Biomedical subjects

D Duval

Publications and source records attributed to D Duval.

At least 55 records · Page 3Linked to original sources

PubMed1990

Comparison of various methods for monitoring hybridoma cell proliferation.

The design of a strategy for the control of large scale cultures of hybridoma cells requires the use of convenient indicators to monitor properly the evolution of the biomass. In order to select appropriate indicators, we have measured in parallel, in bulk cultures of mouse hybridoma cells, the evolution of several metabolic parameters together with those of cell density and viability. We observed that flow cytometry analysis gives an early indication of the proliferative capacity of the cell population. Determination of metabolic rates (i.e. glucose, glutamine, amino acid, consumption, lactic acid or ammonium production) adequately indicates the current metabolic status of the cells. Indeed, a sharp decrease in these metabolic rates occurs rapidly following nutrient deficiency. Finally, measurements of lactate dehydrogenase (LDH) and DNA fragments released into the culture supernatants accurately reflect the kinetics of cell death.

Amino Acids↗

PubMed1990

Effect of isoproterenol on lipid metabolism and prostaglandin production in cultures of newborn rat heart cells, under normoxic and hypoxic conditions.

Catecholamines are known to exert deleterious effects on heart cells and to provoke biochemical alterations similar to those observed during myocardial infarction. In order to investigate the mechanisms of these effects, we have studied in cultures of muscle (M) and fibroblast-like (F) cells derived from newborn rat hearts, the action of isoproterenol on membrane lipid metabolism and on prostaglandin production. We showed in F cells that beta-agonist stimulation produced a striking loss of membrane phospholipids and a moderate hydrolysis of cell triglycerides. In addition, isoproterenol treatment induced a significant stimulation of the secretion of prostacyclin but not of prostaglandin E2 by F cells. None of these effects were potentiated by oxygen deprivation. In contrast, M cells, which are sensitive to ischemia, failed to respond to isoproterenol treatment. These results suggest that catecholamines and hypoxia may exert combined deleterious effects on heart tissue by acting separately on the different target cells in vivo.

Animals↗

PubMed1990

Effect of glucocorticoids on arachidonate metabolism and prostaglandin secretion.

The effects of glucocorticoids on eicosanoid synthesis are briefly summarized and issues that remained unresolved or controversial are described. It is also suggested, in view of the present knowledge about the effects of sex steroids on uterine prostaglandin production, that common features may exist between these actions and those of anti-inflammatory steroids.

Animals↗

PubMed1989

Biochemical identification of two types of phenamil binding sites associated with amiloride-sensitive Na+ channels.

The existence of distinct forms of the epithelium Na+ channel that differ in their sensitivity to amiloride has been repeatedly suggested by physiological data. The biochemical basis for these differences was analyzed by using phenamil, the most potent inhibitor known so far for the epithelium Na+ channel. [3H]Phenamil of high radioactive specific activity (30 Ci/mmol) was prepared and used to titrate [3H]phenamil binding sites in pig kidney membranes. Kinetic experiments, equilibrium binding studies, and competition experiments indicated the presence in crude membrane preparations of two classes of independent binding sites. A first binding site was characterized by a high affinity for phenamil (Kd1 = 0.4 nM) and for amiloride (Kd1 = 0.1 microM). A second binding site recognized phenamil and amiloride with lower affinities [Kd2(phenamil) = 28 nM, Kd2(amiloride) = 4 microM]. The ratio of the respective amounts of low- and high-affinity binding sites was 14 +/- 2 in different membrane preparations (range: 6-22). The two types of binding sites for [3H]phenamil copurified and were still observed after purification of the epithelium Na+ channel to homogeneity. These results indicate that at least two types of pharmacologically distinguishable Na+ channels exist in the kidney. They correspond either to two isoforms of the apical Na+ channel or to one single type of channel under two different states of covalent regulation.

Amiloride↗

PubMed1989

Mouse embryo fibroblast proliferation and prostaglandin production in medium supplemented with fetal bovine serum or serum substitutes (Ultroser SF and G): role of glucocorticoids.

The growth of DBA/2 mouse embryo fibroblasts, as well as their prostaglandin (PG) production, was compared under 3 different culture conditions: RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2% Ultroser SF (steroid-free) or with 2% Ultroser G (containing steroids). The effect of the absence or presence of glucocorticoids on both parameters was more precisely investigated. In FBS-supplemented cultures, dexamethasone had a stimulatory effect on cells characterized by a slow growth rate, whereas it markedly inhibited proliferation in rapidly growing fibroblasts. The experiments carried out with serum substitutes (Ultroser SF and G) strongly corroborated the role of the absence or presence of glucocorticoids on fibroblast proliferation. Manipulations of glucocorticoid concentrations in Ultroser SF by adding 5 x 10(-8) M dexamethasone or in Ultroser G by adding 10(-6) M RU 486 reversed the effect of the absence of glucocorticoid in the first case, or in the latter case the effect of the presence of glucocorticoid on both cell growth and PG production. Progesterone had no effect by itself. Our results emphasize the importance of performing complete kinetic studies to investigate the effect of a given factor on cell proliferation in vitro, since glucocorticoids may have opposite effects on fibroblast proliferation according to their cell growth pattern in vitro.

Animals↗

PubMed1989

Effect of dexamethasone on arachidonate metabolism in isolated mouse thymocytes.

In order to investigate the effects of glucocorticoids on arachidonate release and metabolism in mouse thymocytes, we have studied both the action of dexamethasone on arachidonate release from pre-labeled cells and its effect on tracer uptake and metabolism. Our results indicate, first, that dexamethasone failed in this experimental model to affect phospholipase activity; second, that glucocorticoids are able to inhibit the transformation of the precursor into prostaglandins and to block simultaneously its acylation into phospholipids; finally that isolated thymocytes secrete significant amounts of 12-HETE, and that this secretion is unaffected by steroid treatment.

Animals↗

PubMed1989

Effect of exogenous prostaglandins and nonsteroidal anti-inflammatory agents on prostaglandin secretion and proliferation of mouse embryo fibroblasts in culture.

During wound healing, the positive and negative modulation of fibroblast proliferation may be due, in part, to the high prostaglandin concentration of the inflammatory exudates. In vitro, PGF2 alpha has been shown to stimulate, whereas PGE2 inhibits, the growth of different fibroblast cell lines. Therefore, we have investigated the effect of exogenous prostaglandins (PGs) and of various nonsteroidal anti-inflammatory drugs (NSAIDs) on the proliferation and the prostaglandin (PG) synthesis of normal mouse embryo fibroblasts. PGF2 alpha, 6-keto PGF1 alpha and PGE2 increase fibroblast proliferation. On the other hand, PGF2 alpha increases the synthesis of PGE2 and 6-keto PGF1 alpha while 6-keto PGF1 alpha solely inhibits PGF2 alpha release, PGE2 being inactive. The mouse embryo fibroblasts partially transform the prodrug sulindac sulfoxide in the sulfide form, which completely inhibits PG synthesis, as does indomethacin. In contrast, ibuprofen exerts a differential action, according to the type of PG measured. Among the NSAIDs tested, only sulindac (sulfoxide or sulfide) stimulates fibroblast proliferation and this effect appears independent of an alteration of PG synthesis. Therefore, in this model of normal mouse embryo fibroblasts, while endogenous prostaglandins are not involved in the control of cell proliferation, exogenous PGs have the ability to alter fibroblast growth and PG synthesis.

6-Ketoprostaglandin F1 alpha↗

PubMed1989

A new ex vivo method for the study of nasal drops on ciliary function.

Any pharmaceutical nasal preparation should respect the physiological function of the mucociliary transport system and should undergo testing to this effect. An experimental protocol has been developed using the guinea pig in order to assess the effects of commercial nasal drop preparations on mucociliary function. The method presented here consists of applying in vivo the test solution on the nasal respiratory epithelium. After a specified contact time and following rapid sacrifice of the animal, the mucosa is removed; the beating frequency of the cilia is then recorded ex vivo by micro-photo-oscillography. The method is sensitive to compounds known to diminish mucociliary function as sodium mercurothiolate inhibits ciliary movement of the nasal epithelium ex vivo. This inhibition of ciliary movement is long-lasting, although reversible. This method can be used to test the action of intranasally administered pharmaceutical preparations on mucociliary function. Commercially available solutions of the nasal vasoconstrictors tymazoline, fenoxazoline or oxymetazoline do not alter ciliary movement ex vivo at dose levels equal to or greater than those clinically utilized. ATP significantly enhances nasal ciliary frequency in instances where a low basal rate occurred. Thus, this method can be used for the testing of the maintenance of nasal ciliary function in the presence of compounds and preparations which will be applied into the nostrils. The advantages over previous techniques include a closer approach to the therapeutic utilization and the maintained physiological conditions of the mucosa during drug administration.

Adenosine Triphosphate↗

PubMed1989

Effect of oxygen deprivation on metabolism of arachidonic acid by cultures of rat heart cells.

To investigate the mechanisms responsible for the impairment of phospholipid metabolism observed in ischemic cells, we have studied the effect of conditions simulating ischemia on the metabolism of arachidonic acid (AA) by muscle (M-) and nonmuscle (F-) cells isolated from newborn rat hearts and cultured separately. In muscle cells, oxygen deprivation induces a significant stimulation of the release of [14C]AA from prelabeled cells associated with a preferential redistribution of [14C]AA into cell triglycerides but not formation of radioactive prostaglandins. Moreover, the fatty acid content of phospholipids, as measured by capillary gas chromatography, appears markedly reduced in ischemic myocardial cells. This fact may be related to phospholipase stimulation during ischemia as suggested by the antagonistic effect of mepacrine or p-bromophenacyl bromide. In contrast, oxygen deprivation failed to induce any significant alteration of AA metabolism in fibroblast-like heart cells. Our results indicate that these cultures of newborn rat heart cells, which exhibit many of the features observed in intact organ during ischemia, may represent a useful experimental model to investigate the pharmacological control of the membrane phospholipid turnover.

Aerobiosis↗

PubMed1989

Comparative rheological profile of rat gastric and duodenal gel mucus.

Methods for measuring the adhesiveness, plasticity, viscoelasticity and spinnability of mucus microsamples have been developed. The rheological properties of the rat gastric and duodenal gel mucus have been analyzed and compared. Using a controlled stress rheometer (Carri-Med), flow and creep experiments showed that gastroduodenal mucus exhibits a typically non-newtonian, pseudoplastic and viscoelastic behaviour. The apparent viscosity (7,800 +/- 11,000 Pa.s) and yield stress (24.9 +/- 8.5 Pa) of gastric mucus were significantly higher than the duodenal mucus viscosity (39 +/- 160 Pa.s) and yield stress (12.9 +/- 2 Pa). Spinnability of gastric mucus, measured with a Filancemeter (SEFAM), was significantly lower (4.9 +/- 2.5 mm) in comparison to duodenal mucus (6.9 +/- 1.5 mm). Adhesive properties of gastric mucosa (analyzed with the platinum ring method) were not significantly different in comparison to duodenal mucus (99.9 +/- 31.5 mN/m and 92.8 +/- 11.2 mN/m, respectively).

Adhesiveness↗

PubMed1988

The Na+/Ca2+ antiporter in aortic smooth muscle cells. Characterization and demonstration of an activation by phorbol esters.

The Na+/Ca2+ antiporter is present in aortic smooth muscle cells of the A7r5 cell line. Imposing an outward Na+ gradient to the cells promoted a 45Ca2+ uptake component which was sensitive to amiloride derivatives and insensitive to blockers of the voltage-dependent Ca2+ channel. The Ca2+ uptake system was dependent on intracellular Na+ concentration; it was inactive when Li+ replaced intracellular Na+ and it was electrogenic. Flow cytometric analysis of cells that had been loaded with the Ca2+ indicator indo-1 showed that all conditions that promoted Ca2+ influx led to corresponding increases in the free cytoplasmic Ca2+ concentration. Treatment of the A7r5 cells with phorbol myristate acetate, a known activator of protein kinase C (Ca2+/phospholipid-dependent enzyme), led to a two-fold activation of the system and to larger intracellular Ca2+ transients when cells were shifted to Na+-free solutions. Activation was observed at all intracellular Na+ concentrations. Changing the activity of the Na+/Ca2+ system did not affect the size and duration of intracellular Ca2+ transients elicited by the Ca2+ mobilizing hormone vasopressin. It is concluded that the Na+/Ca2+ antiporter in smooth muscle cells is a target for protein kinase C but that the system is not involved in the regulation of Ca2+ transients induced by vasopressin.

Animals↗

PubMed1988

Effect of cortisol on the plasma and lymphoid tissue distributions of tritiated glucocorticoids in C57BL/6 mice.

The mechanism of action of very high doses of corticosteroids, such as those administered as bolus doses in the treatment of inflammatory and immune diseases or those currently used in rodents to isolate the small proportion of medullary thymocytes considered to be corticoresistant, is still undefined. The possible existence of selective local concentration by some tissues, particularly lymphoid organs, cannot be excluded. Therefore, using C57BL/6 mice, the kinetics of lymphoid tissue and plasma radioactivities after i.p. injection of steroids, either alone or with an excess of non-radioactive cortisol hemisuccinate (up to 10 mg/animal, i.e. 500 mg/kg) were studied. There was a rapid and dose-dependent retention of [3H]corticosterone and [3H]cortisol in the thymuses of cortisol-treated compared with control animals. The spleen also appeared to be capable of accumulating steroids. However, when the tissue/plasma ratio of [3H]steroid concentration and the change in extracellular space in the presence of an excess of nonradioactive cortisol were taken into consideration, only the thymus was able to concentrate steroids above concentrations in the plasma. Moreover, this effect did not appear to be specific for glucocorticoids, since tracer accumulation was also observed when sex steroids were used as tracers. The cells of the reticulo-endothelial system may, in part, be responsible for this phenomenon of steroid concentration in lymphoid organs.

Animals↗

PubMed1988

Mouse embryo fibroblasts in culture: characteristics of arachidonic acid metabolism during early passages.

In a model of mouse embryo fibroblasts in culture, which is characterized by a very rapid decrease in rate of cell proliferation during early passages, we determined the production of prostanoids either by following the transformation of the radioactive precursor [14C]-arachidonic acid or by radioimmunoassays. Our results demonstrate that mouse embryo fibroblasts in culture produce spontaneously substantial amounts of PGE2 and 6ketoPGF1 alpha as well as trace amounts of PGF2 alpha and of lipoxygenase derivatives (Hetes). We investigated the stability of the different types of prostaglandins produced. We showed that 6ketoPGF1 alpha and PGF2 alpha were stable in vitro, whereas PGE2 as a consequence of its solubilization in an aqueous medium was metabolized by 50% over a 24 h period, independently of the presence of the cell, thus leading to a constant underestimation of real PGE2 concentration. However, comparison of the patterns of prostaglandin production among subcultures of different orders was possible, and showed that the total amount of prostaglandin produced as well as the relative proportions are fairly identical during the first three passages, although the cell proliferation pattern rapidly decreases among the serial subcultures. These results suggest that prostaglandin production does not represent in our experimental model an autocrine means of regulating cell growth.

6-Ketoprostaglandin F1 alpha↗

PubMed1987

In vivo competitive autoradiographic study of [3H]corticosterone and [3H]aldosterone binding sites within mouse brain hippocampus.

The binding sites for [3H]corticosterone (3HB) and [3H]aldosterone (3HA) within the hippocampal area of the mouse brain have been studied by autoradiography in competition experiments. Excess unlabelled aldosterone (A) or corticosterone (B) both abolished the nuclear accumulation of radioactivity within neurons observed after injection of either 3HA or 3HB. Experiments where a subcutaneous injection of a "pure glucocorticoid' RU26988 was given before injection of 3HA alone showed a marked accumulation of radioactivity within neuronal nuclei of the hippocampus suggesting the presence of 3HA binding sites distinct from classical type II glucocorticoid receptors. In addition, when RU26988 was given before the injection of 3HA associated with a 30- or 100-fold excess of either A or B, the cell nuclear accumulation of radioactivity was no longer observed. These results showed that in our in vivo experimental conditions, B displayed the same ability as A to occupy 3HA binding sites, supporting the view that in mouse hippocampal neuronal nuclei, the aldosterone-binding and corticosterone-preferring sites represent the same molecular entity.

Aldosterone↗

PubMed1987

Treatment of canine pulmonary hypertension: effects of norepinephrine and isoproterenol on pulmonary vascular pressure-flow characteristics.

Pulmonary vascular flow resistive properties may be described by mean pulmonary arterial pressure (PAP)-cardiac output (CO) plots. The slope of the PAP-CO relationship defines the incremental resistance and the extrapolated pressure intercept defines the effective outflow pressure. We investigated effects of norepinephrine (11 dogs) and isoproterenol (seven dogs) on the pulmonary vascular PAP-CO relationship in a model of pulmonary hypertension produced by injection of autologous blood clots. Multiple PAP-CO coordinates were obtained with and without drug infusion. CO was varied by opening systemic arteriovenous fistulas. PAP-CO relationships were well described by a linear equation (mean r value .964 +/- .032). Isoproterenol increased mean CO by 61% (p less than .01), and calculated pulmonary vascular resistance (PVR) decreased by 31% (p less than .05), corresponding to a 35% decrease (5.1 +/- 2.0 to 3.3 +/- 0.9 mm Hg X liter X min-1; p less than .01) incremental resistance. In the first seven dogs to receive norepinephrine, despite a 25% increase in blood pressure (p less than .01) no significant effects on CO, PAP, PVR, or PAP-CO relationship were observed. In the next four dogs, norepinephrine was infused at a lower dose to increase blood pressure 50% and a higher dose to ensure an increase in CO. In both conditions, calculated PVR fell (p less than .05) compared with that before norepinephrine. However, measured incremental resistance and effective outflow pressure did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Dexamethasone-induced stimulation of arachidonic acid release by U937 cells grown in defined medium.

Since the presence of serum in culture media has been shown to alter prostaglandin production, as well as to interfere with the action of anti-inflammatory drugs, we have studied the effect of dexamethasone, a potent steroidal anti-inflammatory drug, on the metabolism of arachidonic acid by human monocyte-like cells (U937) grown in a fully defined medium. Under these culture conditions, dexamethasone (10(-6) M, 24 h) induced a marked stimulation of the release of unmetabolized arachidonic acid into the culture medium. The steroid also induced an inhibition of cell proliferation which became significant only after 48 h of treatment. The accumulation of arachidonic acid in the medium after steroid treatment was associated with a significant inhibition of cell acyltransferase activity, suggesting that steroids may also act upon arachidonic acid metabolism at sites other than those of phospholipase activity.

Acyltransferases↗

PubMed1986

[Inhibitory action of glucocorticoids on the synthesis of prostaglandins].

In most of the cell types hitherto investigated in vitro, glucocorticoids have been shown to decrease the synthesis of prostaglandins and of lipoxygenase derivatives of arachidonic acid. This effect seems to be related to the induction of protein inhibitors of phospholipase A2. Several of these inhibitors, called lipocortins, have recently been identified in leucocytes, macrophages and rat renomedullary cell cultures. However, systemic treatment with glucocorticoids in animals and in man has generally failed to induce a significant decrease in either urinary excretion or plasma concentration of prostaglandins. These results suggest that inhibition of prostaglandin synthesis is not the only mechanism explaining the anti-inflammatory action of glucocorticoids and raise the question of the physiological role of lipocortins.

Animals↗

PubMed1986

Thymic hormone-containing cells. VII. Adrenals and gonads control the in vivo secretion of thymulin and its plasmatic inhibitor.

The influence of adrenals and gonads on the intrathymic production and the circulating level of thymulin was evaluated in young adult mice. Adrenalectomy (Adx) and gonadectomy (Cx) induce a temporary decrease of thymulin serum level. One simultaneously notes, as a compensatory phenomenon, an increase in the thymic content of the hormone-producing cells. The decrease of serum thymulin levels after Adx and Cx is at least partially due to the appearance of low m.w. thymulin-inhibitory molecules. The fact that thymectomy prevents the appearance of these inhibitors suggests that the effects of Adx and Cx could be explained by a negative control by sex hormones of the synthesis or activity of thymulin inhibitors produced or controlled by the thymus. Specific hormone replacement therapy of castrated/adrenalectomized animals normalized thymulin serum level and thymic content. Such correction was also spontaneously observed after 4 mo, suggesting that other mechanisms (e.g., an influence of the hypothalamus-hypophysis axis) might be involved in the endocrine control of thymic hormone secretion.

Adrenalectomy↗