Cyclic adenosine 3'-5'-monophosphate (cAMP) is a second messenger, which exerts an important role in the control of human first-trimester trophoblast functions. In the present study we demonstrate the existence of a mechanism that is able to extrude cAMP from trophoblast-derived cell lines, and show evidence indicating the involvement of multidrug resistance protein (MRP) 1, a transporter belonging to the ATP-binding cassette family, in cAMP egress. MRP1 is expressed in trophoblast cell lines and cAMP efflux is highly reduced by the MRP1 inhibitor, MK-571. In addition, interleukin-1beta and estrone are able to enhance MRP1 gene expression and influence extracellular cAMP concentration. The occurrence of a MRP1-dependent cAMP efflux is also shown in human first-trimester placenta explants. Extracellular cAMP could represent a source for adenosine formation, which in turn could regulate cAMP-dependent responses in placental tissue. Evidence is provided that adenosine receptor subtypes are present and functional in human trophoblast-derived cells. A role for cAMP egress mechanism in the fine modulation of the nucleotide homeostasis is therefore suggested.

The trophoblast, i.e. the peripheral part of the human conceptus, exerts a crucial role in implantation and placentation. Both processes properly occur as a consequence of an intimate dialogue between fetal and maternal tissues, fulfilled by membrane ligands and receptors, as well as by hormone and local factor release. During blastocyst implantation, generation of distinct trophoblast cell types begins, namely the villous and the extravillous trophoblast, the former of which is devoted to fetal-maternal exchanges and the latter binds the placental body to the uterine wall. Physiological placentation is characterized by the invasion of the uterine spiral arteries by extravillous trophoblast cells arising from anchoring villi. Due to this invasion, the arterial structure is replaced by amorphous fibrinoid material and endovascular trophoblastic cells. This transformation establishes a low-resistance, high-capacity perfusion system from the radial arteries to the intervillous space, in which the villous tree is embedded. The physiology of pregnancy depends upon the orderly progress of structural and functional changes of villous and extravillous trophoblast, whereas a derangement of such processes can lead to different types of complications of varying degrees of gravity, including possible pregnancy loss and maternal life-threatening diseases. In this review we describe the mechanisms which regulate trophoblast differentiation, proliferation, migration and invasiveness, and the alterations in these mechanisms which lead to pathological conditions. Furthermore, based on the growing evidence that proper inflammatory changes and oxidative balance are needed for successful gestation, we explain the mechanisms by which agents able to influence such processes may be useful in the prevention and treatment of pregnancy disorders.

Normal placentation requires a highly coordinated control of proliferation, migration and invasiveness of extravillous trophoblast cells. Since prostaglandin E2 is a major prostanoid synthesized by intrauterine tissues and highly involved in pregnancy homeostasis, we examined the possibility that it modulates extravillous trophoblast cell functions. Here, we report the presence of mRNAs for prostaglandin E2 EP2 and EP4 receptor isoforms and of proteins in both first-trimester human chorionic villi and in the human trophoblast-derived HTR-8/SVneo cells. Moreover we found that: (i) this cell line releases prostaglandin E2 and the output is enhanced by interleukin-1beta; (ii) the prostanoid consistently inhibits serum- or epidermal growth factor-induced cell proliferation and also migration. An involvement of cAMP in the prostaglandin E2 antiproliferative action is suggested by the observation that the prostanoid greatly enhances cAMP level in HTR-8/SVneo cells and that forskolin inhibits cell proliferation; moreover the administration of prostaglandin E2 plus forskolin, a condition which evokes a synergistic enhancement of cAMP, induces a major impairment of cell growth. Provided that our data are applicable to the trophoblast tissue in vivo, we suggest that prostaglandin E2 exerts an important control on extravillous trophoblast cell functions, preventing an excessive proliferation and migration.

We have tested the hypothesis that human early trophoblast is a target for somatostatin (SRIF) regulatory actions. We report for the first time that SSTR2A and 2B transcripts and proteins are present in first-trimester human chorionic villi and the trophoblast-derived HTR-8/SVneo and JAR cells. In both cell lines, SSTR are functional since SRIF inhibits cyclic AMP pathway, stimulates arachidonic acid release and enhances cell proliferation. Moreover, in HTR-8/SVneo cells, considered a good model of first-trimester EVT, SRIF also enhances migration. An involvement of the cyclic AMP pathway in mediating SRIF effects on proliferation and migration is suggested. Our data support the idea that SRIF regulates early trophoblast functions mainly through an interaction with SSTR2.

In the course of pregnancy amnion cells produce a number of factors which include cytokines and prostaglandins (PGs) produced in response to autocrine, paracrine and endocrine signals. Recent studies performed by several researchers contributed to elucidate the mechanism through which amnion tissue is involved in the triggering of physiological labor. However, there are other possible functions to be ascribed to amniotic cells, depending on the high number of factors that they produce as well as on the receptors that enable them to act in turn as target. For instance, it has been demonstrated that amnion cells are able to produce lecithin upon the regulation of several factors, such as glucocorticoids and epidermal growth factor, a finding that suggests a protective role of the tissue on fetal pulmonary function. As regards to triggering the uterine contractions, it is accepted that prostaglandin release by amnion cells represents a key event. It is under the control of hormones, growth factors, cytokines and probably PGs themselves. A striking analogy has been found between the mechanism of inflammation and the onset of myometrial activity in labor. In this context, it has been shown that for-Met-Leu-Phe (fMLP), the prototype of a series of formylated peptides traditionally considered chemotactic agents, is also involved in the regulation of amniotic PG release. The similitude between labor and inflammatory response is enforced by the antiprostaglandin action of some classes of antibiotics observed in amnion tissue, that enable them as effective tools against preterm labor, both in the absence and in the presence of infection. As for the mechanisms responsible for the regulation of PG synthesis, some agents act by influencing protein synthesis, while others exert their effects through the production of intracellular second messengers, mainly represented by phosphatidyl-inositol-4-5 bisphosphate and cyclic AMP. The mechanism whereby second messengers induce PG release is not clear, and a crosstalk between the two transduction pathways could be hypothesized. This interaction has extensively been analysed in "WISH" cells, a human amnion-derived cell line, which represent a model for the in vitro study of amnion functions. In the present review, we intend to report the results of the studies regarding the mechanisms through which the control of the above mentioned functions is executed.

Phagocytes are activated by several extracellular signals, including formyl-peptides derived from bacterial proteins or disrupted cells. The most intensely studied member of the formylpeptide family is the synthetic tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), whose specific receptors have been identified on neutrophil plasma membrane and subsequently cloned. The fMLP-receptor interaction activates multiple transduction pathways responsible for various neutrophil functions such as adhesion, chemotaxis, exocytosis of secretory granules and superoxide anion production, which represent the physiological response to bacterial infection and tissue damage. An unresolved question is whether signaling requirements are identical or specific for each physiological function. The development of fMLP receptor agonists and antagonists has led to an improvement of our knowledge about the above issue. Of particular interest is the possibility that receptorial antagonists, able to transiently inhibit neutrophil responses to formylpeptides, could be therapeutic agents in the treatment of inflammation-related diseases. Aim of this review is, i) to summarise the current understanding of the series of events that begins at the level of formylpeptide-receptor interaction and is responsible for the activation of transduction pathways, which finally determine neutrophil response; ii) to define the state of art regarding the synthesis as well as the biological actions of fMLP receptor agonists and antagonists.

In human amnion-derived WISH cells [(3)H]estradiol-17beta binding sites are not detectable, but they become measurable in cells exposed to cAMP elevating agents such as forskolin or Ro 20-1724. In cells unexposed to these drugs, 17beta-estradiol stimulates prostaglandin (PG)E(2) release but exerts an evident inhibitory effect in cells exposed to Ro 20-1724. Both stimulatory and inhibitory actions are inhibited by the estrogen receptor antagonist, tamoxifen, by cell pretreatment with cycloheximide, or when the hormone is bound to BSA. Our data demonstrate for the first time that 1) 17beta-estradiol modulates PGE(2) release from WISH cells, interacting with specific intracellular receptors and probably evoking new protein synthesis, and 2) WISH cell responsiveness to 17beta-estradiol seems to be modulated by cAMP, whose levels are significantly increased by the steroid hormone in the presence of Ro 20-1724. The nucleotide is presumably responsible for the enhacement of hormone receptor availability and for the inhibition of PGE(2) release observed in the presence of Ro 20-1724.

OBJECTIVE: The effect of antibiotics in the prevention of preterm labor needs to be further investigated. The aim of this study was to determine the effect of ampicillin on prostaglandin E release from amnion as a possible explanation for its ability to retard preterm labor.STUDY DESIGN: The effect of the beta-lactam antibiotic ampicillin on prostaglandin E release from human amnion was tested under basal and stimulated conditions.RESULTS: Ampicillin dose dependently inhibits basal prostaglandin E release from amnion in both static and dynamic conditions. In our experiments, 10(-7) mol/L ampicillin (a concentration able to significantly inhibit prostaglandin E output) leaves the microbiologic features of the medium substantially unmodified up to 5 hours of incubation. Moreover, the drug reversibly counteracts the prostaglandin E elevation induced by arachidonic acid or oxytocin.CONCLUSION: This finding (i.e., that ampicillin inhibits prostaglandin E release from amnion) may offer an explanation for a beneficial response to ampicillin therapy in the case of perterm labor even in the absence of bacterial infection.

The suggested role of cAMP in the regulation of amnionic prostaglandin release was investigated using two adenylate cyclase inhibitors, MDL 12330A and SQ 22536. These substances exhibited a dose-dependent inhibitory effect on both amnionic enzyme and cAMP levels, but they did not influence prostaglandin E (PGE) release. In addition forskolin and IBMX (3-isobutyl-1-methylxanthine), two drugs known to increase cAMP levels, did not affect PGE output, while dibutyryl cyclic cAMP showed a dose-dependent inhibitory effect. On the basis of our data, the suggested role of amnionic adenylate cyclase in triggering prostaglandin release is not confirmed, and the pathway of phospholipase A2 activation at the onset of labor remains to be elucidated.

The effects of guanine nucleotides were tested on basal and agonist-modulated adenylate cyclase in guinea-pig superior cervical ganglion crude membrane preparations. GTPγS and Gpp(NH)p dose-dependently stimulate, while GDPβS inhibits, both the basal and the prostaglandin E2-stimulated enzyme activity. Low GTP doses, up to 10−5M, stimulate, while higher doses inhibit, the ganglionic adenylate cyclase. The GTP-induced diphasic pattern is maintained also in the presence of prostaglandin E2,d-Ala2-Met-enkephalinamide, or a combination of the two drugs. However, the opioid inhibits the enzyme activity, but only at high GTP doses, while the prostaglandin stimulates the enzyme at all GTP concentrations. The effect is potentiated by a combination of prostaglandin and enkephalin. The enhancing effect of the prostaglandin and of the combination with enkephalin is maximally expressed at high, almost physiological, GTP doses.

1. The effects of serotonin, D-ala2met-enkephalinamide, GTP and several non-physiological modulators of adenylate cyclase were examined on cAMP system of the segmental ganglia of the leech Hirudo medicinalis.

The effects of opiates on cyclic adenosine monophosphate (cAMP) levels have been studied in sympathetic ganglia of guinea-pig, rat and rabbit. D-[Ala2]-Met-enkephalinamide inhibits cAMP synthesis in guinea pig and rat but not in rabbit ganglia, while morphine is always ineffective. In the presence of the enkephalin plus prostaglandin E2 (PGE2) a synergistic increase of the nucleotide levels is observed in guinea pig. This effect is not induced by morphine plus PGE2 and is not shared by rat and rabbit. In guinea pig alpha-endorphin inhibits both basal and PGE2-stimulated cAMP synthesis. In the same preparation the enkephalin increases [3H]PGE2 binding. In guinea pig ganglia a cooperative effect of the enkephalin and PGE2 on the cAMP system is suggested.

Biosynthesis of prostaglandins of E series in BK virus-transformed rabbit-kidney cells (RKBK) and mouse hepatoma cells cultured in vitro with and without exogenous arachidonic acid was determined by radioimmunoassay. Elevated concentrations of prostaglandin E2 were observed in both cell types; moreover, tumor and transformed cells were capable to synthesize higher levels of prostaglandin E2 than normal cells. Mouse hepatoma cells produced more prostaglandins compared to RKBK cells; in addition they seemed less sensitive to inhibition by indomethacin. These data suggest that arachidonic acid metabolism may be another one of the intrinsic biochemical properties that differentiate tumor and virus transformed cells.

Some years ago Greengard and coworkers (5–7) put forward a comprehensive hypothesis to explain the genesis of the slow synaptic potentials observed in curarized mammalian sympathetic ganglia after tetanization of preganglionic fibers. Following this hypothesis the slow synaptic potentials were due to an increase of cyclic nucleotides levels brought about by the interaction of either acetylcholine (ACh)(slow EPSP) or dopamine (DA)(slow IPSP) with specific receptors located on ganglion neuron membranes. In particular, according to Greengard’s hypothesis, prostaglandin E1 (PGE1) would act as a DA antagonist reducing the amplitude of the slow IPSP extracellularly recorded from isolated ganglion preparations. Since DA was supposed to increase cAMP levels via activation of adenylate cyclase, PGE1 would exert an inhibitory action on this enzyme.

Publisher Summary This chapter describes the influence of prostaglandins (PGs) of E type on synaptic transmission of the guinea-pig superior cervical ganglion. The chapter discusses a study to test the hypothesis that PGEs are involved in pre-synaptic control of acetylcholine release. The addition of PGE1 10−7–10−8 M to the superfusing medium significantly reduced the efficacy of synaptic transmission in guinea-pig isolated superior cervical ganglion. It is found that when single ganglion neurons were impaled under PGEs treatment, action potentials evoked by liminar orthodromic stimulation disappeared within 2–3 min and were substituted in the tracings by excitatory postsynaptic potentials (EPSPs) of progressively decreasing amplitude. A neuron in which synaptic input was reduced to a single active fiber is examined. In these conditions, electrical orthodromic stimulation gave rise only to long series of evoked EPSPs of fluctuating amplitude. The lower tracing was taken after PGE1 had been added to the superfusing medium. It can be seen that the number of failures in the tracing is clearly increased and the amplitude of the elementary event doesn't appear to be significantly changed. The effect of PGEs on ganglionic synaptic transmission can be even better appreciated in which the distribution of evoked EPSPs amplitude obtained before and after PGE treatment of ganglion preparations is shown.