Implantation: embryonic signals and the modulation of the uterine environment--a review.

Interaction between maternal endometrium and embryo during implantation is mediated through the multiple molecules and signalling cascades, which are still not fully understood. This complex sequence of events results in the transformation of stromal cells into decidual cells (decidualization). The conceptus is essential to regulate these changes in baboon stromal fibroblasts, possibly through production of cytokines by the implanting embryo. The role of interleukin-1 (IL-1) system during implantation and its contribution to decidualization is discussed in this review. Decidualized endometrial stromal cells are thought to contribute to establishment of a successful pregnancy by expressing a number of gene products. One of the major products of decidual cells is insulin-like growth factor binding protein-1 (IGFBP-1). The IGFBP-1 serves as a modulator of insulin-like growth factor (IGF) protein action in the interactions between the decidua and invading trophoblast. The delineation of the mechanisms involved in cooperative action of transcription factors FKHR and HOXA10 leading to IGFBP-1 expression, as well as interrelationship between IL-1 and IGF signalling cascades will contribute to the understanding of events leading to successful implantation.

IL-1beta during in vitro decidualization in primate.

The sequence of biochemical and molecular events associated with decidualization in the primate remain unclear. In the baboon, the sequential changes during this period in vivo are characterized by the downregulation of alpha-smooth actin followed by induction of cyclooxygenase-2 (COX-2) at the implantation site and the expression of insulin growth factor binding protein-1 (IGFBP-1). IGFBP-1 is the predominant protein in decidualized cells and is considered to be biochemical marker of decidualization. In the baboon the expression of IGFBP-1 requires the presence of a conceptus in vivo or N(6), 2'-O-dibutyryladenosine 3:5'-cyclic monophosphate (dbcAMP) in the presence of hormones in vitro. In addition IL-1beta, as a possible conceptus-mediated factor, can induce IGFBP-1 expression in the presence of hormones following 3 days of incubation. However, if IL-1beta and dbcAMP are added together, IGFBP-1 expression is inhibited which resulted in IL-1beta being considered to be "inhibitory" to decidualization. Current data suggest that IL-1beta can activate multiple signaling pathways that either positively (no exogenous cAMP) or negatively (in presence of exogenous cAMP) regulate IGFBP-1 gene expression and decidualization in vitro. Signaling pathways activated by IL-1beta following 10 min of stimulation result in the phosphorylation of mitogen-activated protein kinase (MAPK, specifically p38 MAPK) and also lead to NF-kappaB activation. The expression of COX-2 and matrix metalloproteinase-3 (MMP-3) genes follows after 4-6 h. The steroid hormones, particularly progesterone, which are critical for IGFBP-1 expression, modulate the activity of IL-1beta by down-regulating MMP-3 activity. Disruption of actin filaments enhances IGFBP-1 induction during decidualization. IL-1beta induced MMP-3 may upregulate IGFBP-1 by initiation of cytoskeletal reorganization through degradation of extracellular matrix (ECM). Inhibition of IL-1beta induced pathways leads to reduction of IGFBP-1 expression, suggesting that IL-1beta may be involved in the events leading to decidualization in baboons.

The role of chorionic gonadotropin (CG) in blastocyst implantation.

Implantation is a complex spatio-temporal interaction between the genotypically different embryo and the mother. Success of this event requires the synchronization of development and effective biochemical communications from both sides. Chorionic gonadotropin (CG), which is a major embryonic signal in the primate, is a glycoprotein hormone synthesized and secreted by the trophoblast. Various isoforms exist in plasma, urine, and blastocyst culture medium, a result of posttranslational modifications. The exponential secretion of CG and its long circulatory half-life extends the life span of corpus luteum to maintain the supply of progesterone during the first 6-8 weeks of pregnancy. To study the direct effects of CG in the uterus, we used the baboon (Papio anubis) as a non-human primate model. In vivo stimulation with CG during the window of uterine receptivity results in further morphologic and biochemical modifications of the receptive endometrium. These are characterized by the plaque reaction in the luminal epithelium, an increase in glycodelin expression and secretion by the glandular epithelium, and the differentiation of subepithelial stromal fibroblasts characterized by expression of the alpha smooth muscle actin (alpha SMA). Pretreatment with progesterone receptor antagonist (PRa) completely or partially inhibits these effects. The signal transduction pathway activated by CG in primate endometrial epithelial cells involves the protein kinase A (PKA)-independent phosphorylation of extracellular signal regulated kinase (ERK 1/2). This alternate signal transduction pathway may prevent CG Receptor (R) downregulation at the implantation site and enhance epithelial cell proliferation and differentiation. Thus, our results suggest that CG plays an important role in implantation in addition to its luteotrophic role.

Interleukin-1beta induces the expression of insulin-like growth factor binding protein-1 during decidualization in the primate.

Since interleukin (IL)-1 can modulate fetal/maternal interactions, we hypothesized that IL-1beta is one potential embryonic cytokine that regulates the conceptus-induced decidual response in baboon stromal fibroblasts. Treatment of stromal fibroblasts with IL-1beta (10 ng/ml, 10 min) resulted in the phosphorylation of p38 mitogen-activated protein kinase and IkappaB-alpha. This suggests that IL-1beta induces multiple signaling pathways in stromal cells that result in the activation of mitogen-activated protein kinase cascade and the transcription factor NF-kappaB. After 4 h of stimulation, IL-1beta induced gene expression of cyclooxygenase-2 (COX-2) but not cyclooxygenase-1 (COX-1). PGE2 synthesis paralleled COX-2 messenger RNA expression. The addition of hormones [36 nM estradiol-17beta, 1 microM medroxyprogesterone acetate, and 100 ng/ml relaxin] to IL-1beta-treated cells induced insulin-like growth factor binding protein-1 (IGFBP-1) messenger RNA expression after 3 days of incubation. A specific COX-2 inhibitor, NS 398 (10 nM), partially inhibited IGFBP-1 protein synthesis. In contrast, the induction of IGFBP-1 by N6, 2'-O-dibutyryladenosine 3:5'-cyclic monophosphate (dbcAMP) and hormones was not affected by NS 398 treatment. Both dbcAMP and IL-1beta, in the presence of hormones, can independently induce IGFBP-1 gene expression and decidualization. However, if IL-1beta and dbcAMP were added together, IGFBP-1 expression was inhibited. These data suggest that IL-1beta can activate multiple signaling pathways that either positively or negatively regulate IGFBP-1 gene expression and decidualization.

Complementary mechanisms of enhanced oxytocin-stimulated prostaglandin E2 synthesis in rabbit amnion at the end of gestation.

The up-regulation of oxytocin (OT) receptors in rabbit amnion at the end of gestation is associated with a large increase in the ability of OT to stimulate PGE2 synthesis. The purpose of these investigations was to determine what other factors contribute to this increase. OT enhanced PGE2 synthesis at several levels. The concentrations of cytosolic phospholipase A2, which generates arachidonic acid for PGE2 synthesis, and PGH endoperoxide synthases (types 1 and 2), which catalyze the conversion of arachidonic acid to prostanoids, rose substantially in rabbit amnion at term. OT stimulated translocation of cytosolic phospholipase A2 to the cell particulate fraction, presumably by a Ca2+-mediated process, and phosphorylation of cytosolic phospholipase A2 via the extracellular regulated protein kinase 2/1-mediated pathway. OT-stimulated increases in intracellular Ca2+ concentrations and extracellular regulated protein kinase 2/1 phosphorylation were both mediated by G(q/11) activation. OT also increased the expression of PGH endoperoxide synthase-2 after treatment of amnion cells in culture for 2 h; however, PGE2 release in response to OT was virtually immediate. These findings show that the rapid stimulation of PGE2 synthesis by OT occurs through cytosolic phospholipase A2 activation and PGH endoperoxide synthase-1 activity, both of which, along with OT receptor concentrations, are considerably up-regulated in the amnion at the end of gestation.

Signal pathways mediating oxytocin stimulation of prostaglandin synthesis in select target cells.

A major action of oxytocin is to stimulate prostaglandin production in reproductive tissues. The two major enzyme systems involved are cytosolic phospholipase A2 (cPLA2), which catalyses the formation of arachidonic acid from membrane glycerophospholipids, and prostaglandin endoperoxide-H synthases-1 and -2, which allow conversion of arachidonic acid to prostaglandins. During gestation, the concentrations of all three enzymes rise in the rabbit amnion. Agonists, including oxytocin, increase cPLA2 activity, in part, by elevating intracellular Ca2+ concentration, which causes cPLA2 to be translocated from the cytosol to intracellular membrane binding sites. Cytosolic PLA2 is then activated by a mitogen-activated protein kinase (MAPK)-dependent step. Our studies have elucidated signal pathways involved in oxytocin-stimulated prostaglandin output in both rabbit amnion cells and Chinese hamster ovary cells stably transfected with the rat oxytocin receptor. The two cell types are alike with respect to oxytocin-stimulated intracellular Ca2+ transients, mediation via Gq, and the specific MAPK that catalyses the phosphorylation of cPLA2. However, they differ with respect to the mechanisms of upregulation of key enzymes involved in prostaglandin E2 synthesis. These findings illustrate the tiers of complementary mechanisms involved in oxytocin stimulation of prostaglandin E2, and the extent of the diversity in the cellular signalling pathways involved.

The proximal portion of the COOH terminus of the oxytocin receptor is required for coupling to g(q), but not g(i). Independent mechanisms for elevating intracellular calcium concentrations from intracellular stores.

As the oxytocin receptor plays a key role in parturition and lactation, there is considerable interest in defining its structure/functional relationships. We previously showed that the rat oxytocin receptor transfected into Chinese hamster ovary cells was coupled to both G(q/11) and G(i/o), and that oxytocin stimulated ERK-2 phosphorylation and prostaglandin E(2) synthesis via protein kinase C activity. In this study, we show that deletion of 51 amino acid residues from the carboxyl terminus resulted in reduced affinity for oxytocin and a corresponding rightward shift in the dose-response curve for oxytocin-stimulated [Ca(2+)](i). However, oxytocin-stimulated ERK-2 phosphorylation and prostaglandin E(2) synthesis did not occur in cells expressing the truncated receptor. Oxytocin also failed to increase phospholipase A activity or activate protein kinase C, indicating that the mutant receptor is uncoupled from G(q)-mediated pathways. The Delta51 receptor is coupled to G(i), as oxytocin-stimulated Ca(2+) transients were inhibited by pertussis toxin, and a Gbetagamma sequestrant. Preincubation of Delta51 cells with the tyrosine kinase inhibitor, genistein, also blocked the oxytocin effect. A Delta39 mutant had all the activities of the wild type oxytocin receptor. These results show that the portion between 39 and 51 residues from the COOH terminus of the rat oxytocin receptor is required for interaction with G(q/11), but not G(i/o). Furthermore, an increase in intracellular calcium was generated via a G(i)betagamma-tyrosine kinase pathway from intracellular stores that are distinct from G(q)-mediated inositol trisphosphate-regulated stores.

Demonstration of functional oxytocin receptors in human breast Hs578T cells and their up-regulation through a protein kinase C-dependent pathway.

Oxytocin (OT) receptors (OTRs) have been demonstrated in a number of human breast tumors and tumor cells, but it was not clear whether the receptors were functional. We examined the regulation and function of OTR in a tumor cell line, Hs578T, derived from human breast. These cells expressed moderate levels of OTR when cultured in 10% FBS, as demonstrated by RT-PCR and binding analyses. Serum deprivation resulted in the loss of OTRs, with no effect on cell viability. Restoration of serum and addition of 1 microM dexamethasone (DEX) increased OTR levels by about 9-fold. Up-regulation was blocked by the addition of phospholipase C and PKC inhibitors. Serum/DEX treatment also increased steady state OTR messenger RNA levels. OT increased intracellular Ca2+ in a time- and dose-responsive manner, and the effects of OT were lost when OTRs were down-regulated by serum starvation. Serum/DEX up-regulation of OTR restored the responsiveness to OT. OT also stimulated ERK-2 (extracellular signal-regulated protein kinase) phosphorylation and PGE2 synthesis in Hs578T cells. In addition to showing that OTRs in the breast tumor cells are functional, these studies show that Hs578T cells can be used to study molecular regulation of OTR gene expression and intracellular signaling pathways stimulated by OT.

ERK2 mediates oxytocin-stimulated PGE2 synthesis.

Oxytocin (OT) induces PG synthesis by both uterine endometrial and amnion cells. We showed previously that CHO cells stably transfected with the rat oxytocin receptor (CHO-OTR cells) also synthesize PGE2 in response to OT. In the present work we have demonstrated that OTRs are coupled to both Gi and Gq/11, using immunoprecipitation of solubilized OTR complexes and ADP ribosylation. OT treatment caused the rapid phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2 or p42MAPK), which was partially inhibited by pertussis toxin (PTX), consistent with OTR-Gi coupling. The PTX-insensitive portion of ERK2 phosphorylation was linked to Gq, as inhibitors of both phospholipase C (U-73122) and protein kinase C (GF-109203X) blocked OT-induced ERK2 phosphorylation. OT-stimulated c-fos expression was also mediated by ERK2 phosphorylation. The ERK-c-fos pathway has been shown to be associated with cell proliferation, but OT had no effect on [3H]thymidine uptake by CHO-OTR cells. However, inhibition of OT-induced ERK2 phosphorylation with an ERK kinase inhibitor (PD-98059) markedly reduced OT-stimulated PGE2 synthesis, pointing to the importance of ERK2 activation in OT action.

Coupling of oxytocin receptor to G proteins in rat myometrium during labor: Gi receptor interaction.

Occupancy of oxytocin receptor (OTR) binding sites in pregnant rat myometrial membranes with iodinated oxytocin antagonist (OTA), followed by detergent solubilization and size selection, showed that radioactivity eluted in two distinct peaks: one corresponding in size to the isolated receptor (approximately 60 kDa) and the other ranging from 240 to 320 kDa. The unliganded 240- to 320-kDa fraction contained OTRs coupled to G proteins, as the addition of oxytocin (OT) increased guanosine 35S-labeled 5'-O-(3-thiotriphosphate) binding up to twofold in a dose-dependent manner. The effects of OT were blocked by coincubation with OTA. G protein alpha-subunits associated with OTRs in the 240- to 320-kDa peak were identified by immunoadsorption. Significant amounts of both G alpha q/11 and G alpha i3 were associated with the OTR; a lesser amount of G alpha s was complexed. Using the same approach but with antibodies to effector enzymes, we observed that phospholipase C beta 1 (PLC beta 1) and PLA2 were also associated with the OTR. The results corroborate the well-established interaction of OTR with Gq and further show that Gi coupling might be an important component of OTR signal transduction. To further investigate the interaction of Gi with the OTR, we showed that OT stimulation of guanosine 5'-triphosphatase activity in intact myometrial membranes was inhibited by pertussis toxin. Pertussis toxin-stimulated ADP ribosylation of G alpha i in myometrial membranes was also decreased by OT treatment. These findings with pertussis toxin strongly indicate that OTR is coupled to Gi in rat myometrial membranes. The 60-kDa OTR peak (noncoupled receptor) was demonstrable in the myometrium only before the end of gestation and after parturition and accounted for about one-half the 125I-OTA binding activity. At term, there was about a fivefold increase in binding and almost a complete shift to the 240- to 320-kDa-size complex. Thus the established increased sensitivity of the myometrium to OT at term could be the result of both upregulation of OTRs and an increase in the fraction of receptors coupled to signal transduction components, one of which is Gi.

A new linear V1A vasopressin antagonist and its use in characterizing receptor/G protein interactions.

We characterized a new iodinated, high affinity, linear V1a vasopressin antagonist, phenylacetylD-Tyr(Et)Phe-Gln-Asn-Lys-Pro-Arg-Tyr-NH2. The antagonist bound specifically to the V1a vasopressin receptor in crude rat liver membranes with an apparent Kd value of 0.168 nM. This affinity is approximately 1 order of magnitude greater than that of the natural agonist, vasopressin. The inhibitory activity of the antagonist can be demonstrated by its inability to elicit activation and uncoupling of G proteins from the receptor. Thus, after occupancy of receptor sites in rat liver membranes with labeled antagonist and detergent solubilization, the labeled receptor (approximately 60 kDa) was eluted as a stable 400-kDa complex on size-exclusion chromatography. In contrast, when the receptor sites were occupied by the agonist [3H]vasopressin, the receptor eluted as a 60-kDa peak. Coincubation of membranes with iodinated antagonist and an excess of unlabeled vasopressin caused both reduced antagonist binding and a complete shift from the 400-kDa to the 60-kDa peak. The addition of vasopressin to unliganded 400-kDa fractions resulted in a 75% increase in [35S]guanosine-5'-O-(3-thio)triphosphate binding activity, indicating that the 400-kDa fraction contains complexes between the V1a receptor and G proteins. The vasopressin-elicited increase was inhibited by antagonist. Using specific antibodies and immunoadsorption to protein A/Sepharose columns, we found that G protein isotypes G(alpha q/11), G(alpha i3), and G(alpha s), and effector enzymes PLC-beta1, PLC-gamma2 and PLA-2 were associated with the antagonist-labeled receptor in the 400-kDa fraction. Because the 400-kDa complex was found in the absence of ligand, the V1a receptor and the appropriate G proteins and effector enzymes are likely preassociated with each other and do not aggregate after antagonist addition. The association of V1a receptor with the different specific G proteins and effector enzymes is consistent with the multiple actions of vasopressin on liver cells. Antibodies directed against a portion of the carboxyl-terminal domain of the V1a receptor interacted with 60-kDa antagonist-occupied receptor but not with receptor in the 400-kDa complex. These results suggest that the carboxyl-terminal region of the receptor is sterically hindered when coupled to G proteins. The iodinated linear vasopressin antagonist therefore allows stable receptor/G protein complexes and can be an important tool (along with the antisera) for use in the study of factors that control V1a receptor/G protein coupling.

Molecular cloning and functional characterization of the oxytocin receptor from a rat pancreatic cell line (RINm5F).

Oxytocin (OT) and vasopressin (AVP) stimulate insulin and glucagon release from the pancreas, and evoke insulin secretion from the rat insulinoma cell line, RINm5F. To determine which AVP/OT receptor subtype is expressed in RINm5F cells, we used PCR with degenerate primers to two transmembrane domains of the AVP (V1a, V1b (or V3), V2) and OT receptors (OTRs). The single PCR fragment identified was used to obtain a full length cDNA from a RINm5F cDNA library. Comparison of the deduced amino acid sequence of this clone with uterine OTR sequences from several species (human, sheep, bovine) and to the pig kidney epithelial cell (LLC-PK1) OTR reveals a very high degree of homology. After the RIN cell OTR cDNA was stably transfected into CHO cells (CHO-OTR), the cell membranes bound iodinated oxytocin antagonist with an apparent Kd comparable to that of RIN cell membranes and those from other OT target cells. Comparison of the ligand specificities of CHO-OTR and RIN cells membranes showed that the relative Ki values of a series of OT analogues were approximately equivalent in both preparations. The rank order of apparent Ki values also corresponded to published values for the rat myometrium, where OT elicits intracellular calcium transients, and increases inositol phosphate production. In uterin endometrium and amnion cells, OT stimulates prostaglandin release. Stimulation of CHO-OTR cells with OT caused an increase in cytosolic calcium concentration originating from both intracellular and extracellular sources, and a dose-dependent increase in inositol phosphate levels. Arachidonic acid release and PGE2 synthesis were also stimulated by OT. These findings (amino acid sequence homology, binding specificity, and signal transduction/second messenger production) suggest that OTRs from RINm5F cells are indistinguishable from OTRs that have been described in other tissues. The expression of OTR in pancreatic cells implies that OT plays a role in pancreatic function.

Peroxidase labelled monoclonal antibody against light chains of human cardiac myosin.

Monoclonal antibody against light chains of human cardiac myosin (MLC) was labelled with horseradish peroxidase. The conjugation was performed by two different methods with glutaraldehyde and periodate respectively. The binding activities of the conjugates were tested by enzyme linked immunosorbent assay (ELISA) on the microtitration plates with immobilized MLC (1-1000 ng per well). A comparison of both methods revealed their universal suitability for the preparation of conjugates as well as their applicability. The use of conjugates shortens the time needed and improves the ELISA method for MLC estimation. Specific advantages of the glutaraldehyde and the periodate method concern diverse details.

[A patient with acute hydroxychloroquine poisoning; recommendation for treatment]. / Een patiënt met acute intoxicatie door hydroxychloroquine; een behandelingsadvies.

We treated a 30-year-old man for whom Plaquenil (hydroxychloroquine) had been prescribed for rheumatoid arthritis, and who had taken 4 g orally to end his life. Symptoms of severe intoxication due to (hydroxy)chloroquine are rapid onset of hypoventilation, cardiovascular collapse with bradycardia, peripheral vasodilation, arrhythmias and convulsions. The lethal dose of chloroquine has been estimated at 3-5 g in adults and at 0.75-I g in young children. Acute intoxication should be treated with aspiration of gastric contents, artificial ventilation in case of hypoventilation and intravenous or intratracheal dopamine, noradrenaline or adrenaline in case of cardiovascular depression and peripheral vasodilation. Arrhythmias and convulsions should be treated symptomatically. The patient in our case survived the intoxication and is now under psychiatric treatment.

Effects of forskolin and triiodothyronine on cyclic AMP production in rat thymocytes.

The effects of forskolin, 3,5,3'-L-triiodothyronine, and isobutyl methylxanthine on the accumulation of cyclic AMP were studied in rat thymocytes in vitro. Forskolin was found to stimulate markedly the production of intracellular cAMP which was partially released from the cells into the medium. Isobutyl methylxanthine, an inhibitor of cAMP phosphodiesterase, markedly potentiated the effect of forskolin on intracellular cAMP concentration, while triiodothyronine was ineffective. However, triiodothyronine acted synergically with forskolin and/or isobutyl methylxanthine. It was confirmed that low forskolin concentrations modulate the effects of the hormone.