Intrauterine growth restriction (IUGR) is associated with chronic fetal hypoxia, altered placental vasodilatation and reduced endothelial nitric oxide synthase (eNOS) activity. In human umbilical vein endothelial cells (HUVEC) from pregnancies complicated with IUGR (IUGR cells) and in HUVEC from normal pregnancies (normal cells) cultured under hypoxia l-arginine transport is reduced; however, the mechanisms leading to this dysfunction are unknown. We studied hypoxia effect on l-arginine transport and human cationic amino acid transporters 1 (hCAT-1) expression, and the potential NO and protein kinase C α (PKCα) involvement. Normal or IUGR HUVEC monolayers were exposed (0–24h) to 5% O2 (normoxia), and 1 or 2% O2 (hypoxia). l-Arginine transport and hCAT-1 expression, phosphorylated and total PKCα or eNOS protein and mRNA expression were quantified. eNOS involvement was tested using a siRNA against eNOS (eNOS-siRNA) adenovirus. IUGR cells in normoxia or hypoxia, and normal cells in hypoxia exhibited reduced l-arginine transport, hCAT-1 expression, NO synthesis and eNOS phosphorylation at Serine1177, effects reversed by calphostin C (PKC inhibitor) and S-nitroso-N-acetyl-l,d-penicillamine (SNAP, NO donor). However, NG-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor) reduced hCAT-1 expression only in normal cells in normoxia. Increased Thr638-phosphorylated PKCα was exhibited by IUGR cells in normoxia or hypoxia and normal cells in hypoxia. The effects of hypoxia in normal cells were mimicked in eNOS-siRNA transduced cells; however, IUGR phenotype was unaltered by eNOS knockdown. Thus, IUGR- and hypoxia-reduced l-arginine transport could result from increased PKCα, but reduced eNOS activity leading to a lower hCAT-1 expression in HUVEC. In addition, IUGR endothelial cells are either not responsive or maximally affected by hypoxia. These mechanisms could be responsible for placental dysfunction in diseases where fetal endothelium is chronically exposed to hypoxia, such as IUGR.

Dielectric Barrier Discharges (DBD) operated at atmospheric pressure and working at reduced temperatures (T < 115 degrees C) and a copper-manganese oxide catalyst are combined for the direct decomposition and the steam reforming of methanol (SRM) for hydrogen production and for the preferential oxidation of CO (CO-PROX).

Reduced oxygen level ( hypoxia) induces endothelial dysfunction and release of the endogenous nucleoside adenosine. Human umbilical vein endothelium ( HUVEC) function in an environment with 3% to 5% O(2) and exhibit efficient adenosine membrane transport via human equilibrative nucleoside transporters 1 (hENT1). We studied whether adenosine transport and hENT1 expression are altered by hypoxia in HUVEC. Hypoxia (0 to 24 hours, 2% and 1% O(2)) reduced maximal hENT1-adenosine transport velocity (V(max)) and maximal nitrobenzylthionosine (NBMPR, a high-affinity hENT1 protein ligand) binding, but increased extracellular adenosine concentration. Hypoxia also reduced hENT1 protein and mRNA levels, effects unaltered by N(omega)-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase [ NOS] inhibitor) or PD-98059 ( inhibitor of mitogen-activated protein kinase kinase 1 and 2 [MEK1/2]). Hypoxia reduced endothelial NOS ( eNOS) activity and eNOS phosphorylation at Ser(1177), but increased eNOS protein level. Hypoxia increased (1 to 3 hours), but reduced (24 hours) p42/44(mapk) phosphorylation. Thus, hypoxia-increased extracellular adenosine may result from reduced hENT1-adenosine transport in HUVEC. Hypoxia effect seems not to involve NO, but p42/44(mapk) may be required for the relatively rapid effect (1 to 3 hours) of hypoxia. These results could be important in diseases where the fetus is exposed to intrauterine environments poor in oxygen, such as intrauterine growth restriction, or where adenosine transport is altered, such as gestational diabetes.

Serotonin (5-HT) plays a pivotal role in pregnancy and a hyperserotonomic condition has been documented in pre-eclampsia. We have attempted to elucidate the possible participation of 5-HT as an aetiological factor in pre-eclampsia, by estimating the activity and expression of the 5-HT transporter (SERT) and monoamine oxidase A (MAO-A) in human placenta from full term normal (NG) and severe pre-eclamptic (PES) pregnancies. Uptake of 5-[1,2-3H] hydroxytryptamine binoxalate (specific radioactivity, 30.4 Ci/mmol) was determined in placental brush border vesicles by a rapid filtration technique. 5-HT metabolism in placental homogenate was measured using a HPLC-ECD system. Expression of SERT and MAO-A was determined by Western blot, using specific antibodies against the human SERT and MAO-A in placental tissues obtained from NG and PES. Our results, showed no significant difference in 5-HT uptake between both groups. However, 5-HT metabolism was significantly lower in placental homogenates from PES than in NG placentas, with the pathological preparations showing no MAO-A activity against 5-HT during the first 60 min of incubation (87% and 5% of metabolism of 5-HT initially added, NG and PES respectively). Western blot analysis showed a similar expression of SERT in BBMV from NG and PES. However, unlike for normal pregnancies, the expression of MAO-A in placental homogenates from PES was found to be very low, or almost negligible. These findings confirm our previous results and suggest that the higher plasma free 5-HT levels observed in severe pre-eclampsia could be mainly due to a reduction in placental MAO-A expression and activity and are not limited by the expression and uptake of 5-HT into the placental tissue.

We have attempted to elucidate the possible participation of serotonin as an etiological factor in pre-eclampsia. The transport of serotonin into vesicles from the maternal-facing brush border membrane was measured, as well as the metabolism induced by monoamine oxidase (MAO) in placental homogenate obtained from normal-term and severely pre-eclamptic placentas. Kinetic analysis of serotonin uptake by the placental brush border membrane of the syncytiotrophoblast between normally pregnant and severely pre-eclamptic subjects showed no significant difference (similar Vmax and Km values). However, the metabolism of serotonin was significantly higher in placental homogenate from normal pregnancies than in placentas from severely pre-eclamptic pregnancies. These findings suggest that the higher plasma-free serotonin levels observed in severe pre-eclampsia are mainly due to a reduction in MAO-A activity and not limited by the rate of serotonin uptake into the cells.

Isolated human placental cotyledons from normal term (37-40 weeks of gestation) and preterm (26-36 weeks of gestation) labor were perfused in vitro, and the effect of angiotensin II (ANG II) and its interaction with prostanoids was measured. In the preterm group, ANG II caused greater maximal increases in perfusion pressure than in normal term pregnancies without affecting sensitivity. Also, preparations from normal term pregnancies showed a marked development of tachyphylaxis compared to placentae from preterm pregnancies. Indomethacin (10(-6) M) increased the maximum pressor response to ANG II by 33.6% in normal term, however, in preterm placentas a 39.2% reduction was observed. Infused ANG II 10(-6) M) decreased the concentrations of thromboxane B(2) and 6-keto-PGF(1alpha) in both pregnancy groups, but this effect was not statistically different from the baseline values. In the current study, we show that the placenta of preterm pregnancies in basal conditions produce 7.6 times as much thromboxane as the normal term placenta (2,800+/-470 vs. 366.5+/-62 pg/min, respectively), without significant change in prostacyclin levels (preterm 88.6+/-11.0 vs. Term 100.6+/-30.7 pg/min). These observations provide evidence that the contribution of basally released thromboxane from placental tissue appears to contribute to abnormalities in the regulation of fetoplacental hemodynamics in premature pregnancies.

This study characterizes the reactivity of human chorionic plate vein in full-term (39.4±0.3 weeks of gestation) and preterm (34.4±0.6 weeks of gestation) pregnancy to endothelin-1 (ET-1) and attempts to characterize ET-1 receptor subtype, and the contribution of nitric oxide and cyclooxygenase products in these responses. In placental veins from full-term and preterm pregnant women, cumulative addition of ET-1 (10−10–10−6 M) caused marked and long-lasting concentration-dependent contractile responses. The mean EC50 and Emax values for ET-1-induced venoconstriction did not differ between the full-term and preterm pregnancy groups. In the veins from preterm placental preparations, the ETA receptor-selective antagonist cyclo(d-α-aspartyl-l-propyl-d-valyl-l-leucyl-d-tryptophyl (BQ123) reduced the ET-1-induced contraction by 28.6±2.4%, compared to a decline in tension of 51.2±4.2% in the full-term placental vessels. The ETB receptor-selective antagonist, N-[N-[N-[2,6-dimethyl-1piperidinyl)carbonyl]-4-methyl-l-leucyl]-1-(methoxycarbonyl)-d-tryptophyl]-d-norleucinemonosodium (BQ788), did not influence ET-1-induced contraction in placental vein from both pregnancy groups in terms of maximal contraction and sensitivity. Pretreatment with the cyclooxygenase inhibitor, indomethacin (1 μM) and the nitric oxide synthase inhibitor Nw-nitro-l-arginine (NOLA, 100 μM) did not significantly affect either the EC50 or the maximum contraction to ET-1 in veins from normal full-term and preterm preparations. The results of this study suggest that there is no correlation between ET-1-induced vasoconstriction and gestational age and that this vasoconstriction is mediated predominantly via ETA receptor subtype in both groups of pregnant women, independent of NO and eicosanoids.

1. In isolated human placental chorionic vein segments, 5-hydroxytryptamine (5-HT; 10(-8) to 5 x 10(-5) M) elicited concentration-dependent contractions with EC50 = 5.5 (5.2-5.7) x 10(-8) M) and Emax = 93.1 +/- 7.3% of 75 mM KCl-induced contraction. 2. The agonist of 5-HT2 receptors, alpha-methyl-5-hydroxytryptamine, and the selective agonist of 5-HT1 receptors, N,N-dipropyl-5-carboxamidotryptamine and 5-carboxamidotryptamine, induced pronounced concentration-related contractions, which reached 71.1 +/- 6.0%, 53.0 +/- 5.0% and 75.0 +/- 7.8% at the highest dose tested, respectively. The agonist of 5-HT3 receptor, 2-methyl-5-hydroxytryptamine, reached a maximum averaging 36.7 +/- 5.1% of the maximal response to KCl. 3. The 5-HT1 and 5-HT3 receptor antagonists, methiothepin and metoclopramide (10(-7) to 10(-6) M) did not alter the response to 5-HT. However, ketanserin (10(-7) to 10(-6) M), a 5-HT2 receptor antagonist, induced significant inhibition of the concentration-response curve to 5-HT. 4. Contractile responses to 5-carboxamidotryptamine and 2-methyl-5-hydroxytryptamine were not affected by methiothepin and metoclopramide, respectively, whereas ketanserin significantly attenuated the contractile response to these agonists. 5. In conclusions, our study shows that 5-HT2 receptors mediate contraction of the human placental vein with no obvious role for 5-HT1-like, or 5-HT3 receptors.

Pharmacologic and methodologic advances over the last decade have resulted in a body of information implicating serotonin as a mediator in the genesis of preeclamptic hypertension. Platelets contain the largest storage of serotonin in peripheral blood and have the ability to take up this amine from sourroundings, store and release it by several mechanisms. Plasma and platelet serotonin concentrations and platelet serotonin uptake have been measured in 8 non-pregnant women, 12 normal pregnant women and 8 women with severe pre-eclampsia. Plasma serotonin concentration was significantly higher in severely preeclamptic women, compared with age and gestation matched normal pregnant women. In addition, plasma serotonin concentration was directly related to systolic and diastolic blood pressure with severity of the syndrome. Furthermore, platelet serotonin concentration in women with pre-eclampsia was significantly higher than in non-pregnant controls, but it was not significantly different from the normal pregnant women. Moreover, serotonin is effectively taken up by platelets through a saturable transport process. The calculated apparent Km for serotonin uptake process did not differ significantly among non-pregnant women, normal pregnant women and women with pre-eclampsia. However, Vmax values were significantly higher in women with pre-eclampsia than in the normotensi ve pregnant women. As the actions of serotonin in the periphery could be terminated primarily by active uptake system by platelets and placenta, significant alterations in the rate of transport could result in physiologically significant changes in serotonin levels. These data raise the possibility that abnormal regulation of transporter function is involved in the etiology of pre-eclampsia.

The possibility that prostanoids mediate the contractile response of serotonin on placental vessels was investigated. Rings of chorionic plate arteries and veins with and without endothelium were suspended in an organ bath for recording isometric mechanically activity. Serotonin caused dose-dependent contractions that were significantly attenuated by indomethacin (cyclo-oxygenase inhibitor, 10 μm) and SQ29,548 (thromboxane receptor antagonist, 1 μm). Pretreatment of placental venous and arterial rings with indomethacin decreased sensitivity (EC50) to serotonin of 2.3- and 1.9-fold, respectively. Pretreatment with SQ29,548 decreased sensitivity to serotonin of twofold in veins and 2.1-fold in arteries. In the endothelium-denuded placental arteries and veins, pretreatment with indomethacin and SQ29,548 reduced the serotonin-induced contraction in a similar way to that obtained in the endothelium-intact vessels. In isolated perfused cotyledon through the fetal circulation, serotonin caused a significant increase in perfusion pressure and stimulated thromboxane release 1.9-fold compared with basal values. Therefore, serotonin-induced vasoconstriction in the human fetoplacental circulation appears to be mediated in part by thromboxane release or action. This effect is not dependent on mediators released from the endothelium. The present study provides evidence for the participation of thromboxane A2 in the contractile response to serotonin in the human placental circulation. The ability of serotonin to release thromboxane A2 which is also a potent vasoconstrictor agent, may be important in increase fetoplacental resistance, one of the features of pre-eclampsia.