The placenta is simply a neuroendocrine parasite.

This paper will document the early scientific observations that kindled my neuroendocrinological interest in pre-eclampsia, a life-threatening disease that affects both mother and baby. My interest in this subject started with the placental origin of melanotrophin activity, moving on, through corticotrophin-releasing factor and its binding protein, to a tachykinin modified specifically in the placenta by phosphocholine, a post-translational moiety normally used by parasites to avoid immune surveillance and rejection. This work may finally have led to an understanding of the identity of the elusive placental factor that, whilst attempting to compensate for the poor implantation of the placenta, causes the many symptoms seen in the mother during pre-eclampsia.

Maternal plasma corticotropin-releasing factor (CRF) and CRF-binding protein (CRF-BP) levels in post-term pregnancy: effect of prostaglandin administration.

OBJECTIVE: Placental corticotropin-releasing factor (CRF) affects myometrial contractility and the secretion of several uterotonins such as prostaglandins (PGs); however, the activity of CRF is counteracted by CRF-binding protein (CRF-BP). At term and pre-term labor, CRF levels in maternal plasma are highest whereas those of CRF-BP are falling, and the cause of this fall is unknown. Thus, in this study, we investigated the effect of PG administration for labor induction on maternal plasma CRF and CRF-BP concentrations. DESIGN: Maternal plasma CRF and CRF-BP levels were assayed before and after (2 h later) induction of labor by intracervical administration of prostaglandin E(2) (PGE(2)), and at delivery in a group of healthy post-term pregnancies (n=18). Controls were women at term out of labor (n=22), who subsequently progressed to deliver a healthy singleton baby. METHODS: CRF was measured by two-site immunoradiometric assay, and CRF-BP was assayed by radioimmunoassay. RESULTS: Maternal plasma CRF levels were significantly (P<0.0001) lower and CRF-BP significantly (P<0.0005) higher in post-term than in term pregnancies. With respect to induction of labor, 2 mg PGE(2) were sufficient to increase maternal plasma CRF levels at delivery (P<0.005). While 0.5 mg PGE(2) significantly decreased maternal plasma CRF-BP levels at delivery (P<0.001), 2.0 mg PGE(2) significantly reduced CRF-BP concentrations both after 2 h (P<0.05) and at delivery (P<0.0001). CONCLUSIONS: In the light of the well-known stimulation of prostaglandin release by CRF, these data suggest a positive feedback effect of PGE(2) on maternal CRF release during induced labor.

The measurement of maternal plasma corticotropin-releasing factor (CRF) and CRF-binding protein improves the early prediction of preeclampsia.

In the present study we measured maternal plasma concentrations of two placental neurohormones, corticotropin-releasing factor (CRF) and CRF-binding protein (CRF-BP), in 58 at-risk pregnant women consecutively enrolled between 28 and 29 wk of pregnancy to evaluate whether their evaluation may predict third trimester-onset preeclampsia (PE). The statistical significance was assessed by t test. The cut-off points for defining altered CRF and CRF-BP levels for prediction of PE were chosen by receiving operator characteristics curve analysis, and the probability of developing PE was calculated for several combinations of hormone testing results. CRF and CRF-BP levels were significantly (both P < 0.0001) higher and lower, respectively, in the patients (n = 20) who later developed PE than in those who did not present PE at follow-up. CRF at the cut-off 425.95 pmol/liter achieved a sensitivity of 94.8% and a specificity of 96.9%, whereas CRF-BP at the cut-off 125.8 nmol/liter combined a sensitivity of 92.5% and a specificity of 82.5% as single markers for prediction of PE. The probability of PE was 34.5% in the whole study population, 93.75% when both CRF and CRF-BP levels were changed, and 0% if both hormone markers were unaltered. The measurement of CRF and CRF-BP levels may add significant prognostic information for predicting PE in at-risk pregnant women.

Has the mechanism by which the endocrine placenta scavenges the mother whilst sparing the foetus been unmasked?

The endocrine placenta has a dilemma; it shares the foetal circulation and yet it needs to secrete active peptide hormones into the maternal circulation to control her metabolism to meet the demands of the growing foetus. Simultaneously, it needs to allow the endocrine systems of the foetus to develop independently. This Article will describe how peptide hormones are processed from inactive intermediates and will propose a hypothesis of how the placenta has revised this process to protect the foetus from the potentially damaging affects of these products.

Neurokinin B is a paracrine vasodilator in the human fetal placental circulation.

Neurokinin (NK) B is a member of the tachykinin family of neurotransmitters, exerting hypotensive or hypertensive effects in the mammalian vasculature through synaptic release from peripheral neurons, according to either NK(1) and NK(2) or NK(3) receptor subtype expression, respectively. There is recent evidence that NKB is expressed by the syncytiotrophoblast of the human placenta, an organ that is not innervated. We hypothesized that NKB is a paracrine modulator of tone in the fetal placental circulation. We tested this hypothesis using the in vitro perfused human placental cotyledon. Our data show that NKB is a dilator of the fetal vasculature, causing a maximal 25.1 +/- 4.5% (mean +/- SEM; n = 5) decrease in fetal-side arterial hydrostatic pressure (5- microM NKB bolus; effective concentration in the circulation, 1.89 nM) after preconstriction with U-46619. RT-PCR demonstrated the presence of mRNA for NK(1) and NK(2) tachykinin receptors in the placenta. Using selective receptor antagonists, we found that NKB-induced vasodilation is through the NK(1) receptor subtype. We found no evidence for the involvement of either nitric oxide or prostacyclin in this response. This study demonstrates a paracrine role for NKB in the regulation of fetal placental vascular tone.

Identification of stimulatory and inhibitory inputs to the hypothalamic-pituitary-adrenal axis during hypoglycaemia or transport in ewes.

This study used the novel approach of statistical modelling to investigate the control of hypothalamic-pituitary-adrenal (HPA) axis and quantify temporal relationships between hormones. Two experimental paradigms were chosen, insulin-induced hypoglycaemia and 2 h transport, to assess differences in control between noncognitive and cognitive stimuli. Vasopressin and corticotropin-releasing hormone (CRH) were measured in hypophysial portal plasma, and adrenocorticotropin hormone (ACTH) and cortisol in jugular plasma of conscious sheep, and deconvolution analysis was used to calculate secretory rates, before modelling. During hypoglycaemia, the relationship between plasma glucose and vasopressin or CRH was best described by log10 transforming variables (i.e. a positive power-curve relationship). A negative-feedback relationship with log10 cortisol concentration 2 h previously was detected. Analysis of the "transport" stimulus suggested that the strength of the perceived stimulus decreased over time after accounting for cortisol facilitation and negative-feedback. The time course of vasopressin and CRH responses to each stimulus were different However, at the pituitary level, the data suggested that log10 ACTH secretion rate was related to log10 vasopressin and CRH concentrations with very similar regression coefficients and an identical ratio of actions (2.3 : 1) for both stimuli. Similar magnitude negative-feedback effects of log10 cortisol at -110 min (hypoglycaemia) or -40 min (transport) were detected, and both models contained a stimulatory relationship with cortisol at 0 min (facilitation). At adrenal gland level, cortisol secretory rates were related to simultaneously measured untransformed ACTH concentration but the regression coefficient for the hypoglycaemia model was 2.5-fold greater than for transport. No individual sustained maximum cortisol secretion for longer than 20 min during hypoglycaemia and 40 min during transport. These unique models demonstrate that corticosteroid negative-feedback is a significant control mechanism at both the pituitary and hypothalamus. The amplitude of HPA response may be related to stimulus intensity and corticosteroid negative-feedback, while duration depended on feedback alone.

Adrenal growth is controlled by expression of specific pro-opiomelanocortin serine protease in the outer adrenal cortex.

As previous work had shown that extreme N-terminal fragments of the ACTH precursor pro-opiomelanocortin (POMC) not containing gamma-melanotropin (gamma-MSH) were active adrenal mitogens but an antiserum raised against gamma-MSH paradoxically also inhibited adrenal growth we proposed that the adrenal mitogen is processed from pro-gamma-MSH by a neurally controlled protease at the growing adrenal. To this end we have characterised a novel serine protease (named adrenal secretory protease (AsP) as Psort predicted a leader motif) which is expressed at the glomerulosa/fasciculata boundary where mitosis takes place. The expression of AsP was also found to be essential for mitosis of the adrenal cortical tumor Y1 cell-line in POMC containing media and 3D homology modeling revealed the presence of a catalytic pocket flanked by the classical His/Asp/Ser motifs. An usual feature of the model was a cluster of arginine residues on the underside of the protease suggesting that this basically charged face would tend to retain it on the cell surface on secretion-immunocytochemistry using an antiserum raised against a synthetic peptide spanning residues 1-25 of AsP showed that this was the case for Y1 cells. Specificity of AsP (affinity purified from Y1 media) was demonstrated by its inability to cleave model substrates for either trypsin or pro-hormone converting enzymes but was able to cleave an internally quenched POMC (44-55) model peptide. Interestingly mass spectral analysis of products of the latter predicts that the protease cleaves between the bond between Val52 and Met53 suggesting the natural adrenal mitogen is POMC (1-52).

Hox gene expression in adult tissues with particular reference to the adrenal gland.

In comparison to the embryo, very little work has been carried out on the expression and role of Hox genes in the adult animal. An expression profile of all 39 vertebrate Hox genes on a select panel of adult human tissues reveals that in fact these genes are widely expressed throughout the adult human and a colinear pattern of expression is displayed similar to that of the developing embryo. Of particular interest is the abundance of Hox genes that are expressed within the adult adrenal gland. Adrenal cortical cells are continuously renewed to sustain production of zonal steroids. Cell proliferation occurs at the periphery of the cortex and cells are then displaced centripetally, phenotypically switching as they migrate through the gland before undergoing apoptosis at the zona reticularis/medullary boundary. It is still unclear which mechanisms cause the cells to differentiate as they cross the zonal boundaries and we hypothesise that Hox genes may be involved in the phenotypic switching of the adrenocortical cells. In situ hybridisation experiments were carried out on adult rat adrenal gland sections and Hox gene expression was localized within the zonal borders, coinciding with the localization of cells that undergo phenotypic differentiation, and thus supporting our hypothesis that Hox genes may be involved in the phenotypic switching of the adrenocortical cells. As in the developing embryo, the genes display colinear expression with the 3' Hox genes being expressed within the outer gland and the 5' genes within the inner zones.

The characterization of pregnancy associated plasma protein-E and the identification of an alternative splice variant.

We have performed differential display and bioinformatic database mining of the placenta, in an attempt to find novel diagnostic markers of pathological pregnancies. We have identified a full-length cDNA encoding the preproprotein of pregnancy associated plasma protein-E (PAPP-E); a putative metalloprotease, of 1790-residues with a putative 21-residue signal peptide. An alternatively spliced mRNA was found to encode an 826-residue precursor protein corresponding to the N-terminus of PAPP-E. Both PAPP-E variants were found to be co-expressed abundantly in the placenta and non-pregnant mammary gland with low expression in the kidney, foetal brain and pancreas. Analysis of the predicted proteins suggests that the longer variant be targeted to the nucleus while the shorter variant is secreted extracellularly. Gene structure analysis revealed that PAPP-E was encoded on 23 exons on chromosome 1 and its splice variant on the first five same exons. The discovery of the PAPP-E variants will help in the deciphering of the physiology of this new family of metzincins in not only the placenta during pregnancy but also the mammary gland in breast cancer. The new PAPP-E variants could have the potential for the diagnosis of pathological pregnancies including trisomies such as Down's syndrome.

The role of neurokinin B in pre-eclampsia.

Pre-eclampsia, a life-threatening disease unique to pregnancy, has been called a disease of theories. To date, there has been no widely accepted predictive test or therapeutic intervention to prevent or delay pre-eclampsia. The discovery of a new placental hormone, neurokinin B, may finally help to answer some of the past mysteries.

Characterization of a serine protease that cleaves pro-gamma-melanotropin at the adrenal to stimulate growth.

The adrenal gland requires stimuli from peptides derived from the ACTH precursor, pro-opiomelanocortin (POMC), to maintain its tonic state. Studies have proposed that a specific postsecretional cleavage of the nonmitogenic N-terminal 16 kDa fragment, also known as pro-gamma-melanotropin (pro-gamma-MSH), is required, releasing shorter fragments that promote adrenal growth. Here, we provide evidence for this hypothesis by the cloning and characterization of a serine protease that is upregulated during growth of the adrenal cortex. It is expressed exclusively in the outer adrenal cortex, the site of cell proliferation, and in the Y1 adrenal cell line. We also show that it is required for growth of Y1 cells, remains bound to the cell surface, and cleaves its substrate, pro-gamma-MSH, at a specific bond.

The human apolipoprotein L gene cluster: identification, classification, and sites of distribution.

We report the cloning of a new gene family encoding six apolipoprotein L (apoL-I to -VI) proteins. The genes were identified as a cluster spanning a region of 619 kb on chromosome 22. Each apoL was found to share significant identity in its predicted amphipathic alpha helices while phylogenetic tree mapping showed the genes to be evolutionarily conserved. Tissue distribution by semiquantitative PCR revealed expression in all tissues, but consistently higher levels in the placenta were observed, except for apoL-V, which had a restricted expression. A comparison of tissue distribution with apoA-I, the major structural component of high-density lipoprotein, suggests that the apoL proteins may play a general and fundamental role in lipid biochemistry. In situ hybridization for expression of apoL-I in the placenta revealed expression throughout this tissue. The pathological expression of the apolipoproteins during pregnancy is implicated in fetal growth retardation, preeclampsia, and the onset of adult atherosclerosis.

A regulatory role for neurokinin B in placental physiology and pre-eclampsia.

Tachykinin dogma has assumed, so far, that neurokinin B (NKB) is a neuropeptide that is not produced in any peripheral tissue even though its endogenous receptor, NK3, has been found in a number of locations throughout the human body. We have found an abundant source of peripheral NKB in the human and rat placenta. In this review we describe the discovery of NKB in the placenta and examine its possible role in placental physiology and pre-eclampsia (PE). Excessive secretion of placental NKB into the maternal circulation during the third trimester of pregnancy has been found in women suffering from PE. This may provide the key to the cause of the multiple and complex symptoms associated with this potentially life-threatening illness. We also reveal the structural organisation of the human NKB gene for the first time as well as discussing putative mechanisms for its control.

Agouti related protein in the rat adrenal cortex: implications for novel autocrine mechanisms modulating the actions of pro-opiomelanocortin peptides.

Agouti related protein (AgRP) is a recently discovered melanocortin receptors (MCR) antagonist implicated in the control of feeding behaviour. Expression of AgRP has been shown to be localized by in situ hybridization to the arcuate nucleus and median eminence of the brain, where it acts as an antagonist to the MC3 and MC4 receptors, while in the periphery the only significant expression was located in the adrenal medulla. As AgRP is only a weak antagonist of the MC2 and MC5 receptors, which are expressed principally by adipocytes and in the adrenal cortex, the question arizes as to the function of peripheral AgRP. In this study, we investigated the expression of AgRP in the rat adrenal and suggest that it is expressed in the adrenal cortex and not as previously described in the medulla. We also show that AgRP mRNA expression is upregulated in the adrenal during fasting and in the contralateral gland following unilateral adrenalectomy but not during chronic stress. These results indicate an as yet undefined role for AgRP in the periphery and are supportive of the suggestion that a further melanocortin receptor exists.

mRNA expression profiles for corticotrophin-releasing factor (CRF), urocortin, CRF receptors and CRF-binding protein in peripheral rat tissues.

The expression and activities of corticotrophin-releasing factor (CRF), urocortin (UCN), the CRF-binding protein (CRF-BP) and CRF receptors in rat brain have been well documented; however, information regarding their peripheral distributions remains incomplete. Given the multiple immunomodulatory effects of peripherally administered CRF and UCN and the high levels of CRF receptor type 2 (CRF-R2) mRNA and protein expressed in the heart, the lymphoid organs and heart have become targets for some of the latest CRF-related research. Here we demonstrate the presence of UCN mRNA in both the rat spleen and human Jurkat T-lymphoma cells using 3'-RACE (rapid amplication of cDNA ends) PCR. Following on from these initial results, we used semi-quantitative RT-PCR to carry out a comprehensive study assessing the relative amounts of CRF, UCN, CRF-R1, CRF-R2 and CRF-BP mRNAs in the brain, thymus, spleen and heart of normal, untreated rats. The rank orders of mRNA abundance in each of the tissue types were as follows: for CRF, brain>>thymus=spleen=heart; for UCN, heart>/=brain>thymus>spleen; for CRFR1, brain>>thymus>spleen (absent in heart); for CRF-R2, brain=heart>thymus>spleen; and CRF-BP was only detectable in the brain. We have provided evidence for the existence of CRF, UCN, CRF-R1 and CRF-R2 expression in resting immune cells, with UCN expression being particularly predominant in the rat thymus and human Jurkat cells. Additionally, the high levels of UCN mRNA detected in heart corresponded to the high expression of CRF-R2 mRNA, suggesting an important role for UCN/CRF-R2 coupling in this tissue.

Excessive placental secretion of neurokinin B during the third trimester causes pre-eclampsia.

Pre-eclampsia is a principal cause of maternal morbidity and mortality, affecting 5-10% of first pregnancies worldwide. Manifestations include increased blood pressure, proteinuria, coagulopathy and peripheral and cerebral oedema. Although the aetiology and pathogenesis remain to be elucidated, the placenta is undoubtedly involved, as termination of pregnancy eradicates the disease. Here we have cloned a complementary DNA from human placental messenger RNA encoding a precursor protein of 121 amino acids which gives rise to a mature peptide identical to the neuropeptide neurokinin B (NKB) of other mammalian species. In female rats, concentrations of NKB several-fold above that of an animal 20 days into pregnancy caused substantial pressor activity. In human pregnancy, the expression of NKB was confined to the outer syncytiotrophoblast of the placenta, significant concentrations of NKB could be detected in plasma as early as week 9, and plasma concentrations of NKB were grossly elevated in pregnancy-induced hypertension and pre-eclampsia. We conclude that elevated levels of NKB in early pregnancy may be an indicator of hypertension and pre-eclampsia, and that treatment with certain neurokinin receptor antagonists may be useful in alleviating the symptoms.

Detection of N-terminal pro-corticotrophin releasing hormone (CRH) and a 'novel' CRH in human maternal plasma and placenta.

Corticotrophin-releasing hormone (CRH) is released from the human placenta in high concentrations towards the end of the third trimester of pregnancy, but relative concentrations of other cleavage products derived from the CRH prohormone are unknown. We have measured CRH and N-terminal (1-100) and (1-127) proCRH peptides in maternal plasma in the second and third trimesters of pregnancy and in term placental extract using immunoradiometric assays (IRMAs) specific to different regions of the CRH precursor. Levels of N-terminal proCRH (amino acid residues 1-100) rose from 24+/-4 pmol/l (mean+/-s.e.) in the second trimester to 378.8+/-65 pmol/l (mean+/-s.e.) at term. Levels of intact proCRH and/or (1-127) proCRH remained relatively constant throughout the second and third trimesters, with a concentration of 29.3+/-3.8 pmol/l (mean+/-s.e.). In the course of this work a novel form of CRH that cross-reacts within specific CRH immunoassays was observed. The use of two IRMAs developed for CRH (1-41) having different C-terminal epitope specificities provided evidence for two types of CRH coexisting in maternal plasma. Separation of term placental extract by HPLC and application of the two CRH IRMAs revealed two peaks of immunoreactivity one of which coeluted with synthetic CRH (1-41).

Urocortin is the principal ligand for the corticotrophin-releasing factor binding protein in the ovine brain with no evidence for a sauvagine-like peptide.

To purify novel ligands for the corticotrophin-releasing factor binding protein (CRF-BP) from ovine brain, whole brain was homogenised in methanol and the supernatant extracted on Sep-pak C18 cartridges followed by a preliminary HPLC step. Three peaks of ovine CRF-BP ligand activity were detected in the HPLC fractions, the first two of which were also detected by a specific corticotrophin-releasing factor two-site immunoradiometric assay, the third peak being detected by a human CRF-BP ligand assay, which will not detect ovine CRF. Human CRF-BP ligand-containing fractions were further purified by affinity chromatography on a human recombinant CRF-BP column with two additional HPLC steps. The human CRF-BP ligand was found to: (a) possess a molecular mass of 4707 Daltons, (b) have an N-terminal amino acid sequence (5 residues) identical to rat urocortin, (c) be detected by a specific urocortin radioimmunoassay, (d) have high affinity for both the human and ovine CRF-BPs and (e) be present in many regions of the ovine brain. Additionally, a 300 bp cDNA fragment sharing 83% homology with the rat urocortin gene was cloned from ovine brain, the product of which was predicted to have an identical amino acid sequence to that of rat urocortin. These pieces of information confirmed the identity of the human CRF-BP ligand as an ovine urocortin. The specially developed CRF-BP ligand assays showed that the rank orders of affinity of the CRF family members for human CRF-BP were: carp urotensin-1>>human CRF=rat/ovine urocortin>human urocortin>>frog sauvagine>>ovine CRF, and those for the ovine CRF-BP were: carp urotensin-1> human CRF=rat/ovine urocortin>human urocortin> frog sauvagine>>ovine CRF. This study describes a successful technique for the purification and detection of peptide ligands for the CRF-BP. We conclude that urocortin is the principal ligand for the CRF-BP in ovine brain and we could find no evidence for a centrally located mammalian sauvagine-like peptide.

Cleavage of recombinant human corticotropin-releasing factor (CRF)-binding protein produces a 27-kilodalton fragment capable of binding CRF.

CRF is both a peripheral and a central mediator of inflammation, the activity of which is modified by the presence of a 37-kDa binding protein (CRF-BP). The objective of this study was to measure and characterize this protein in the synovial fluid of rheumatoid arthritis patients and to observe the effects of this inflammatory condition on its structure and properties. Measured by immunoradiometric assays, the mean CRF-BP concentration in synovial fluid from 27 arthritic patients was 0.51 nmol/L (SD = 0.24 nmol/L); that for CRF was 6.31 pmol/L. The mean plasma concentration of CRF-BP in 24 control subjects was 1.38 nmol/L (SD = 0.35 nmol/L) and that for 10 arthritic patients was 2.89 nmol/L (SD = 0.84 nmol/L). Synovial fluids were found by immunoblotting to contain intact CRF-BP and a 10-kDa C-terminal CRF-BP fragment; synovial fluid from healthy controls was not examined. We previously reported that after purification of recombinant CRF-BP, spontaneous cleavage frequently occurs, resulting in a 27-kDa N-terminal and a 10-kDa C-terminal fragment. Because concentrations of native CRF-BP in synovial fluid were insufficient to study the effects of cleavage on ligand binding, they were determined using recombinant human CRF-BP. Tryptophan excitation fluorescence spectra of intact and cleaved recombinant CRF-BP revealed that cleavage was accompanied by conformational change in the N-terminal fragment, leading to exposure of the sole tryptophan residue to polar molecules (emission peak shift from 310 to 250 nm). Using gel filtration chromatography to separate the N- and C-terminal fragments, it was found that the N-terminal fragment of the recombinant protein bound human CRF, although dimerization was somewhat impaired. The C-terminal fragment did not bind CRF. Scatchard analysis confirmed that the affinity of both intact and cleaved CRF-BP for CRF was 1 x 10(10) L/mol. We conclude that synovial fluid contains intact CRF-BP in molar excess to CRF and fragmented CRF-BP. The significance of cleavage and the role of cleavage products have yet to be determined, although they may represent the generation of a novel bioactivity.