrenocorticotropin, beta-endorphin and corticosterone in conscious rats: a possible link between the immune and nervous systems.

To investigate whether platelet-activating factor (PAF) exerts an indirect action on immune cells by altering the secretion of hypothalamic-pituitary-adrenal (HPA) axis products, the effects of intracerebroventricular (i.c.v.) PAF on adrenocorticotropic hormone (ACTH), beta-endorphin and corticosterone blood levels were examined in adult male rats. Hormones were radioimmunoassayed on blood samples from conscious or ether-anesthetized rats after i.c.v. injection of PAF or vehicle into the left lateral ventricle. PAF induced significant increases in these stress-related hormones under both, basal and ether-induced stress conditions. The analysis of the time course response to PAF of hormone release into the blood of unrestrained rats revealed that: i.c.v. injection of 5.4 nmol PAF resulted in rapid increases in ACTH and beta-endorphin, at the latest within 15 min after the onset of injection. The maximal response of both hormones was reached within 45 min after the onset of injection and was followed by an elevation of plasma corticosterone. Hormone release is related to the PAF dose infused, the lowest effective PAF concentration was 1 nmol. The stimulatory effect of PAF on ACTH and beta-endorphin secretion was strongly decreased in rats previously treated with purified anti-rat corticotropin-releasing factor (CRF) antibody. These results, associated with the in vitro demonstration that PAF increases CRF release from incubated rat median eminence, strongly support the hypothesis that the stimulatory action of PAF on the secretion of HPA axis products is mediated at least partly, by stimulating hypothalamic CRF release.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗

PubMed1990

Epoxygenase products of arachidonic acid are endogenous constituents of the hypothalamus involved in D2 receptor-mediated, dopamine-induced release of somatostatin.

The epoxyeicosatrienoic acids (EETs) were discovered as products of a cyclooxygenase/lipoxygenase-independent, cytochrome P-450 catalyzed metabolism of arachidonic acid (AA) termed the "epoxygenase" pathway. The rat hypothalamus is able to synthesize EETs from exogenous AA, and 5,6-EET has been found to release the neuropeptide somatostatin (SRIF) from hypothalamic nerve terminals of the median eminence (ME). In the present study, hypothalami from male rats were examined for the presence of endogenous EETs, using chemical, chromatographic, and mass spectral analysis procedures. The samples were initially separated in a C18 Sepralyte column, fractionated on TLC plates, and purified by reverse phase HPLC. Thereafter, they were esterified (pentafluorobenzyl esters) and subjected to negative ion chemical ionization/gas chromatography (GC)/mass spectral (MS) analysis. The GC retention time and the MS fragmentation patterns revealed the presence of a mixture of 8,9-, 11,12- and 14,15-EETs; instability of 5,6-EET during the isolation protocol precluded its identification. Total hypothalamic EET concentration was estimated to be 120 ng/g wet tissue. The 8,9-regiosomer released SRIF from ME nerve terminals with an ED50 of 5 x 10(-12) M; Dopamine (DA) and the D2 receptor agonist PPHT, but not the D1 receptor agonist SKF-38393, induced SRIF release from the ME. This effect was blocked by clotrimazole and ketoconazole, two inhibitors of microsomal cytochrome P-450 function and AA epoxygenase in particular. In contrast, the inhibitors failed to affect the increase in SRIF release induced by 8,9-EET. These results indicate that: 1) in addition to cyclooxygenase and lipoxygenase products, epoxygenase metabolites of AA are endogenous compounds of the hypothalamus, and 2) EETs may mediate the increase in SRIF release from hypothalamic neurons induced by the interaction of DA with D2 receptors.

Animals↗

PubMed1989

Adrenocortical response following acute neurogenic stimuli is mediated by CRF-41.

The present study examined whether neurogenic stimuli activate the pituitary-adrenal axis via CRF-41. Adult male rats were exposed to photic, acoustic or sciatic nerve stimulation. At 4, 15, and 30 min following the onset of stress, animals were sacrificed, trunk blood collected and the median eminence removed. At 4 min following the stress onset, there was a significant decrease in CRF-41 content of the median eminence, which persisted for 30 min. Concomitant with the decrease in CRF-41 content, serum adrenocorticotropic hormone (ACTH) and corticosterone levels increased. Thus, this study demonstrates that CRF-41 released from the median eminence plays a dynamic role in mediating the ACTH and corticosterone response to neurogenic stimuli.

Adrenal Cortex↗

PubMed1989

Triton X-100 eliminates plasma proteins interference in a radioimmunoassay for luteinizing hormone-releasing hormone (LHRH) and LHRH analogues.

A method is described for the radioimmunoassay of native LHRH and DTrp6-LHRH, an LHRH analogue which does not require extraction of plasma samples. Interference by binding proteins normally present in plasma is removed by addition of Triton X-100 to the binding buffer at a concentration of 1% for LHRH and 0.15% for the LHRH analogue. This approach permits a direct estimation of the peptide level in unextracted plasma with quantitative recoveries for concentrations ranging from 0.015 to 10 ng/ml. Although the antiserum titre is reduced, the affinity of the antibody does not change at the detergent concentrations used in this study. This procedure is recommended for peptide assays in which the non-specific effects of plasma prevent a direct assay.

Animals↗

PubMed1989

Release of luteinizing hormone-releasing hormone: interrelations between eicosanoids and catecholamines.

The in vitro release of luteinizing hormone-releasing hormone (LHRH), prostaglandin (PG) E2 and leukotriene (LT) C4 from male rat median eminences (ME), was estimated by radioimmunoassay (RIA) in the presence of the catecholamines (CA), norepinephrine (NE) and dopamine (DA). NE increased the release of PGE2 in the presence and in the absence of the Ca2+ ionophore A23187 (5 x 10(-6) M), but it did not modify the A23187-induced release of LTC4 from endogenous precursors or radiolabelled arachidonic acid. DA also stimulated the A23187-induced release of PGE2 but inhibited that of LTC4. However, while NE increased both the basal and the A23187-induced release of LHRH, DA increased the basal release of LHRH and inhibited the A23187-induced LHRH release. Exogenous LTC4 cancelled the inhibitory effect of DA on LHRH release. Blockade of dopaminergic receptors with haloperidol suppressed the effects of DA on PGE2, LTC4 and LHRH release. Neither eicosanoid affected the K+-evoked [3H]DA release, whereas only PGE2 inhibited the K+-evoked [3H]NE release. We conclude that LTC4 does not interact with the noradrenergic pathway and that the stimulatory effect of both catecholamines on LHRH release involves PGE2, but the inhibitory effect of DA is associated with reduced LTC4 production.

Animals↗