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.

Loss of myometrial oxytocin receptors during oxytocin-induced and oxytocin-augmented labour.

Oxytocin is used widely for the induction and augmentation of labour, but there is little information about the dynamics of oxytocin receptors in human myometrium during parturition, and the possible effect of oxytocin infusion. This information is important because G protein-coupled receptors, such as the oxytocin receptor, undergo desensitization after prolonged or repeated stimulation. The concentration of myometrial oxytocin receptors and the steady state of its mRNA were measured in patients undergoing Caesarean sections before or during spontaneous or induced labour. The concentration of receptors before labour was 477 (175-641) fmol mg(-1) protein (median, quartile range), and decreased to 140 (72-206; P < 0.05) and 118 (69-75; P < 0.01) fmol mg(-1) protein during prolonged oxytocin-augmented and oxytocin-induced labour, respectively. The corresponding oxytocin receptor mRNA concentrations decreased by 60- and 300-fold, respectively. The decrease in receptor binding and mRNA in women receiving oxytocin infusion indicates that homologous receptor desensitization occurs in vivo.

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.

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.

Expression of corticotrophin-releasing hormone receptor mRNA and protein in the human myometrium.

The reported effects of corticotrophin-releasing hormone (CRH) on human myometrium support the existence of specific receptors for the hormone in this tissue. We have used the reverse transcriptase-polymerase chain reaction (RT-PCR) technique to study the expression of mRNA coding for the CRH R1 and R2 receptors. RT-PCR of total RNA from both nonpregnant and pregnant myometrium using specific primers resulted in amplification products of the expected sizes for the R1 alpha and R2 alpha CRH receptors. The identity of these amplification products was confirmed by specific restriction digests and sequencing. Immunohistochemistry using a rabbit antibody raised against a specific domain of the CRH R1 receptor demonstrated that the R1 mRNA is translated into protein and confirmed that it is the uterine smooth muscle cells from both nonpregnant and pregnant women that bear this receptor. Our results suggest that CRH may play a role in human pregnancy at the myometrium.

Pyridazines. XIII. Synthesis of 6-aryl-5-oxygenated substituted-3(2H)-pyridazinones and evaluation as platelet aggregation inhibitors.

Several 6-aryl-5-oxygenated substituted pyridazinones have been synthesized and evaluated in vitro for inhibition of platelet aggregation induced by adenosine 5'-diphosphate (ADP), thrombin and collagen. All the tested compounds (except 8 and 9) inhibited platelet aggregation in a dose-dependent manner. The IC50 of the most active substance, compound 2b, was around 60 microM against ADP and collagen as inducers. The inhibition of platelet aggregation caused by test compounds was dependent on the level of oxidation of the function at the 5-position, with the order of IC50 values being R-OH (2a, b, 5) < R-CHO (6, 7) < < R-COOH (8, 9). None of the tested compounds increased the intracellular levels of cAMP, indicating a lack of inhibitory activity on cAMP phosphodiesterase (PDE III) in intact cells. These results suggest that the group present at the 5 position of 6-aryl-5-substituted pyridazinones determines the platelet aggregation-inhibitory activity, and that a mechanism other than PDE inhibition is responsible for this effect.

Characterization of Grb2-binding proteins in human platelets activated by Fc gamma RIIA cross-linking.

Glutathione-S-transferase (GST)-Grb2 fusion proteins have been used to identify the potential role of Grb2-binding proteins in platelet activation by the platelet low-affinity IgG receptor, Fc gamma RIIA. Two tyrosine phosphoproteins of 38 and 63 kD bind to the SH2 domain of Grb2 following Fc gamma RIIA stimulation of platelets. Both are located in the particulate fraction following platelet activation and are also able to bind to a GST-construct containing the SH2 and SH3 domains of phospholipase C gamma 1. p38 also forms a complex with the tyrosine kinase csk in stimulated cells and is a substrate for the kinase. The SH3 domains of Grb2 form a stable complex with SOS1 and two proteins of 75 kD and 120 kD, which undergo tyrosine phosphorylation in Fc gamma RIIA stimulated cells. The 75-kD protein is recognized by antibodies to SLP-76, which has recently been isolated from T cells and sequenced. Tyrosine phosphorylation of p38 and p63 is also observed in platelets stimulated by the tyrosine kinase-linked receptor agonist collagen and by the G protein-coupled receptor agonist thrombin, although phosphorylation of SLP-76 is only observed in collagen-stimulated platelets. p38 and p63 may provide a docking site for Grb2, thereby linking Grb2 SH3-binding proteins SOS1, SLP-76, and p120 to downstream signalling events.

Thrombopoietin potentiates activation of human platelets in association with JAK2 and TYK2 phosphorylation.

Thrombopoietin (TPO), also known as the c-mpl ligand, stimulates rapid tyrosine phosphorylation of multiple proteins in human platelets including the Janus family kinases JAK2 and TYK2. On its own, TPO has no effect on platelet aggregation and dense-granule secretion but induces a general potentiation of these responses by other stimuli. The most dramatic effect is observed against threshold concentrations of agonists for aggregation. Shape change or weak reversible aggregation induced by low concentrations of thrombin, collagen and the thromboxane mimetic, U46619, are converted into irreversible aggregation in the presence of TPO. A similar result is obtained in the presence of the ADP scavenger apyrase and cyclo-oxygenase inhibitor indomethacin. TPO also induces potentiation of dense-granule secretion measured through release of 5-hydroxy[3H]-tryptamine. This effect is most striking against low concentrations of stimuli and is independent of aggregation as it is observed in the presence of chelation of extracellular Ca2+ with EGTA. TPO potentiates activation of phospholipase C and elevation of intracellular Ca2+, providing a molecular explanation for potentiation of functional responses. TPO may have an important physiological role in priming platelet activation in thrombocytopenia, an action that may help to compensate for the reduced platelet density.

In-vitro platelet responses to arachidonic acid in the rat.

Using both the turbidimetric and the conductive methods to study aggregation of platelets, we found that arachidonic acid stimulated rat washed platelets in a dose-dependent manner (40 microM-0.5 mM). Although a high concentration of arachidonic acid (0.5 mM) produced an increase in light transmission both in the presence of 2 mM CaCl2 and EGTA (45.8 +/- 2.8 and 50.4 +/- 0.8% respectively) no changes in impedance were detected. Lysis caused by this concentration of arachidonic acid was very high at all the concentrations of calcium used (mean of 81.3%). In addition, the turbidimetric response induced by 0.5 mM arachidonic acid implied an initial decrease in light transmission but did not correlate with a real shape change. Forty micromolar arachidonic acid induced a calcium-dependent aggregation measured both by aggregometry and impedance. Morphology of aggregates induced by both concentrations was also studied. These results suggest that the optimal concentration for studying rat platelet activation by arachidonic acid is 40 microM; high concentrations (0.5 mM) cause aspecific effects not correlated to a physiological activation response.

Phosphorylation of JAK2 in thrombin-stimulated human platelets.

We show the presence of the tyrosine kinase JAK2 in human platelets and demonstrate that it undergoes phosphorylation on tyrosine residues on challenge with the G protein receptor stimulus, thrombin, or the tyrosine phosphatase inhibitor, peroxovanadate. Thrombin-induced phosphorylation of JAK2 is inhibited by two structurally distinct inhibitors of tyrosine kinases, staurosporine and the tyrphostin ST271. The protein kinase C (PKC) inhibitor, Ro 31-8220, and intracellular Ca2+ chelator, BAPTA-AM, also inhibit thrombin-induced phosphorylation of JAK2, while the phorbol ester, phorbol dibutyrate (PDBu), and Ca2+ ionophore, A23187, induce tyrosine phosphorylation of JAK2. These results suggest that tyrosine phosphorylation of JAK2 stimulated by thrombin may be mediated downstream of phosphoinositide metabolism.

The diacylglycerol kinase inhibitor, R59949, potentiates secretion but not increased phosphorylation of a 47 kDalton protein in human platelets.

We have investigated the action of a novel inhibitor of DG-kinase, R59949. This agent was found to produce partial inhibition of formation of phosphatidic acid in human platelets challenged with thrombin, DC8 or OAG. However, this effect was not associated with enhanced phosphorylation of a 47 kDa protein, a known substrate for protein kinase C. We therefore believe that this compound does not represent a major advance on its earlier prototype, R59022.