Estrogen sulfotransferase in human placenta

Human placental estrogen sulfotransferase (ESFT) was partially purified from the term placental cytosol by (NH4)2SO4 precipitation and agarose gel chromatography. Additional purifications caused a rapid loss of the enzyme activity. The activity was abolished by isoelectrofocusing but partially retained by chromatofocusing. The value of pI of human placental ESFT is 5.8 and the same value was obtained for bovine adrenal ESFT. The enzyme protein was able to bind to the affinity resin, estradiol-17-hemisuccinyl-1,2-diaminododecane sepharose 4B, but difficult to be extracted by estradiol (E2). The extract of the affinity resin showed one major protein band at 68,000 dalton on SDS-polyacrylamide gel electrophoresis. Kinetic studies using partially purified ESFT revealed that E2 is the best substrate for this enzyme. The relative rate of sulfurylation of E2, estrone, estriol and dehydroepiandrosterone at 4 microM (Km for E2) is 1, 0.3, 0.08 and 0.08, respectively.     

Effects of prostaglandins on cyclic nucleotide phosphodiesterase activity in the human term placenta

Slices of human full-term placentas, obtained by elective cesarean section, were incubated in the absence or presence of prostaglandins (PGs) and the cyclic AMP phosphodiesterase (cAMP PDE) activity was measured. PGE1 and PGI2 were shown to stimulate cAMP PDE activity. The effect of PGE1 is related to an increase in the Vmax of the low Km activity without alteration of this apparent Km. Several findings suggest that the cAMP PDE is activated by its own substrate; PGE1 and PGI2, promote an increase of cAMP formation which is observed before the cAMP PDE activation. Dibutyryl cAMP or theophylline also activate cAMP PDE. In contrast, PGF2 alpha does not influence either adenylate cyclase or AMP PDE. In addition, we found that the ability of the placenta to degrade cAMP, increases after parturition. PG levels are higher in the foeto-placental unit during labor, and a causal relationship between these two phenomena is possible. Our data supporting the concept of hormonal control of cAMP PDE is consistent with the hypothesis that an accelerated cAMP metabolism in placenta contributes to the maintenance of a constant equilibrium of the cyclic nucleotide levels in the foeto-placental unit.     

AMP-deaminase from human term placenta

AMP-deaminase from human term placenta was chromatographed on a phosphocellulose column and physico-chemical and immunological properties of the purified enzyme were investigated. At physiological pH7.0, in the absence of regulatory ligands (control conditions) studied AMP-deaminase manifested sigmoid-shaped substrate saturation kinetics, with half-saturation parameter (S_0.5) value of about 7 mM. Addition of important allosteric effectors (ATP, ADP or orthophosphate) modified kinetic properties of studied AMP-deaminase, influencing mainly the value of S_0.5 parameter. Micromolar concentrations of stearylo-CoA inhibited potently the enzyme making it no longer sensitive towards 1 mM ATP-induced activation. SDS-PAGE electrophoresis of the purified enzyme revealed presence of 68 kDa protein fragment, reacting with anti-(human) liver AMP-deaminase antibodies. Experimental results presented indicate that liver type of AMP-deaminase is an enzyme form present in human term placenta.     

Dopamine-stimulated adenylate cyclase in human term placenta

The effect of dopamine on adenylate cyclase activity was investigated in slices of human term placentas. Dopamine elicited a dose-dependent stimulation of cAMP formation with a ED50 value of about 1 X 10(-6)M dopamine and an increase of 110% over the control with 1 X 10(-4)M dopamine. (-)-Epinephrine and (-)-norepinephrine also increased placental cAMP formation. Apomorphine displayed a slight but non-significant stimulatory effect while bromocriptine was not effective. SCH 23390, a selective antagonist of dopamine D1 receptors caused a dose-dependent decrease of the dopamine activation. In contrast, the dopamine increase of cAMP was unaffected by beta- and alpha-adrenergic blocking drugs and by the D2 selective antagonist, (-)-sulpiride. These data indicate that dopamine stimulates cAMP formation in human term placenta through a specific mechanism via D1 dopaminergic receptors positively coupled to adenylate cyclase.     

HMG-CoA reductase activity in the microsomal fraction from human placenta in early and term pregnancy

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) mevalonate: NADP oxidoreductase (CoA acylating; EC 1.1.1.34) in microsomes from early- and term-pregnancy placenta has been found to be 24 +/- 2 and 6 +/- 3 pmol/min per mg protein, respectively. Inactivation of the enzyme required the addition of ATP and Mg2+ and was dependent on the time of preincubation. Reactivation of the enzyme was also dependent on the incubation time and prevented by the presence of fluoride--a phosphoprotein phosphatase inhibitor. These data suggest that (despite a low activity) placental HMG-CoA reductase is covalently modulated via the phosphorylation-dephosphorylation system. The conversion of [14C]acetate and [3H]mevalonate into digitonin precipitable placental sterols indicates that the lower reductase activity in term, than in early, placental microsomes is accompanied by a less active conversion of [14C]acetate in this tissue.     

Oxytocinase-immunohistochemical demonstration in the immature and term human placenta

Summary Oxytocinase (cystine aminopeptidase) was purified from human retroplacental serum by a combination of fractional precipitation, hydroxylapatite chromatography and gel exlusion chromatography on Sephadex G-200. The purified enzyme possessed a specific activity of 980 mIU/mg using L-cystine-di-p-nitroanilide as substrate. This represented a 3200 fold concentration from the starting material in an overall yield of 12%. Antibodies against oxytocinase were raised in rabbits and the -globulin fraction labelled with fluorescein isothiocyanate prior to its use in the immunofluorescence histochemical localization of the enzyme in human placental tissue. Oxytocinase was confined to the syncytiotrophoblastic cells of normal term, and immature placentas as well as in placentas from patients suffering from severe toxaemia. Specific immunofluorescence was also present in the outer margins of the chorion and to a lesser extent in the amnion.This work was financed by a grant from The Medical Research Council of New Zealand.     

Nitric oxide synthase activity in human trophoblast, term placenta and pregnant myometrium

To investigate the possible role of nitric oxide (NO) produced locally or intramurally in the quiescence of the pregnant myometrium, nitric oxide synthase (NOS) activity was measured in samples from first trimester (villous, and non villous-trophoblast), term placenta and pregnant myometrium. Trophoblast tissue was obtained from psychosocial termination of pregnancy (9 – 12 weeks' gestation) whereas placenta and myometrium, from the same patient, at deliveries by Caesarean section. NOS activity was measured in both cytosolic and particulate fractions by the formation of ¹⁴C-citrulline from ¹⁴C-arginine. Western immunoblotting was used to identify the endothelial NOS (eNOS) and neuronal (nNOS) isoforms. The activity of NOS in particulate fractions from all preparations was considerably higher than the cytosolic fractions. Activity in all fractions except the myometrium was highly Ca-dependent. More than 50% of particulate NOS from the myometrium was Ca-independent. NOS activity was highest in the villous trophoblast and there was a significant difference between the villous and non-villous trophoblast. In placenta and myometrium, NOS was 2–4 fold and 20–28-fold lower than the villous trophoblast, respectively. Western blot analysis showed clearly eNOS in the particulate fraction and a weak eNOS band in the cytosolic fractions, whereas nNOS was not detectable in any of the fractions. In view of the marginal activity of NOS in the myometrium, NO produced by the trophoblast and placenta could play a significant role in maintaining uterine quiescence by paracrine effect.     

The cytoplasmic 60 kDa progesterone receptor isoform predominates in the human amniochorion and placenta at term

Background  

The mechanism that initiates human parturition has been proposed to be 'functional progesterone withdrawal' whereby the 116 kDa B-isoform of the progesterone receptor (PR-B) switches in favour of the 94 kDa A-isoform (PR-A) in reproductive tissues. Recently, other PR isoforms, PR-S, PR-C and PR-M generated from the same gene have been identified and partially characterised.     

Characterization of parathyroid hormone-related protein in the human term placenta.

To characterize parathyroid hormone-related protein (PTHrP) in the human placenta, we measured PTHrP-like immunoreactivity (PRP-LI) in the term placenta and studied the elution profiles of placental tissue extracts on Sephadex G-75 chromatography with a specific RIA. We also examined the gene expression of PTHrP mRNA by Northern blot analysis and the localization of PRP-LI in the placenta by immunohistochemistry. The amount of PRP-LI in placental extracts (n = 7) was 20.9 +/- 2.2 pg/g wet tissue (mean +/- SE). Dilution curves of placental tissue ran parallel to those of synthetic PTHrP (1-34) standards. Sephadex G-75 gel chromatography demonstrated two major PRP-LI peaks; the first peak was eluted around the molecular size between 10 kilodaltons (Kda) and 20 Kda and the other around 5 Kda. Northern blot analysis of PTHrP mRNA extracted from placental tissues showed a major hybridization signal around 18S. PTHrP immunohistochemistry showed PRP-LI staining in the cytoplasm of syncytiotrophoblasts and stroma cells (Hofbauer cells) in the term placenta. These results suggest that syncytiotrophoblasts and stroma cells in the term placenta synthesize PTHrP in two major molecular forms, 10 Kda-20 Kda and around 5 Kda.     

The activity of glutaminase in the human placenta

Homogenates and extracts of human placenta are able to desamidate glutamine by means of an enzyme which has the properties of glutaminase. Placental glutaminase is activated by phosphate. Its pH optimum lies at 9.0.A method for its assay in placental homogenate is described. It was found that the glutaminase activity decreases toward the end of pregnancy. At this time, the activity, expressed as Q_NH3 (N), amounts to 23.7 ± 6.7.Some quantitative aspects of glutaminase activity in the human placenta and kidney are discussed.