Calreticulin in human pregnancy and pre-eclampsia.

Pre-eclampsia is a disorder of human pregnancy that involves pregnancy-induced maternal hypertension and proteinuria. Evidence indicates that pre-eclampsia involves widespread activation of maternal endothelial cells. Calreticulin is a ubiquitously expressed, multi-functional protein that has been shown to have both pro- and anti-inflammatory effects on cultured endothelial cells in vitro and in whole animals. In order to clarify the role of this protein in normal human pregnancy and in pre-eclampsia, this study has measured expression of calreticulin in maternal blood and in placenta in patients with pre-eclampsia and in control pregnancies. There was a significant increase (approximately 5-fold) in calreticulin in plasma in term pregnant women compared with women who were not pregnant. There was no difference, however, in calreticulin in plasma from women who were sampled at first trimester, second trimester and at term. In addition, there was a significant increase (approximately 50%) in calreticulin in plasma from pre-eclamptic women compared to controls. Calreticulin mRNA and protein expression in placenta were not changed between pre-eclampsia and control pregnancies. These novel results indicate that calreticulin is increased in peripheral maternal blood early in pregnancy and remains elevated throughout normal gestation and that there is a further increase in calreticulin in pre-eclampsia.

Homeobox genes are differentially expressed in macrovascular human umbilical vein endothelial cells and microvascular placental endothelial cells.

Angiogenesis is fundamental to normal placental development. Aberrant angiogenesis within the placental terminal villi is a characteristic of significant placental pathologies and includes structural and vascular abnormalities as well as altered endothelial cell function, which substantially impacts on maternal-fetal exchange. Homeobox gene transcription factors regulate vascular development in embryonic and adult tissues, but their role in the placental microvasculature is not well known. In this study, we isolated and enriched human placental microvascular endothelial cells (PLEC) by a perfusion-based method and compared homeobox gene expression between PLEC and macrovascular human umbilical vein endothelial cells (HUVEC). Reverse transcriptase PCR detected mRNA expression of homeobox genes DLX3, DLX4, MSX2, GAX and HLX1 in both PLEC and HUVEC. DLX4 and HLX1 have not been previously detected in PLEC and with the exception of GAX, none of these homeobox genes have been previously identified in HUVEC. There was lower expression of HLX1 mRNA in HUVEC compared with PLEC. Using real-time PCR analysis PLEC HLX1 mRNA expression relative to housekeeping gene GAPDH was 0.9+/-0.06 fold of the calibrator (n=6) versus 0.2+/-0.06 (n=6) for HUVEC, p<0.001. These data provided evidence of heterogeneity in homeobox gene expression between microvascular PLEC and macrovascular HUVEC that most likely reflects significant differences in endothelial cell function in the two different cellular environments.

Expression and cellular localisation of chloride intracellular channel 3 in human placenta and fetal membranes.

Chloride channels regulate the movement of a major cellular anion and are involved in fundamental processes that are critical for cell viability. Regulation of intracellular chloride is achieved by multiple classes of channel proteins. One class of putative channels are the chloride intracellular channel (CLIC) family. Evidence suggests that several CLICs are expressed in human placenta, although their roles in this tissue are not certain. Northern blot analysis has shown that CLIC3 is highly expressed in placenta relative to other human tissues; however, its cellular distribution is not known. This study used microarray expression profiling to clarify which CLICs are expressed in human placenta and RT-PCR, Western blot and immunohistochemistry to determine the expression pattern of CLIC3 in human placenta and fetal membranes. Placentas and fetal membranes were obtained from term pregnancies after delivery and placental tissue was obtained from first trimester following either chorionic villous sampling or elective pregnancy termination. Trophoblast cells were isolated from first trimester and term placentas and placental endothelial cells were isolated from term placentas. Microarray expression profiling identified high expression of mRNA for CLICs 1, 3 and 4 in the isolated first trimester and term trophoblast cells. High mRNA expression in the isolated endothelial cells was also found for CLICs 1 and 4, but not CLIC3. Low expression was found for CLIC5 in all three types of isolated cells. RT-PCR confirmed that CLIC3 mRNA was expressed in trophoblast cells at both gestational ages, but was not present in endothelial cells. CLIC3 mRNA was also identified in whole placental extracts at both gestational ages and in term amnion and choriodecidua. Immunohistochemistry using a chicken anti-human CLIC3 antibody localised strong CLIC3-specific staining to the syncytiotrophoblast and villous cytotrophoblast cells in both first trimester and term placentas, and weaker staining in extravillous trophoblast cells in first trimester. In fetal membranes at term strong CLIC3-specific staining was localised to chorionic trophoblast cells, with weaker staining in amniotic epithelial and decidual cells. It was previously shown that chloride uptake was increased into cells that had been transfected with CLIC3. CLIC3 may facilitate chloride ion movement and the regulation of cellular processes associated with the movement of chloride in the placental and fetal membrane cells in which it is expressed.

Increased biological oxidation and reduced anti-oxidant enzyme activity in pre-eclamptic placentae.

Oxidative stress occurs when cellular levels of reactive oxygen species exceed anti-oxidant capabilities and has been implicated in the pathogenesis of pre-eclampsia. In this study we have examined the tissue levels of endogenous anti-oxidant proteins (superoxide dismutase, glutathione peroxidase, thioredoxin reductase and thioredoxin) and the level of lipid and protein oxidation in placental samples from normal and pre-eclamptic pregnancies. Pre-eclamptic tissue homogenates demonstrated significantly increased levels of lipid peroxidation (20.68 +/- 7.811 microM protein versus 5.33 +/- 4.03 microM/mg protein, P < 0.001) and a trended increase in protein carbonyl concentration (248.1 +/- 97.71 units/mg protein versus 209.7 +/- 82.6 U/mg protein) when compared to controls. The levels and activities of the anti-oxidant proteins superoxide dismutase (2.48 +/- 0.6 U/mg protein versus 2.02 +/- 0.51 U/mg protein, P <0.02), thioredoxin reductase (19.25 +/- 9.81 U/mg protein versus 13.02 +/- 5.66 U/mg protein,P = 0.02), thioredoxin (107.00 +/- 18.11 ng/mg protein versus 91.12 +/- 21.18 ng/mg protein, P = 0.02) and glutathione peroxidase (17.33 +/- 6.63 mmol/min/mg protein versus 11.50 +/- 3.11 mmol/min/mg, P < 0.02) were all found to be significantly reduced when comparing pre-eclamptic placental tissue homogenates to gestational age-matched control placentae from non-pre-eclamptic pregnancies. The results of this study demonstrate a decreased enzymatic anti-oxidant capacity and increased oxidation in placental tissue from pre-eclamptic women, which may contribute to the pathogenesis of this complex disorder.

Fetal growth restriction is associated with increased apoptosis in the chorionic trophoblast cells of human fetal membranes.

The aim of the study was to determine the incidence and spatial distribution of apoptotic cell in fetal membranes obtained from human pregnancies complicated with fetal growth restriction (FGR) for which there was no established cause. Fetal membrane samples from normal (n=10) and FGR-affected (n=10) pregnancies were collected and stored following delivery. The incidence of apoptosis and the number of apoptotic cells in normal and FGR-affected fetal membranes were determined using immunohistochemistry and a monoclonal antibody for neo-epitope of cytokeratin-18, M30. The level of apoptotic proteins in FGR-fetal membranes compared to the normal tissue was determined using Western immunoblotting analysis. Multiple labeling of trophoblast cells using immunofluorescence markers was used to investigate regional differences in localization of apoptotic cells between normal and FGR-affected fetal membranes. Apoptosis was detected in both normal and FGR-affected fetal membranes. However, quantitative analysis of apoptotic cells by immunohistochemistry showed a significant increase in FGR-affected fetal membranes compared to normal (p<0.005). Furthermore, it was observed that apoptotic cells were predominantly localized to chorio-decidual layer of the fetal membrane. By using semi-quantitative analysis of Western immunoblotting, a significant increase in the levels of the apoptotic marker proteins poly-ribo (ADP) polymerase (PARP) and the neo-epitope of cytokeratin-18 were observed in FGR-affected fetal membranes compared to normal (p<0.005). Immunofluorescence studies further confirmed the restriction of the apoptotic cells to the chorionic trophoblast cells in FGR-affected fetal membranes. Our results document for the first time an increased incidence of apoptosis in FGR-affected fetal membranes, with the apoptotic cells restricted primarily to the chorionic trophoblast layer of the fetal membranes. Increased apoptosis in FGR-affected fetal membranes may impair functions of the fetal membranes that are important for normal fetal development and growth. Elucidation of the molecular mechanisms involved in the control of apoptosis in the chorionic trophoblast layer in the FGR-affected fetal membranes may provide further insights into the etiology of FGR.

Expression of GLUT12 in the fetal membranes of the human placenta.

The aim of this study was to characterize the expression of the novel glucose transporter GLUT12 in the fetal membranes of the human placenta. RT-PCR and Western blotting of extracts of amnion and choriodecidua from four normal term placentas identified GLUT12 mRNA and protein expression. In all four samples the signals for GLUT12 were markedly stronger in the choriodecidua than in the amnion, whereas the signals for GLUT1, a glucose transporter know to be expressed in fetal membranes, were similar for the two tissues. In further studies, paraffin sections of fetal membranes were analyzed by immunohistochemistry with GLUT12 and GLUT1-specific polyclonal antibodies. GLUT12 immunoreactivity was localized predominantly to the trophoblast cells in the chorion and to a lesser extent to decidual cells and to epithelial and fibroblast cells of the amnion. GLUT1 was localized to chorionic trophoblast cells and amniotic epithelial and fibroblast cells. GLUT12 expression was predominantly cytoplasmic, whereas GLUT1 was associated with the membrane of the cells. These results show that GLUT12 is expressed in cells of human fetal membranes and suggest that GLUT12 may play a role in the facilitation of glucose transport into these cells.

Atrial natriuretic factor production by the human placenta.

Atrial natriuretic factor (ANF), a 28 amino acid peptide, is primarily produced and secreted by cardiac atrial myocytes to modulate cardiovascular and renal functions. Although ANF is also produced in tissues other than the heart, it remains uncertain whether the peptide is synthesised by human placentae. We provide here evidence from in vitro studies suggesting de novo production of ANF by the human placenta. Placental tissues were collected from normal term pregnancies by elective Cesarean section and acid extracted for ANF radioimmunoassay. The level of placental immunoreactive (ir) ANF was 186 +/- 33 pg/gm wet tissues (mean +/- SE, n = 6). Perfusates from in vitro perfusion of the fetoplacental compartment of placental lobules yielded 26 +/- 6 pg/min/gm wet tissue (n = 3) of irANF. Furthermore, pro-ANF mRNA signals were localised by colorimetric in situ hybridization in a subpopulation of placental cytotrophoblast-like cells, but not in syncytiotrophoblasts nor in chorionic cells of placental sections. Northern blot analysis of placental tissue extracts showed a single band of pro-ANF mRNA signals (-0.85 Kb) similar in size to that found in the rat heart. Our findings suggest that ANF is expressed and produced by a small population of human placental cytotrophoblast-like cells. The possibility that placental ANF may be secreted locally or into the fetoplacental circulation to exert paracrine, autocrine or both effects now needs to be considered.

Acetylcholine output and foetal vascular resistance of human perfused placental cotyleda.

The foetal villous vessels of single cotyleda of human placentae have been perfused with a constant flow of Krebs solution, recording inflow pressure and passing the venous perfusate in cascade over guinea-pig ileum and rat stomach strip preparations in vitro. Each cotyledon released for at least 4 h a substance that was probably acetylcholine. The perfusate caused contractions of both preparations which were inhibited by atropine or hyoscine and potentiated by physostigmine. Contractile activity was destroyed after incubation at 37 degrees C of perfusate with acetylcholinesterase but not with acetylcholinesterase plus physostigmine. When the perfusion temperature was lowered to 34 degrees C or below, acetylcholine output was reduced, the extent depending on the fall in temperature. No change in resistance of the villous vessels occurred during the changes in temperature or in the presence at 37 degrees C of atropine, hyoscine, hexamethonium, (+)-tubocurarine, hemicholinium-3 or bretylium. Submaximal vasoconstrictor responses of the villous vessels to the thromboxane A2-mimetic U46619 were not affected by reduction of the perfusion temperature to 30 degrees C, which lowered acetylcholine-like output by approximately 70%. Responses to U46619, at 37 degrees C, were unchanged during the presence of atropine or hyoscine. Acetylcholine is released into the foetal circulation of the human placenta but no evidence could be obtained that it affects villous vascular smooth muscle tone or vasoconstrictor responses.

Effects of opioids on acetylcholine output of perfused human placental cotyleda.

(1) Output of acetylcholine (ACh) from fetal vessels of Krebs-perfused human placental cotyleda was estimated by bioassay using the rat stomach strip. (2) Infusion of high concentrations of the opioids pethidine, morphine or ethylketocyclazocine, but not [D-Ala2]-methionine enkephalinamide (0.1-300 mumol/l), reduced ACh output. Fifty per cent inhibition of output occurred in the presence of concentrations of greater than or equal to 300 mumol/l (morphine and ethylketocyclazocine) and 338 (317, 353; 95 per cent c.l., n = 6) mumol/l (pethidine). (3) ACh output was inhibited by infusion of 100 mumol/l naloxone or 10 mumol/l naltrexone, but was not affected by lower concentrations of either antagonist. (4) These results suggest that the therapeutic concentrations of morphine and pethidine likely to occur in vivo would not affect placental ACh output into fetal vessels. The finding that high concentrations of synthetic opioids or opioid antagonists were required to inhibit output suggests that they may not be acting specifically, and provides no evidence for the hypothesis that endogenous opioids play a role in control of ACh release into fetal vessels of human placentae.

Evidence for inhibition by endothelium-derived relaxing factor of thromboxane A2 receptor-mediated vasoconstriction in the fetal vessels of the human perfused placenta.

Three inhibitors of the release or effects of endothelium-derived relaxing factor (EDRF), N-nitro-L-arginine, methylene blue and oxyhemoglobin, caused further increases in perfusion pressure during vascular constriction with submaximal concentrations of the thromboxane A2-mimetic, U46619 in fetal vessels of human placental lobules perfused in vitro. The results suggest the EDRF, released during constriction of fetal placental vessels in response to thromboxane A2 receptor stimulation, attenuates the vasoconstrictor response. Hence, impairment of EDRF release or function could contribute to the reduced placental blood flow observed in various disease states associated with increased thromboxane A2 production such as pre-eclampsia.

GLUT12 expression in human placenta in first trimester and term.

The aim of this study was to characterize the expression of a novel glucose transporter protein GLUT12 in human placenta. GLUT12 mRNA expression was identified by RT-PCR in extracts from five normal term placentae and in extracts from cultured cells of the JAR, JEG-3 and HTR-8Svneo cell lines. In further studies, paraffin sections of first trimester tissue from chorionic villus sampling and term tissue obtained after delivery were analysed by immunohistology with a GLUT12 specific polyclonal antibody. GLUT12 immunoreactivity was expressed predominantly in the syncytiotrophoblast and in extra-villous trophoblast cells in first trimester tissues at 10, 11 and 12 weeks' gestation. In term tissue, however, GLUT12 staining was not detected in syncytiotrophoblast and was found predominantly in villous vascular smooth muscle cells and villous stromal cells. These results suggest that there is a dynamic spatial and temporal expression pattern for the novel glucose transporter GLUT12 in human placenta.

Changes in the biological activity of autacoids during passage through the human perfused fetoplacental lobule.

Changes in the biological activities of a number of autacoids after a single passage through the human perfused fetoplacental lobule have been assessed by bioassay, whilst recording fetal vascular resistance. Bradykinin did not affect vascular resistance, but its biological activity on the superfused bioassay tissues fell by approximately 98%, whereas des-Asp1-angiotensin I activity increased at least 80-fold and the vascular resistance rose. All these effects were inhibited by captopril. Angiotensin II increased vascular resistance but its activity on the bioassay tissues was not changed. 5-Hydroxytryptamine activity was reduced by 67-90% and resistance to flow was not affected. The activities of prostaglandins D2, E2, and F2 alpha were slightly reduced. Prostaglandins D2 and F2 alpha caused vasoconstriction, their maximum effects being greater than those of either of the angiotensins. The TxA2-mimetic U46619 was approximately 90 times more potent than PGF2 alpha, as a vasoconstrictor, but the maximal effects were comparable. Thus, autacoid activity can be reduced, augmented or not affected during passage through the human perfused fetal placental vasculature.

Endothelin: release by and potent constrictor effect on the fetal vessels of human perfused placental lobules.

Human placental lobules were bilaterally perfused with a modified Krebs solution at constant flow rates of 5 mL min-1, and fetal inflow perfusion pressure was recorded. The effect of infusions of endothelin-1 and endothelin-3 (ET-1 and ET-3) on the perfusion pressure was assessed and compared with that for the thromboxane A2-mimetic U46619 and prostaglandin F2 alpha (PGF2 alpha). All substances caused significant increases in pressure, ET-1 being the most potent, followed in order by U46619, ET-3 and PGF2 alpha. In addition, ET-like immunoreactivity was identified in the fetal effluent of placental lobules during 4 h of basal perfusion. The mean ET-1 equivalent immunoreactivity at 1 h of perfusion was 0.6 +/- 0.2 fmol min-1 g-1 of wet lobule weight for 10 placentae. These data suggest that human fetal placental endothelial cells are capable of synthesizing ETs and that ETs are potent constrictors of the fetal placental vessels. Thus, endothelins may play a role in the control of fetal vascular tone in the human placenta in normal and/or pathological conditions.

Regulation of human placental fetal vessel tone: role of nitric oxide.

Factors affecting fetal vessel resistance have been studied in vitro in bilaterally perfused lobules of human placentae. Potent and efficacious constrictors in this preparation (in order of potency) include endothelin-1 > the thromboxane mimetic U46619 > endothelin-3 > prostaglandin F2 alpha. Inhibitors of eicosanoid synthesis did not affect fetal vessel basal perfusion pressure, nor did they potentiate the effects of the vasoconstrictor U46619. In contrast, the nitric oxide inhibitors N omega-nitro-L-arginine (NOLA), haemoglobin and methylene blue all increased fetal vessel basal perfusion pressure and also increased U46619-induced constriction. Similarly, NOLA markedly potentiated the constrictor effects of endothelin-1, angiotensin II, 5-hydroxytryptamine and bradykinin. These studies therefore provide evidence that NO is important in the maintenance of low basal fetal vessel impedance and also reduces the effects of a number of vasoconstrictor autacoids. Nitric oxide synthase (NOS) activity of human placental homogenates has been measured and shown to be mainly calcium-dependent. Human placental NOS activity was not affected by labour state but was reduced in pre-eclampsia. No evidence was found that in pre-eclampsia raised concentrations of the endogenous NOS inhibitor asymmetric dimethylarginine were responsible for the reduced placental NOS activity. Hence, these studies provide evidence that NO is an important endogenous dilator of the fetal vessels of the human placenta and that reduced NOS activity could contribute to the pathogenesis and/or effects of pre-eclampsia.

Human placental and fetal membrane nitric oxide synthase activity before, during and after labour at term.

The aim of this study was to determine whether any labour-associated changes in nitric oxide synthase (NOS) activity occur in human placenta and fetal membranes. NOS activity in amnion, choriodecidua, and placenta obtained from women before (at Caesarean section, not in labour), during (at Caesarean section, in labour) and after (spontaneous onset labour, normal vaginal delivery) labour was assessed by measuring conversion of radio-labelled L-arginine to L-citrulline. NOS activity, as judged by its inhibition by the specific NOS inhibitor N omega-nitro-L-arginine, was present in placental and amnionic tissues, but not in choriodecidual tissue specimens. Activity detected in choriodecidua was significantly blocked during incubation with a high concentration of valine, suggesting that L-arginine was being consumed by reactions other than NOS under the experimental conditions in that tissue. There were no significant differences among the labour groups in either amnion or placental NOS activities measured in the presence of 1 microM L-arginine. Amnion NOS activity was significantly less than that in placenta. Placental V(max) and Km values (determined after removal of endogenous L-arginine) did not differ significantly among the different labour groups.

Effect of asymmetric dimethyl arginine on nitric oxide synthase activity in normal and pre-eclamptic placentae.

An endogenous inhibitor of nitric oxide synthase (NOS), NG,NG dimethylarginine (asymmetric dimethylarginine, ADMA), which is present in human plasma and urine, has been reported to be elevated in the plasma of women with pre-eclampsia. As ADMA inhibition may contribute to reduced placental NOS activity observed in pre-eclampsia, the aim of this study was to compare the effects of ADMA on placental NOS activity from pre-eclamptic and normal pregnancies (gestational ages 38.4 +/- 0.9 and 38.3 +/- 0.3 weeks respectively). NOS activity was determined by measuring the conversion of [3H]L-arginine to [3H]L-citrulline in homogenates of normal and pre-eclamptic placentae in the absence and presence of increasing concentrations of ADMA (1-100 microM). The IC50 for ADMA for the pre-eclamptic placentae (22.1 +/- 2.1 microM, n = 6) was not significantly different from that for the normal placentae (18.8 +/- 1.4 microM, n = 6). When ADMA and L-arginine in homogenates was removed by ion exchange chromatography and exogenous L-arginine replaced (32 microM), the IC50 for the pre-eclamptic placentae (19.5 +/- 1.8 microM, n = 6) was not significantly different than that for the normal placentae (20.9 +/- 1.0 microM, n = 6), and NOS activity in the absence of endogenous and exogenous ADMA was still reduced in pre-eclamptic placentae. These results provide no evidence that the sensitivity of placental NOS to ADMA is affected by pre-eclampsia, or that placental ADMA contributes to the reduction of placental NOS in pre-eclampsia.

Analysis of the responses of the fetal vessels of human perfused placental lobules to acute infusions of arachidonic acid.

The vasodilatory response to arachidonic acid by the fetal vessels of human perfused placental lobules, in which tone had been increased by infusion of the thromboxane A2 mimetic U46619, was significantly reduced in the presence of the prostacyclin (PGI2) synthetase inhibitor tranylcypromine compared with saline infusion controls. HPLC analysis of the fetal effluent from human perfused placental lobules, in which radiolabelled arachidonic acid had been infused, identified two peaks of activity. The first peak displayed an elution profile similar to that of a 6-keto-PGF1 alpha standard; the presence of 6-keto-PGF1 alpha in this peak was confirmed by RIA. The second peak had an elution profile similar to that of an arachidonic acid standard. These results suggest that the vasodilatory response to the fetal vessels of human perfused placental lobules to acute infusions of arachidonic acid is mediated, at least in part, by the synthesis of PGI2. These data are consistent with the hypothesis that PGI2 may be involved in the maintenance of low vascular resistance of the fetal placenta in utero.

Human placental acetylcholine.

The human placenta contains both acetylcholine (ACh) and choline acetyltransferase, and in vitro bilaterally perfused placental lobules release ACh. The function of this placental cholinergic system has not yet been clearly defined, although changes occur in it during parturition and it may be linked to placental prostaglandin generation at this time. It has also been suggested that ACh may regulate placental amino-acid transport and/or blood flow. It has been found that ACh release from fetal vessels of bilaterally perfused placental lobules is reduced during preeclampsia but is not necessarily correlated with any change in perfusion pressure or materno-fetal transfer of the nonmetabolizable amino acid alpha-aminoisobutyric acid. However, a correlation has been found between releases from human placental explants of ACh (when inhibited by (2-benzoylethyl)trimethylammonium or vesamicol) and of prostaglandins E2 and F2 alpha. Thus, although the evidence for a role of ACh in the control of placental amino-acid transfer or vascular tone is not conclusive, inhibition of the human placental cholinergic system has been shown to be associated with reduced output of prostaglandins from this tissue.

Effects of ATP, ADP, thrombin, vasopressin and U46619 on human placental nitric oxide synthase activity.

The human placenta contains nitric oxide synthase (NOS) activity, which had been previously found to be localized in the syncytiotrophoblast and the endothelial cells of stem villous vessels. Nitric oxide produced by the syncytiotrophoblast could affect maternal platelet aggregation. The aim of the present study was to examine the effects of substances known to cause platelet aggregation (arginine vasopressin (AVP), the thromboxane A2 mimetic U46619, adenosine diphosphate (ADP) and thrombin) on NOS activity of human placental explants in vitro. Placentae were obtained at term from women with obstetrically uncomplicated deliveries. NOS activity was measured in explants by determining the conversion of [3H]L-arginine to [3H]L-citrulline during 60 min incubations. Either the presence of N-omega-L-arginine or the omission of calcium (in the presence of EDTA) significantly inhibited NOS activity. Adenosine triphosphate (ATP) at concentrations of 100-1000 microM significantly stimulated NOS activity, and was used as a positive control. ADP at a concentration of 1000 microM was found to significantly stimulate NOS activity, however, the other platelet aggregating substances investigated, thrombin (0.1, 10 U/ml), AVP (1, 10, 20 microM) and U46619 (3, 30, 300 nM), had no significant effect on NOS activity. These results show that ATP and ADP can stimulate human placental NOS activity, but provide no evidence that platelet aggregating agents other than ADP can affect the production of NO.

No change in calreticulin with fetal growth restriction in human and rat pregnancies.

INTRODUCTION: Calreticulin is a ubiquitously expressed protein that was detected in the circulation and is significantly increased in maternal blood during human pregnancy compared to the non-pregnant state. Calreticulin is further increased in the plasma of women with the pregnancy-related disorder pre-eclampsia compared to normotensive pregnancy. The aims of this study were to compare calreticulin in human pregnancy with calreticulin in rat pregnancy, and to compare calreticulin during fetal growth restriction with normal control pregnancies. METHODS: Women were recruited who either had normal pregnancies or had pregnancies complicated with fetal growth restriction; maternal blood samples and placentas were collected. Blood was also taken from women who were not-pregnant. Growth restriction was induced in pregnant rats by uterine vessel ligation; blood and placental samples were collected. Blood was also taken from non-pregnant rats. Western blot was used to quantify the placental expression of calreticulin and the concentrations of calreticulin in plasma. RESULTS: Although calreticulin was significantly increased in maternal plasma during human pregnancy compared to the non-pregnant state; it did not increase in plasma during rat pregnancy. These results suggest that there may be differences in the role of extracellular calreticulin in human compared to rat pregnancy. Calreticulin was not significantly altered in either placental extracts or maternal plasma in both the human and rat pregnancies complicated by fetal growth restriction compared to gestational matched control pregnancies. CONCLUSION: This study found that there was no change in calreticulin during human pregnancy complicated with fetal growth restriction or when growth restriction is induced in rats.