Vitamins C and E inhibit apoptosis of cultured human term placenta trophoblast.

Preeclampsia can be lethal to both mother and baby. The prominent symptoms of this syndrome are hypertension, proteinuria and oedema, resulting from an exaggerated aseptic systemic inflammatory response, triggered by placental factors shed into the maternal circulation. Syncytiotrophoblast microparticles (STBM) are one possible factor, shed when the placenta is exposed to stressors such as hypoxia/reperfusion. These can disrupt mitochondria, triggering apoptosis and necrosis, placental pathologies which are increased in preeclampsia. We tested the effects of antioxidant vitamins C (50 microM) and E (50 microM) on trophoblast in culture, using term villous cytotrophoblast preparations. Following Percoll gradient centrifugation and MHC class I expressing cell depletion of placenta digests, syncytial fragments were removed using anti-placental alkaline phosphatase antibody. This yielded cytotrophoblasts of consistently high purity. EGF (10 ng/ml) stimulated syncytialisation and hCG and progesterone production. However, mitochondrial induced apoptosis (MIA) was evident 96h post-isolation, as mitochondrial membrane potential loss and caspase 9 and caspase 3 activation. ROCK-1 cleavage and syncytiotrophoblast particle shedding increased concurrently with apoptosis induction. Vitamins blocked MIA and syncytiotrophoblast particle shedding and significantly increased hCG (p<0.005) and progesterone (p<0.02) concentrations in culture supernatants, reflecting the increased survival rates. Although more cells survived in culture, syncytialisation rate (%) was significantly reduced (p<0.005). We conclude that vitamins C and E can significantly reduce mitochondrial damage generated following syncytialisation in vitro. However, further work is required to determine whether antioxidant vitamins interfere with normal fusion processes.

Caveolin-1 and lipid rafts in confluent BeWo trophoblasts: evidence for Rock-1 association with caveolin-1.

Lipid rafts are detergent-insoluble, low-density membrane domains that are rich in cholesterol and sphingolipids; caveolae are a subdomain of the biochemically defined glycolipid raft whose expression is associated with the protein caveolin-1. This protein associates with numerous signalling molecules, regulating their activity by holding them inactive. Human villous cytotrophoblasts contain caveolin-1, but levels reduce greatly during their differentiation into syncytiotrophoblast. Since caveolin-1 is a known regulator of apoptosis and trophoblast syncytialisation involves the apoptotic cascade, we hypothesised that cytotrophoblast caveolin-1 may also play a role in regulating fusion events involved in syncytium formation. The BeWo choriocarcinoma cell line has previously proved valuable for studying trophoblast syncytialisation, hence the present work was carried out to determine whether BeWo cells could be used as a model for the exploration of caveolin-1's role in regulating the syncytialisation process. Undifferentiated BeWo cells were found to express caveolin-1 in similar amounts to villous cytotrophoblasts isolated from term placenta. Lipid raft fractions prepared from these BeWo cells at confluence contained the raft-associated proteins caveolin-1 and -2, flotillin-1 and -2, stomatin and the heterotrimeric G protein, Galphaq. Confocal immunofluorescence studies revealed that caveolin-1 is internalized to the mitochondria, but not to the Golgi or endoplasmic reticulum, in subconfluent BeWo and that the protein relocates to the plasma membrane upon confluence, an observation confirmed by caveolin-1 and cytochrome c Western blotting of lipid raft fractions and mitochondria purified from confluent and subconfluent cells. Western blotting and immunofluorescence experiments comparing undifferentiated cells and those induced to differentiate using the cAMP analogue, dibutyryl cAMP, showed that BeWo syncytialisation was accompanied by a reduction in caveolin-1 levels, similar to the situation in primary villous cytotrophoblasts. Confluent, undifferentiated BeWo cultures were then used to investigate the cellular localisation of Rock-1, a protein which promotes cytoskeletal re-organisation important for syncytialisation and apoptosis. Its association with caveolin-1 was evidenced by the demonstration that the 160kDa proenzyme form of Rock-1 co-immunoprecipitates with caveolin-1 and vice versa, as well as by the co-localisation of the two proteins at the plasma membrane, as shown in immunofluorescence studies. A proportion of the total cell Rock-1 content was found in BeWo lipid raft fractions, confirming its membrane presence in confluent cells. This close association of plasmalemmal caveolin-1 with Rock-1 protein raises the possibility that caveolin-1 may regulate Rock-1 in these trophoblasts. We conclude that cell-cell contact is required for BeWo trophoblast to exhibit plasmalemmal caveolin-1; BeWo cells at confluence offer a useful model for the study of trophoblast raft behaviour during syncytialisation and for the exploration of the potential Rock-1-regulating role of caveolin-1 in this process.

High fetal urocortin levels at term and preterm labor.

CONTEXT: Placental urocortin has a role in the cascade of events leading to parturition by stimulating myometrial contractility and placental uterotonins secretion. OBJECTIVE: The objective of this study was to evaluate urocortin levels in maternal and fetal [umbilical cord artery (UCA) and vein (UCV)] plasma at term and preterm labor. DESIGN: The study design was a controlled cross-sectional study performed from November 2003 to June 2004. SETTING: This study was performed at the Division of Obstetrics and Gynecology, University of Siena (Siena, Italy). PATIENTS: Plasma samples were collected at term in the absence of labor (TNL; n = 27; 39.3 +/- 0.1 gestational weeks), at term spontaneous vaginal delivery (TL; n = 24; 40.1 +/- 0.2 gestational weeks), and at preterm labor (PTL; n = 19; 32.4 +/- 0.4 gestational weeks). Changes in urocortin mRNA expression were also evaluated in placentas collected from TNL (n = 11), TL (n = 11), and PTL (n = 10). INTERVENTION: Urocortin levels were measured by specific RIA. Changes in placental mRNA expression were determined by real-time quantitative RT-PCR analysis. RESULTS: Maternal and UCA plasma urocortin levels were significantly (P < 0.0001 for all) higher in TL and PTL than in TNL. Furthermore, UCA concentrations were significantly (P < 0.0001 for all) higher than and correlated with maternal concentrations (TNL: r = 0.45; P < 0.05; TL: r = 0.959; P < 0.0001; PTL: r = 0.7719; P < 0.0001). UCV levels were significantly (P < 0.001) higher in TL and PTL than in TNL and were significantly (P < 0.0001 for all) higher than and significantly (P < 0.0001 for all) correlated with maternal values, but were significantly (P < 0.0001 for all) lower than and correlated with UCA values (TNL: r = 0.9548; P < 0.0001; TL: r = 0.927; P < 0.0001; PTL: r = 0.838; P < 0.0001). Placental urocortin mRNA expression did not differ among TNL, TL, and PTL samples. CONCLUSIONS: Fetal urocortin secretion is increased in term and preterm labor. Whether these changes are a consequence rather than a cause of human parturition remains to be addressed.

Caveolae and caveolin-1 in human term villous trophoblast.

Caveolae are flask-shaped invaginations of the plasma membrane found in many cell types, particularly endothelium. A major structural component is the membrane protein caveolin-1 which associates with numerous signalling molecules, including endothelial nitric oxide (eNOS). Caveolin-1, which co-immunoprecipitates with eNOS in preparations from endothelial cells, regulates eNOS activity, holding it inactive. Controversy now exists regarding the presence of caveolae and caveolin-1 in trophoblasts, hence this study was carried out to examine whether the high levels of eNOS expressed in human syncytiotrophoblast are associated with caveolin-1, and to find out if caveolae are present in villous cytotrophoblasts and syncytiotrophoblast. Immunohistochemistry of term placentae revealed only weak labelling for caveolin-1 in the syncytiotrophoblast although the endothelium of the terminal villus vessels stained strongly. By electron microscopy, numerous caveolae were identified in the villus capillary endothelium but were extremely rare in the syncytium. Caveolin-1 staining was extensive in purified, isolated term villous cytotrophoblasts, with the purity of these cytokeratin positive cells confirmed by cytospin analysis and flow cytometry. Caveolae were clearly demonstrated in ultrastructural sections of the purified cytotrophoblasts. The time course of expression of caveolin-1 and eNOS during differentiation of villous cytotrophoblast into syncytiotrophoblast in culture was studied. Western analysis showed that caveolin-1 expression evident in day 1 whole cell lysates decreased at day 3 when the cells had syncytialized and declined further by day 6, while the levels of actin (control) remained high. eNOS expression in the same samples followed a different pattern, with the low levels in day 1 cells increasing substantially by 3 days in culture, subsiding again by day 6. eNOS association with caveolin-1 in day 1 and day 3 trophoblast cultures was evidenced by the demonstration that eNOS co-immunoprecipitates with caveolin-1 and vice versa. We conclude that human villous cytotrophoblasts express caveolin-1, which assembles into caveolae. Differentiation into syncytium results in a decrease, but not disappearance, of expression of caveolin-1 and a marked reduction of the caveolae.

Matrix metalloprotease-9, placental syncytiotrophoblast and the endothelial dysfunction of pre-eclampsia.

The maternal syndrome of pre-eclampsia is caused by generalized maternal endothelial cell dysfunction, arising directly or indirectly from factors of placental origin. Syncytiotrophoblast membrane microvesicular particles are shed from the placental surface into maternal blood in increased amounts in pre-eclampsia and, in vitro, both inhibit endothelial cell proliferation and cause marked changes in the morphology of the cultured cell monolayers. Because there is evidence that proteolytic activation and degradation of the underlying matrix can cause the same morphological changes, we tested the hypothesis that proteases intrinsic to syncytiotrophoblast microvillous membranes (STBM) are the cause of the in vitro endothelial changes. Purified STBM were analysed by zymography and western blotting. Although we could confirm the presence of urokinase plasminogen activator (uPA) in STBM we could demonstrate no intrinsic activity presumably because of its association with the plasminogen activator inhibitor-2 (PAI-2) which is also a component of STBM. We detected gelatinase activity and showed that it was due to the matrix metalloproteinase-9 (MMP-9). Its presence was confirmed in this location by immunohistocytochemistry. Protease inhibitors caused a small reversal of the effects of STBM on morphology and no effect on inhibition of proliferation. We conclude that the effect of STBM on endothelial cells is unlikely to be caused by intrinsic proteases.

ED(27) trophoblast-like cells isolated from first-trimester chorionic villi are genetically identical to HeLa cells yet exhibit a distinct phenotype.

ED(27) trophoblast-like cells were prepared from human chorionic villus samples obtained at 9 weeks gestation and have been grown continuously in vitro without phenotypic drift for nearly a decade. These cells express many trophoblast markers, including cytokeratin, placental alkaline phosphatase (PLAP), secretion of 17beta-estradiol, and a microvillous apical surface. The ED(27) cell line is a useful model system for studies of placental cell biology and has been distributed to laboratories world-wide. However, experiments to investigate their relationship to primary villous cytotrophoblast have shown that these cells do not secrete detectable amounts of human chorionic gonadotropin in culture and, when digested with trypsin, disperse into individual cells. Furthermore, immunocytochemical studies demonstrated that, unlike villous cytotrophoblasts, ED(27) cells were immunoreactive with monoclonal antibodies recognizing some HLA Class I antigens. This was not HLA-G, however, as would be expected if these cells originated from extravillous cytotrophoblasts, but rather classical HLA-A, B which is thought not to be expressed by any trophoblast subpopulations. These inconsistencies prompted us to question the authenticity of the continuous cell line as it now exists. Genetic haplotype analysis using the polymerase chain reaction (PCR) revealed that ED(27) was genetically identically to the HeLa cell line. Inasmuch as HeLa cells have never been grown in the laboratory (DAK), the only possible origin of HeLa cell contamination of ED(27) cells was the WISH cell line, and further PCR analysis revealed that this cell line was also genetically identical to HeLa. Like ED(27) cells, HeLa cells and WISH cells synthesized small amounts of estrogen and were found to express PLAP and antigens recognized by the monoclonal antibodies ED822, directed against the syncytiotrophoblast, and J1B5 directed against villous cytotrophoblast. These results point out the need for adherence to rigorous and consistent quality control measures to assure the authenticity of cell lines used as in vitro model systems.

Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors.

Studies in mammalian skin have shown expression of the genes for corticotropin-releasing hormone (CRH) and the related urocortin peptide, with subsequent production of the respective peptides. Recent molecular and biochemical analyses have further revealed the presence of CRH receptors (CRH-Rs). These CRH-Rs are functional, responding to CRH and urocortin peptides (exogenous or produced locally) through activation of receptor(s)-mediated pathways to modify skin cell phenotype. Thus, when taken together with the previous findings of cutaneous expression of POMC and its receptors, these observations extend the range of regulatory elements of the hypothalamic-pituitary-adrenal axis expressed in mammalian skin. Overall, the cutaneous CRH/POMC expression is highly reactive to common stressors such as immune cytokines, ultraviolet radiation, cutaneous pathology, or even the physiological changes associated with the hair cycle phase. Therefore, similar to its central analog, the local expression and action of CRH/POMC elements appear to be highly organized and entrained, representing general mechanism of cutaneous response to stressful stimuli. In such a CRH/POMC system, the CRH-Rs may be a central element.

Corticotrophin releasing hormone: its potential for a role in human myometrium.

Aside from its role as a hypothalamic stress hormone, corticotrophin releasing hormone (CRH) is also a placental hormone, at least in primates. Although the function of placentally derived CRH remains to be fully elucidated, elevated CRH levels have been associated with premature labour, suggesting that the hormone may be involved in regulating the duration of pregnancy. Indeed, pregnant human myometrium expresses functional CRH receptors (CRH R1 and CRH R2 subtypes) thought to signal predominantly via the second messenger cAMP. Thus, like other cAMP-producing hormones in the myometrium such as beta(2) agonists, CRH may play a part in maintaining uterine quiescence. However, several of the CRH receptor isoforms identified to date have a reduced ability to activate adenylate cyclase, raising the question as to whether they are linked to other signal transduction pathways. Here, we discuss critically the evidence for the peptide's role in regulating contractility, both directly at the myometrium and indirectly via the fetal membranes and decidua. The possibility of a role in myometrial growth modulation is also described. Experimental Physiology (2001) 86.2, 273-281.

Adhesion molecules of syncytiotrophoblast microvillous membranes inhibit proliferation of human umbilical vein endothelial cells.

It has been shown previously that syncytiotrophoblast microvillous membranes (STBM), isolated from normal or pre-eclampsia placentae, specifically inhibit the proliferation of cultured human umbilical vein endothelial cells (HUVEC) and disrupt the cell monolayer without causing cell death. We have previously shown that this anti-proliferative activity resides in a self-aggregating complex in which eight proteins, namely integrins alpha(5)(CD49e) and alpha(V)(CD51), dipeptidyl peptidase IV (DPP IV, CD26), alpha-actinin, transferrin receptor (TfR, CD71), transferrin, placental alkaline phosphatase (PLAP) and monoamine oxidase A (MAO-A) were identified. In the present study, we investigated which of these components causes the anti-proliferative activity of STBM. Antibodies against integrin alpha(5)and alpha(V)and DPP IV all reduced the STBM-induced inhibition of proliferation of HUVEC, which was also reversed by added fibronectin. A preparation of PLAP inhibited endothelial proliferation, but this was not due to enzymatic activity. The preparation was shown to be impure with more than 12 bands present on Coomassie blue stained SDS-PAGE gels. These included integrins alpha(5)and alpha(V), which could account, at least in part, for the inhibitory activity. We could not exclude, however, the possibility of other unidentified factors being involved. We conclude that adhesion molecules account for a major part of the anti-proliferative activity of STBM; these appear to compete for ligands in the extracellular matrix or serum with the appropriate receptors on HUVEC.

Processing of procorticotropin-releasing hormone (pro-CRH): molecular forms of CRH in normal and preeclamptic pregnancy.

This study examined the different molecular forms of CRH in normal and preeclampsia maternal plasma and protease-blocked placental extracts using antibodies to different regions of the CRH precursor, pro-CRH. In the absence of protease inhibitors, chromatographed normal placental extracts contained four peaks of immunoreactivity corresponding to unprocessed approximately 19-kDa pro-CRH, its approximately 8-kDa intermediate metabolite, pro-CRH125-194, its approximately 2.8-kDa midportion fragment, pro-CRH125-151, and 4.75-kDa CRH1-41. However, if protease inhibitors were included in the extraction medium, only pro-CRH and pro-CRH125-194 were found. Pro-CRH processing was more extensive in protease-blocked preeclampsia placentas than in those from normal pregnancy, with three peaks corresponding to pro-CRH, proCRH125-194, and mature CRH1-41 peptide found. Using quantitative competitive PCR, the messenger ribonucleic acid levels of CRH precursor in preeclampsia placentas were 1.7-fold higher than those in normal placentas (37.83 +/- 3.48 vs. 21.83 +/- 2.59 attomoles/microg total ribonucleic acid, respectively; P < 0.005). Preeclampsia placentas contained significantly more CRH1-41 cross-reactivity (4.72 +/- 1.22 pmol/g) than normal term placentas (1.52 +/- 0.39 pmol/g; P < 0.048) extracted in medium containing protease inhibitors. The content of pro-CRH(125+/-151)-reactive species in these extracts followed the same pattern, with more immunoreactivity detected in preeclampsia placentas (4.23 +/- 1.39 pmol/g) than in those from normal term pregnancies (1.44 +/- 0.32 pmol/g; P < 0.01). Sequential plasma samples from 10 women with normal pregnancy and 5 women with preeclampsia were assayed for pro-CRH(125-151)- and CRH(1-41)-immunoreactive species In normal pregnancy, maternal plasma CRH(1-41) immunoreactivity rose with increasing gestational age, reaching 460 +/- 48 pmol/L at term. In women with preeclampsia, CRH(1-41) levels at each gestational age point were higher than those at the equivalent stage of normal pregnancy. In contrast, the levels of pro-CRH(125-151)-immunoreactive species remained barely detectable throughout normal and preeclamptic pregnancy. Both pro-CRH and CRH(1-41), but not pro-CRH(125-151), were shown to bind to the plasma CRH-binding protein. Our findings highlight the importance of protection of placental tissue from degrading enzymes during extraction and show that most of the CRH in the human placenta exists as unprocessed pro-CRH, with very little in the form of CRH(1-41) except in preeclampsia. Our studies using maternal plasma indicate that CRH(1-41) is the only one of the pro-CRH fragments studied to be maintained in significant amounts in the maternal circulation and also the only fragment studied for which a specific plasma binding protein exists.

Activation of peripheral leukocytes in rat pregnancy and experimental preeclampsia.

OBJECTIVE: The aim of this study was to search for activation markers of peripheral leukocytes in experimental preeclampsia in the rat. STUDY DESIGN: Experimental preeclampsia was induced in 14-day-pregnant rats by infusion of endotoxin (1.0 microg/kg body weight). For comparison, rats with normal pregnancies that were infused with sodium chloride solution and cyclic rats that were infused with either endotoxin or sodium chloride solution were used. At various points before and after the infusion, blood samples were withdrawn and analyzed by means of whole-blood flow cytometry to evaluate expression of inflammation-associated adhesion molecules (CD11b, CD11a, CD49d, and CD62L) and CD14 on the leukocytes. RESULTS: Normal pregnancy was associated with increased CD11b (granulocytes and monocytes), CD11a (monocytes and lymphocytes), and CD49d (granulocytes, monocytes, and lymphocytes) expression. In addition to these changes found in normal pregnancy, reduced CD62L and increased CD11a and CD49d expression was found on granulocytes after endotoxin treatment of pregnant rats. No effect of endotoxin was observed in cyclic rats. CONCLUSION: Leukocytes of rats with experimental preeclampsia and, to a lesser extent, those of rats with normal pregnancies had an activated phenotype. These results are consistent with our previous findings in human subjects and suggest that (experimental) preeclampsia results from a generalized inflammatory response.

Effects of corticotrophin releasing hormone on contractile activity of myometrium from pregnant women.

OBJECTIVE: To determine whether corticotrophin releasing hormone plays a role in the regulation of tone in term nonlabouring human myometrium. SETTING: A teaching hospital research laboratory. SAMPLE: Thirty-seven women undergoing elective nonlabour caesarean section under regional anaesthesia. METHODS: Human corticotrophin releasing hormone (1, 10, 100 nmol/L) was added to strips of term, nonlabouring myometrium mounted in an organ bath, and the effect on spontaneous, oxytocin (1 nmol/L) or prostaglandin F2alpha (100 nmol/L) stimulated contractions determined. Cyclic adenosine monophosphate (cAMP) content of the tissue was also determined by enzyme immunoassay. RESULTS: Corticotrophin releasing hormone did not affect myometrial tension development in any of the experimental protocols. cAMP increased transiently after addition of corticotrophin releasing hormone (166.7 +/- 12.7% at 1 minute) but this was not reflected by any change in tension. CONCLUSIONS: This study suggests that despite high maternal plasma concentrations of corticotrophin releasing hormone in pregnancy at term, this peptide is unlikely to play a direct role in the control of myometrial contractility in nonlabouring myometrium.

Purification and characterization of a complex from placental syncytiotrophoblast microvillous membranes which inhibits the proliferation of human umbilical vein endothelial cells.

The signs of pre-eclampsia are thought to arise from maternal endothelial dysfunction caused by circulating factors of placental origin. Syncytiotrophoblast microvillous membranes (STBM) cause endothelial disruption and inhibit proliferation in vitro. Significantly increased amounts of STBM can be detected in blood from pre-eclamptic women and could contribute to endothelial dysfunction in vivo. This study purified a complex from STBM which inhibits the proliferation of cultured human endothelial cells. Integral membrane proteins were solubilized with sucrose monolaurate. Anion exchange chromatography yielded two peaks of anti-proliferative activity. Only the second peak was specific to STBM and was subjected to further separation by Sephacryl S-200 gel filtration chromatography (GFC). A single peak of specific activity eluted close to the void volume, at a position unaltered by added denaturing agents, guanidium chloride or urea. On Sephacryl S-300 GFC, two peaks were obtained of 410 and 820 kDa, with similar anti-proliferative activity and protein components (by SDS-polyacrylamide gel electrophoresis). The major protein bands were as integrins alpha5 and alpha v, dipeptidyl peptidase IV, alpha-actinin, transferrin, transferrin receptor, placental alkaline phosphatase and monoamine oxidase A.

Generation of a recombinant herpes simplex virus type 1 expressing the rat corticotropin- releasing hormone precursor: endoproteolytic processing, intracellular targeting and biological activity.

We describe the generation of a recombinant herpes simplex virus type 1 (HSV1) vector, tsK/CRH10, derived from the temperature-sensitive mutant tsK, expressing rat pre-procorticotropin-releasing hormone (ppCRH). In hypothalamic neurons, within the paraventricular and supraoptic nuclei, this neuropeptide precursor is processed to mature CRH (1-41), the key modulator of the hypothalamic-pituitary-adrenal stress response. We used the recombinant HSV1 tsK/CRH10 to study posttranslational processing, intracellular localization and biological activity of proCRH (pCRH) within neuronal, glial and epithelial cell lines. We showed that CRH-like immunoreactivity expressed in neuronal, glial and epithelial cells infected with tsK/CRH10 was biologically active, could be detected intracellularly and was also secreted. Our data also show that within Neuro2a and NG115 cells, the CRH precursor is cleaved to yield a CRH-like immunoreactive fragment of approximately 4.75 kD which could account for mature CRH (1-41). No endoproteolytic processing of the precursor takes place within the astrocytic 1321 NI cell line. Using immunocytochemistry techniques we detected CRH-like immunoreactivity within the endoplasmic reticulum-Golgi region in all cells and within secretory vesicles of Neuro2a and NG115 cells, suggesting correct targeting to the regulated secretory pathway within these cells. Our results demonstrate that the HSV1 recombinant vector expressing the full-length CRH precursor molecule constitutes an excellent delivery system for both cell lines and postmitotic neurons in vitro, which has enabled the study of targeting, endoproteolytic processing and biological activity of this neuropeptide precursor. Furthermore, it can also be used to generate transient transgenesis of the CRH precursor in vivo, to study neuroendocrine-immune interactions within the mammalian central nervous system.

Pharmacokinetics, cortisol release, and hemodynamics after intravenous and subcutaneous injection of human corticotropin-releasing factor in humans.

OBJECTIVE: Two clinical trials investigated the pharmacokinetics of human corticotropin-releasing factor (hCRF), resulting cortisol release, and associated hemodynamic changes. METHODS: In a 3 x 3 Latin square design, subjects were randomized to receive a single dose of 5 microg x kg(-1) hCRF as a 10-minute intravenous infusion, a 180-minute infusion, and a subcutaneous injection in separate study sessions 7 days apart. Twelve additional subjects obtained a subcutaneous dose of either 300, 600, or 1200 microg hCRF on 3 consecutive days. Noncompartmental and compartmental pharmacokinetic analysis was performed. Hemodynamic response was characterized with use of pharmacodynamic models. RESULTS: The volume of distribution at steady state was 9.81 +/- 3.0 and 15.61 +/- 2.9, and the clearance was 256 +/- 40 mL x min(-1) and 345 +/- 90 mL x min(-1) for the 10-minute and 180-minute intravenous infusion, respectively (P < .05). Corresponding elimination half-life was 45 +/- 7 minutes and 37 +/- 10 minutes. Two-compartment and 1-compartment models adequately described the 10-minute and 180-minute infusions, respectively. The bioavailability of hCRF after subcutaneous administration was 67% +/- 17%. Apparent clearance remained unchanged for different subcutaneous doses. Peak plasma cortisol concentrations were similar after subcutaneous and intravenous administration of hCRF. Repetitive administration of hCRF did not result in accumulation but produced a reduced plasma cortisol response. A sigmoidal model related plasma hCRF concentrations to increase in heart rate (maximum, 39 beats x min(-1)). The relationship between the modest decrease in diastolic blood pressure and plasma hCRF concentrations was linear. CONCLUSION: The pharmacokinetics of intravenously administered hCRF were nonlinear, but apparent clearance was constant for various subcutaneous doses. An excellent bioavailability and preserved bioactivity make the subcutaneous route an attractive choice. Repetitive administration of hCRF probably caused tolerance of the cortisol response.

Circulating markers of oxidative stress are raised in normal pregnancy and pre-eclampsia.

OBJECTIVE: To determine whether circulating markers of oxidative stress are elevated in pre-eclampsia when appropriate precautions are taken to prevent in vitro oxidation DESIGN: A prospective study. SETTING: Nuffield Department of Obstetrics and Gynaecology, Oxford and The William Harvey Institute, London. SAMPLE: Three groups of women: those with pre-eclampsia (n = 19), control pregnant women (n = 19) matched for gestation, age and parity and a group of non pregnant individuals of reproductive age (n = 7). METHODS: Citrated plasma was stored at -80 degrees C with 20 micromol beta hydroxytoluene to prevent auto-oxidation. Plasma samples were assayed for levels of 8 epi-prostaglandin F2alpha, lipid hydroperoxides, malondialdehyde and also the lipid soluble antioxidant vitamin E. RESULTS: There were no differences in 8 epi-prostaglandin F2alpha, lipid peroxide or malondialdehyde levels between the groups of women with pre-eclampsia and those acting as pregnant controls. However, lipid hydroperoxides and malondialdehyde were significantly raised in both pre-eclampsia and normal pregnancy, compared with nonpregnant women. Vitamin E levels were similar in women with pre-eclampsia and those with a normal pregnancy, but in both groups levels were significantly higher than in nonpregnant women. CONCLUSION: Circulating markers of oxidative stress are raised in normal pregnancy and pre-eclampsia.

Levels of corticotrophin-releasing hormone receptor subtype 1 mRNA in pregnancy and during labour in human myometrium measured by quantitative competitive PCR.

It is suggested that corticotrophin-releasing hormone (CRH) is involved in parturition. We have previously reported the presence of the CRH receptor subtype 1 (CRH R1) in human uterine myocytes. The aim of the present study was to investigate whether expression of the CRH R1 in myometrial tissue changes in pregnancy and labour. We used a quantitative competitive PCR method to measure the mRNA levels of this receptor in non-pregnant and in term pregnant myometrium before and at different stages of labour. The levels of mRNA for the housekeeping gene for glucocerebrosidase (GCB) were also determined. The results were expressed as a ratio of CRH R1 and GCB mRNA levels. We have found that in pregnancy the CRH R1 is down-regulated from a ratio of 0.093+/-0.011 in non-pregnant myometrium to 0.012+/-0.005 (P<0.001) in term non-labouring myometrium. No significant changes were observed in the CRH R1:GCB ratio in tissues sampled within 13 h (0.013+/-0.004) from the start of labour. In summary, normalised levels of CRH R1 are down-regulated in pregnancy and do not change during labour. We speculate that our results do not support a direct role for the CRH R1 receptor in myometrial stimulation.

Serum from preeclamptic women induces vascular cell adhesion molecule-1 expression on human endothelial cells in vitro: a possible role of increased circulating levels of free fatty acids.

OBJECTIVE: The object was to determine whether serum from preeclamptic women induces expression of vascular cell adhesion molecule-1 on cultured endothelial cells. STUDY DESIGN: Endothelial cells were incubated with medium containing 20% serum (volume/volume) either from women with preeclampsia (n = 15) or from women with normal pregnancies (n = 15) matched for maternal age, gestational age, and parity. A further matched set of samples (n = 10) was exposed to endothelial cells that had previously been incubated in the presence or absence of vitamin E (40 micromol/L final concentration). Free fatty acids were determined in each sample. A mixture of free fatty acids (linoleic, oleic, and palmitic acids, 1:1:1) was added to serum from control subjects in increasing concentrations (70-280 micromol/L final concentration) to emulate preeclamptic serum and the preparation was exposed to endothelial cells. In each experiment vascular cell adhesion molecule-1 expression was determined after 16 hours of exposure by an enzyme-linked immunosorbent assay technique performed on the cell monolayer. RESULTS: Preeclamptic serum had higher levels of free fatty acids than did that of control subjects (0.71 mmol/L, 95% confidence level 0.5-0.93, vs 0.36 mmol/L, 95% confidence level 0.28-0.43). There was a statistically significant increase in vascular cell adhesion molecule-1 expression on the endothelial cells exposed to preeclamptic serum compared with those exposed to control serum (optical density 0.17 vs 0.11). Vitamin E reduced the vascular cell adhesion molecule-1 expression of endothelial cells exposed both to preeclamptic and to control serum samples in a nonspecific manner. Addition of free fatty acids to normal pregnancy serum to mimic the effect of preeclampsia resulted in increased expression of vascular cell adhesion molecule-1 on the cells. CONCLUSION: Preeclamptic serum induces vascular cell adhesion molecule-1 expression on human endothelial cells in vitro, an effect also produced by fatty acids. The elevated level of free fatty acids in women with preeclampsia may contribute to increased vascular cell adhesion molecule expression in vivo.

Procorticotrophin-releasing hormone: endoproteolytic processing and differential release of its derived peptides within AtT20 cells.

Procorticotrophin-releasing hormone (proCRH) is expressed mainly in the hypothalamus and in the placenta, where it undergoes tissue-specific endoproteolysis. Our results show that within stably transfected AtT20/D16V cells proCRH is cleaved to generate two fragments of approximately 8 and 3 kDa which could account for proCRH(125-194) and proCRH(125-151), respectively, and a 4.5 kDa product which could account for mature IR-CRH(1-41). The immunofluorescence staining patterns for IR-CRH and IR-ACTH and their response of secretagogues indicate targeting of proCRH and POMC to the secretory pathway in transfected AtT20 cells. In this work, we have used a unique set of specific RIAs and IRMAs to the full length POMC and proCRH molecules and several products of endoproteolytic processing to assess if they could be released differentially in response to stimulation. Although the release of both IR-ACTH and IR-CRH peptides from transfected AtT20 cells is stimulated in response to exposure to high potassium stimulation (51 mM KCl/SmM CaCl2), the sorting index (SI) suggests that mature ACTH is sorted to the regulated secretory pathway 2.1-fold more efficiently than mature CRH(1-41). Mature ACTH is also sorted to the regulated secretory pathway 9-fold more efficiently than IR-proCRH(125-151). Also, mature CRH(1-41) is sorted to the regulated secretory pathway 3-fold more efficiently than IR-proCRH(125-151). These results therefore indicate that the intracellular mechanisms for the storage and release of POMC, proCRH and their endoproteolytic products differ and would sustain the hypothesis that within mammalian peptidergic cells, different biologically active peptides originating from the same or different precursor molecules, could be differentially released in response to specific stimuli. This would give these cells the capacity to finely regulate neurotransmitter release in response to environmental and physiological demands.

Urocortin in pregnancy.

OBJECTIVES: The aim of this study was to investigate the possibility that urocortin is the ligand that displaces corticotropin-releasing hormone from its binding protein in the maternal circulation during pregnancy and, if so, to determine whether urocortin, like corticotropin-releasing hormone, is synthesized in substantial quantities in the placenta. STUDY DESIGN: A radioimmunoassay specific for urocortin was developed and used for measurement of the peptide in chorionic villi and fetal membranes (amnion and chorion) from normal and preeclamptic pregnancies. These tissues were also assayed for corticotropin-releasing hormone. Assays for urocortin were also carried out on normal term pregnant and nonpregnant myometrium and on plasma from nonpregnant individuals, and assays for both peptides were performed on sequential normal pregnancy plasma samples taken from mid gestation until term. RESULTS: Corticotropin-releasing hormone was present in normal term (1904 +/- 489 pg/g) and preeclamptic placentas (5897 +/- 1526 pg/g) and in normal term fetal membranes (645 +/- 155 pg/g, n = 6 in all cases). Urocortin was not detected in any of the tissues studied, nor was it found in the normal human plasma samples. Unlike the situation for corticotropin-releasing hormone, no pregnancy-related pattern was seen for urocortin in the plasma from pregnant women. CONCLUSIONS: Urocortin is not translated to any great extent in the pregnancy tissues investigated, nor is it present in the circulation of pregnant women in detectable amounts. Furthermore, it is unlikely that urocortin is responsible for the high maternal plasma levels of free corticotropin-releasing hormone circulating in the latter stages of pregnancy, but this does not preclude the possibility that another, as yet uncharacterized, corticotropin-releasing hormone-like peptide may be.