A. Valette

Specific binding for opiate-like drugs in the placenta

Abstract The properties of human placental opiate binding sites were analysed using [3H]etorphine in terms of kinetic parameters, specificity, subcellular distribution and effect of ionic environment. In vitro these sites display a pharmacological profile similar to that of the pituitary opiate receptor site. In particular, they exhibit lower affinities for opioid peptides than do brain opiate receptors. They were detected exclusively in human placenta, in which they appeared during the first half of pregnancy.

Modulation of estrogen receptors by phorbol diesters in human breast MCF-7 cell line

Treatment of MCF-7 cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in an inhibition of cell proliferation and a reduction in the number of estrogen receptors (ER), shown by binding studies and immunoassay. The decrease in ER concentration induced by phorbol ester derivatives parallels their growth inhibitory effect. Moreover, the estrogen receptor of TPA-resistant RPh4 cells (which are insensitive to the antiproliferative and morphological effects of TPA) is not affected by TPA treatment. The reduction in ER concentration appear to be a specific phenomenon since it contrasted with the 2-fold increase in total cell protein content which included an increase in progesterone receptor (PgR). We also found that addition of TPA does not affect estrogen induction of PgR.

Placental kappa binding site: Interaction with dynorphin and its possible implication in hCG secretion

We have previously demonstrated that human placenta contains a homogenous population of kappa binding sites. The selective interaction of dynorphin with these opiate binding sites suggests a possible physiological implication of this endogenous opioid system in the placenta physiology. Ethylketocyclazocine stimulate K+-induced hCG release. This fact favours the hypothesis of a participation of placental opiate receptor on hCG secretion.

Interaction with lectin of kappa opioid binding sites solubilized from human placenta

Kappa opioid binding sites from human placenta, prelabeled with 3H-etorphine and solubilized, were retained on wheat germ agglutinin (WGA) agarose and specifically eluted with N-acetylglucosamine. No significant retention was found with other immobilized lectins, including Concanavalin A (Con A), soybean seed lectin (SBA), Pisum sativum lectin (PsA), Lens culinaris Medik. lectin (LcA), and Lathyrus tingitanus lectin(LtA). About 23% of applied kappa sites were specifically eluted from WGA agarose, less than half of the proportion of rat brain opioid binding sites eluted from the same lectin (55%). Receptors from placental extracts were compared with those from other tissues enriched in either kappa or mu sites. The proportion of applied kappa sites from guinea pig cerebellum eluted specifically from WGA agarose was 36%, whereas elution of binding sites from rat thalamus and rabbit cerebellum (enriched in mu sites) was at a level of 55%. This difference in the level of retention on and elution from WGA may reflect differences in the sugar composition of the glycoproteins of the two types of receptors. Succinylation of WGA abolished its ability to retain opioid binding sites, consistent with involvement of sialic acid. However, currently available evidence suggests that differences in retention on WGA between kappa and mu sites may be due to differences in either sialic acid or N-acetylglucosamine content or both.

Phosphorylation of the Oncofetal Variant of the Human Bile Salt-dependent Lipase IDENTIFICATION OF PHOSPHORYLATION SITE AND RELATION WITH SECRETION PROCESS

In this paper, we report, for the first time, the localization of the phosphorylation site of the fetoacinar pancreatic protein (FAPP), which is an oncofetal variant of the pancreatic bile salt-dependent lipase. Using Chinese hamster ovary (CHO) cells transfected with the cDNA encoding FAPP, we radiolabeled the enzyme with 32P, and then the protein was purified by affinity chromatography on cholate-immobilized Sepharose column and submitted to a CNBr hydrolysis. Analysis of peptides by high pressure liquid chromatography, associated with the radioactivity profile, revealed that the phosphorylation site is located at threonine 340. Site-specific mutagenesis experiments, in which the threonine was replaced by an alanine residue, were used to invalidate the phosphorylation of FAPP and to study the influence of the modification on the activity and secretion of the enzyme. These studies showed that CHO cells, transfected with the mutated cDNA of FAPP, kept all of their ability to synthesize the protein, but the loss of the phosphorylation motif prevented the release of the protein in the extracellular compartment. However, the mutated enzyme, which was sequestrated in the transfected CHO cells, remains active on bile salt-dependent lipase substrates.

Thyrotropin controls dolichol-linked sugar pools and oligosaccharyltransferase activity in thyroid cells

We previously showed that thyroglobulin (Tg) glycosylation is enhanced 1.5-fold under thyrotropin (TSH) stimulation, corresponding to an increased number of oligosaccharide chains per molecule of Tg. Now the steps involving dolichol components and oligosaccharyltransferase activity have been studied. Porcine thyroid cells were cultured on porous bottom filters with or without TSH and incubated with [14C]mevalonate. Under TSH regulation, the level of the whole of dolichol components was increased 1.25-fold without modifying their distribution. Dolichol, and free and monosaccharide-linked dolichyl-phosphate, represented respectively 40% and 45% of total dolichol components while dolichyl-pyrophosphate-oligosaccharide represented 3% only. A marked enhancement (4.2-fold) of oligosaccharyltransferase activity occurred in stimulated cells, which could correspond to the addition of the two TSH effects: stimulation of Tg synthesis (3-fold) and of Tg glycosylation (1.5-fold). The amount of lipid carriers appeared to be insufficiently increased but no component is a limiting step, suggesting that the turnover of dolichol derivatives may be increased under TSH control through their use by higher amounts of Tg.

Effect of ethanol intake on lipoprotein lipase activity in rat heart

We examined the effects of the in vivo administration of ethanol on lipolytic activities assayed in rat post-heparin heart effluents, that hydrolyse tri-, di- and monoacylglycerol. Properties of triacylglycerol lipase (TAGL) are typical of lipoprotein lipase (LPL) whereas diacylglycerol (DAGL) and monoacylglycerol (MAGL) lipase activities hydrolyse sequentially the products of LPL action. After 15 days of ethanol intake, TAGL, DAGL and MAGL activities in post-heparin heart effluents were decreased respectively by 25, 38 and 22%; after 30 days, the decreases amounted to 81, 79 and 71%. After 30 days, but not after 15 days, ethanol increased the levels of triacylglycerol in plasma. Ethanol intake concomitantly decreased TAGL and DAGL activities in post-heparin effluents and in heart tissue extracts, whereas MAGL activity was decreased only in the latter extracts. We conclude that ethanol intake causes a marked impairment in heart LPL and in two closely-related heparin-releasable activities, seemingly by altering the production of a catalytically active enzyme. A distinct heparin-unreleasable MAGL appears to exist in heart, that could be ethanol-insensitive. Overall, the results suggest that a LPL-related alteration in fatty acid supply could contribute to the toxicity of ethanol in heart.

Imbalanced triacylglycerol metabolism in fat cells from estrogen-treated rats

Abstract We investigated the effects of ethinyl estradiol (EE2) on body weight, weights of fat depots, triglyceridemia and lipoprotein lipase (LPL) activity of fat cells isolated from the parametrial fat pads of female rats. LPL activity was assayed at the cell surface, using a suspension of intact fat cells as enzyme source. The cellular uptake of fatty acids liberated during hydrolysis was also evaluated. EE2 was administered daily for 10 days by s.c. injections of 0.06 0.6 6.0 and 60 μg of hormone per animal. EE2 at and above 0.6 jug/rat reduced body weight gain and weights of fat depots. Triglyceridemia increased at doses of EE2 up to 6 μg and showed a relative reduction at 60 μg. In parallel, LPL activity assayed both as cell-bound and heparin-released enzyme and expressed relative to cell DNA, responded to the hormone according to a biphasic pattern: EE2 increased LPL activity in the dose range 0.6-6.0 μg of EE2, whereas a decrease occurred at higher doses. Fat cells incorporated as acylglycerol 56.5 ± 6.2% (mean + SD) of the amount of fatty acid liberated enzymatically during incubation, and this percentage was essentially unchanged by EE2. EE2 at 60 μg/rat caused decreases in triglyceridemia and LPL activity and, as a consequence, decreases in fat depots. By contrast, EE2 at doses up to 6 μg created a paradoxical situation where concomitant increases in triglyceridemia and LPL activity, which should have favoured fat deposition, coexisted with fat depletion. Estrogen hormones at doses below toxicity increase the secretion of triglyceride-rich lipoproteins by liver. Whereas the increase in LPL activity oberved in this study at low doses of EE2 may reflect the LPL responses to liver-borne hypertriglyceridemia, the mechanism for the concomitant fat depletion is unclear. These data emphasize the complex effects of estrogen hormones on lipid metabolism.

Pattern of response to feeding and fasting of heparin-releasable lipoprotein lipase in rat cardiomyocytes

Abstract The regulatory events whereby the amount of secreted heart lipoprotein lipase decreases post-prandially and increases during fasting are unclear. We examined whether the nutritional state influenced the lipolytic activities that hydrolyze tri-, di-, and monoacylglycerol as membrane-associated enzyme in rat cardiomyocytes. Properties of triacylglycerol lipase are typical of lipoprotein lipase whereas diacylglycerol and monoacylglycerol lipase activities hydrolyze the products of lipoprotein lipase action. We observed that: (1) membrane-bound activity levels assayed at the cell boundary were high for MAGL and much lower for TAGL and DAGL, regardless of whether cells originated from fasted or fed rats; (2) the stimulatory effects of serum were likewise similar in the fasted and the fed states; (3) isolated cardiomyocytes exhibited no constitutive secretion of active enzyme; and (4) factors determining the variations in amounts of heparin-releasable enzyme in response to nutritional changes appeared to be related to the pre-existing high (in the fasted state) or low (in the fed state) intracellular content in enzymatic activities, supporting the proposal that the secretion of active lipoprotein lipase involves disruption of intracellular vesicles and exocytosis of the enzyme, without its accumulation in the plasma membrane. On a functional basis, the results emphasize the heterogenous nature of the LPL enzymatic complex.

SPECIFIC OPIATE BINDING SITES IN HUMAN PLACENTA

Human placenta contains specific binding sites for 3H-etorphine and 3H-ethylketocyclazocine, which pharmacological characteristics correspond to the kappa receptors. These opiate binding sites, specifically found in the human species are located on brush border membranes of the syncytiotrophoblaste and their number increases between the 2nd and the 5th month of gestation.