Immunoreactive corticotropin-releasing factor is present in human maternal plasma during the third trimester of pregnancy

Immunoreactive (IR) corticotropin-releasing factor (CRF)-like activity was detectable in the majority of plasma samples obtained from women in the third trimester of pregnancy (68.7 +/- 23.6 pg/ml (14.4 +/- 4.9 fmol/ml); mean +/- SE, n = 15), but not in plasma (less than 10 pg/ml) from first (n = 9) or second (n = 11) trimester of pregnancy, 1 day post partum (n = 7), non-pregnant women (n = 10), or in plasma obtained from patients with Cushing's disease (n = 2) or Nelson's syndrome (n = 1), or in basal (n = 6) or ether-stressed (n = 6) rat plasma. Gel filtration of third trimester pooled plasma revealed that the majority of such material eluted with Kav of rat CRF (1-41). The IR CRF (1-41)-sized material eluted with the identical retention time as rat CRF in a reverse phase high performance liquid chromatography (HPLC) system. The detection of IR CRF exclusively in third trimester maternal plasma, together with our previous demonstration that material physicochemically indistinguishable from it is present in human term placental extracts, suggests that the placenta may be the source of plasma IR CRF.     

Increased myeloperoxidase in the placenta and circulation of women with preeclampsia

Myeloperoxidase (MPO) is a hemoprotein normally released from activated monocytes and neutrophils. Traditionally viewed as a microbicidal enzyme, MPO also induces low-density lipoprotein oxidation, activates metalloproteinases, and oxidatively consumes endothelium-derived NO. The elevated plasma MPO level is a risk factor for myocardial events in patients with coronary artery disease. Patients with preeclampsia display evidence of the inflammation and endothelial dysfunction associated with oxidative stress in the circulation, vasculature, and placenta. We hypothesized that MPO levels in the circulation and placental extracts from women with preeclampsia would be greater than levels in women with normal pregnancies. Placental extracts were prepared from placental villous biopsies from preeclamptic (n=27) and control (n=43) placentas. EDTA plasma samples were obtained from gestationally age-matched preeclamptic and control normal pregnancies. MPO concentrations were measured by ELISA. Immunohistochemistry was used to determine MPO localization in the placenta. MPO levels in placental extracts from women with preeclampsia were significantly higher than the levels in normal control subjects (546+/-62 versus 347+/-32 ng/mL; P=0.025). MPO was found in the floating villi and basal plate of placentas with a greater staining in the basal plates from preeclampsia placentas compared with normal pregnancies. Plasma MPO levels were 3-fold higher in patients with preeclampsia compared with normal control subjects (36.6+/-7.6 versus 11.0+/-3.1 ng/mL; P=0.003). In conclusion, MPO levels are significantly increased in the circulation and placenta of women with preeclampsia. We speculate that MPO may contribute to the oxidative damage reported in the endothelium and placenta of women with preeclampsia.     

Corticotropin-releasing hormone and oxytocin stimulate the release of placental proopiomelanocortin peptides

Human placenta contains the POMC-derived peptides ACTH, alpha MSH, and beta-endorphin, and CRH. High concentrations of immunoreactive (IR) CRH have been recently demonstrated in maternal plasma during pregnancy. To determine if the human placenta secretes CRH and POMC-derived peptides, we developed an in vitro human placental fragment perifusion system. The perifused tissue released IR-CRH and POMC-derived peptides at a constant rate for at least 5 h. The mean IR-CRH concentration in the effluent (under basal conditions) was 158 +/- 26 (+/- SD) pg (34.5 +/- 5.8 fmol)/5-min fraction.g tissue. IR-alpha MSH, IR-beta-endorphin, and IR-ACTH were also released into the perifusion medium; the mean concentration of alpha MSH released was 24.6 +/- 8 pg (14.8 +/- 4.8 fmol)/fraction.g, that of ACTH was 2.9 +/- 1.9 pg (0.65 +/- 0.43 fmol)/fraction.g, and that of beta-endorphin was 12.9 +/- 6 pg (3.8 +/- 1.7 fmol)/fraction.g. We examined the effects of human CRH, oxytocin, vasopressin, and dexamethasone on placental POMC peptide secretion. Five-minute pulses of 10(-8) or 10(-6) mol/L human CRH or oxytocin produced an immediate and dose-dependent increase in all POMC peptides in the effluent. A 5-min pulse of 10(-8) or 10(-6) mol/L vasopressin had no effect. A continuous 4-h exposure to 10(-6) mol/L dexamethasone had no effect on either basal IR-CRH or POMC-derived peptide or their KCl-induced release. In conclusion, we found that 1) human placenta releases IR-CRH and POMC-derived peptides in vitro; this phenomenon seems to be independent of glucocorticoid control; 2) placental CRH may have a paracrine effect on placental POMC peptide release in addition to its possible action on maternal pituitary hormone release; and 3) oxytocin, but not vasopressin, stimulates placental POMC peptide release.     

Plasma beta-endorphin, free fatty acids and blood lipid changes in type 2 (non-insulin dependent) diabetic patients

A lipolytic activity for beta-endorphin (beta EP) has been recently suggested both in vitro and in vivo. In our study we evaluated the relationship between beta EP and blood lipid pattern in Type 2 (non-insulin dependent) diabetic patients. Plasma beta EP, together with plasma beta-lipotropin (beta LPH), ACTH, cortisol and plasma insulin (IRI), was measured by RIA after silicic acid plasma extraction and Sephedex G-75 column chromatography. Although reduced beta EP (7.12 +/- 3.8 fmol/ml) and increased beta LPH (9.3 +/- 3.7 fmol/ml) levels were found in diabetic patients, compared to controls (8.53 +/- 3.3 fmol/ml, p less than 0.05 and 8.34 +/- 2.6 fmol/ml, p less than 0.05, respectively), higher plasma beta EP concentrations were demonstrated in hyperlipidemic diabetic patients (10.3 +/- 3.9 fmol/ml) than in patients with normal blood lipid pattern (4.85 +/- 1.45 fmol/ml, p less than 0.001). Several positive correlations between beta EP, plasma free fatty acids (r = 0.75, p less than 0.001), triglycerides (r = 0.84, p less than 0.001) and VLDL (r = 0.80, p less than 0.001) were found in our patients independently of overweight, hypoglycemic treatment, plasma IRI levels and of the degree of metabolic control. A higher prevalence of micro- and macrovascular complications was demonstrated in hyperlipidemic than in normolipidemic patients. Blood lipid disorders might therefore be associated with increased plasma beta EP levels in Type 2 diabetes.     

Localization and endogenous concentration of endothelin-like immunoreactivity in human placenta.

The aim of this study was to investigate the localization of endothelin-like immunoreactivity (ET-IR) in human placenta, chorion and amnion and to compare the endogenous concentration of immunoreactive endothelin (ET) in these tissues before and after the onset of labour. ET-IR was detected in the endothelium of stem vessels in placental villi, as well as in decidual stromal cells in the basal maternal plate, by immunocytochemistry using primary polyclonal rabbit antibody. A specific radioimmunoassay was used to detect endogenous concentration of ET in homogenized placental tissues. The endogenous concentration of ET-IR was significantly greater in amnion than in chorion and placenta (amnion 249 +/- 13 fmol/g; chorion 190 +/- 11 fmol/g; placenta 169 +/- 14 fmol/g; means +/- S.E.M.; n = 12; P less than 0.01). No significant difference was seen before or after the onset of labour. The detection of ET-IR in placenta, chorion and amnion suggests that the ETs may play a role in the paracrine control of human uterine function.     

PLASMA IMMUNOREACTIVE β-ENDORPHIN IS ELEVATED IN URAEMIA

Plasma immunoreactive-(IR) beta-endorphin (beta-EP) and beta-lipotrophin (beta-LPH) levels were measured in 15 adult uraemic patients on chronic haemodialysis. The presence of immunoreactivity eluting in the position of beta-EP was demonstrated following submission of pooled extracts of uraemic plasma to gel permeation chromatography on Sephadex G-50. To separate beta-EP from beta-LPH, pre-dialysis plasma extracts from six individual patients, and three pools of three patients each, were submitted to sequential immune-affinity chromatography and levels were measured by radioimmunoassay. In all cases, plasma IR beta-EP concentrations were markedly increased compared with normal subjects (m +/- SEM fmol/ml; 64.4 +/- 13.7 vs. 2.3 +/- 0.2). IR beta-LPH concentrations were also increased (m +/- SEM fmol/ml; 55.7 +/- 13.2 vs. normal 6.1 +/- 0.8). In addition, post-dialysis concentrations of plasma IR beta-EP and beta-LPH were lower than pre-dialysis levels (n = 4).     

Immunoreactive adrenomedullin (AM) concentration in maternal plasma during human pregnancy and AM expression in placenta

Adrenomedullin (AM) is a novel vasorelaxant peptide, isolated from human pheochromocytoma. Although AM may be involved in the regulation of the cardiovascular system, a number of other mechanisms are also involved. The present study was undertaken to confirm the presence of AM in human maternal circulation and in placental function during pregnancy. Immunoreactive (ir) AM concentrations in maternal plasma were 3.4+/-0.7fmol/ml (mean+/-s.e. m.) in the first trimester, 3.3+/-1.1fmol/ml in the second trimester, 7.3+/-2.8fmol/ml in the third trimester, 4.1+/-1.9fmol/ml in early puerperium and 3.0+/-0.4fmol/ml in non-pregnant periods; the concentration in the third trimester was significantly greater than those in other periods. Plasma concentrations of estradiol (E(2)), progesterone, human placental lactogen (hPL) and human chorionic gonadotropin (hCG) were also measured, using RIA kits. Significant correlations have been demonstrated between the concentrations of irAM and those of E(2), progesterone and hPL. We therefore examined the expression of AM within the placental tissues using immunohistochemistry and northern blot analysis in order to demonstrate a correlation between the presence of AM in the placenta and maternal plasma. Using immunohistochemistry, we detected AM in the amnion at term and the expression of AM mRNA in human placental tissues using cloned human (h) AM complementary DNA as a probe. This study demonstrates the immunoreactivity of human hAM in maternal plasma during pregnancy, and suggests that hAM in maternal plasma is generated partly from placental tissue.     

Endothelin in human brain and pituitary gland: presence of immunoreactive endothelin, endothelin messenger ribonucleic acid, and endothelin receptors.

The presence of immunoreactive (IR) endothelin, endothelin mRNA, and endothelin receptors in human brain and pituitary gland has been studied by RIA, Northern blot hybridization, and receptor assay. IR endothelin was detected in all five brain regions examined (cerebral cortex, cerebellum, brain stem, basal ganglia, and hypothalamus) (6-10 fmol/g wet wt) and spinal cord (22 +/- 6 fmol/g wet wt, n = 7, mean +/- SEM). Higher concentrations of IR endothelin were found in the pituitary gland (147 +/- 30 fmol/g wet wt). Fast protein liquid chromatographic analysis of the IR endothelin in pituitary gland showed a large IR peak in the position of endothelin-3 and a smaller peak in the position of endothelin-1, whereas IR endothelin in the hypothalamus and brain stem was mainly endothelin-1. Endothelin messenger RNA was detected by Northern blot hybridization in the pituitary but not in hypothalamus. The receptor assay showed that 125I-endothelin-1 binding sites were present in large numbers in all five brain regions but were much less abundant in the pituitary gland. Binding capacity and dissociation constant were 5052 +/- 740 fmol/mg protein and 0.045 +/- 0.007 nM in brain stem and 963 +/- 181 fmol/mg protein and 0.034 +/- 0.009 nM in hypothalamus. In the pituitary gland, there were two classes of binding sites for endothelin with dissociation constants of 0.059 +/- 0.002 nM (binding capacity = 418 +/- 63 fmol/mg protein) and 0.652 +/- 0.103 nM (binding capacity = 1717 +/- 200 fmol/mg protein). Endothelin-1, -2 and -3 were almost equipotent in displacing the binding (IC50 approximately 0.04 nM). These findings are in accord with the possibility that endothelin acts as a neurotransmitter, neuromodulator or neurohormone in man.     

Expression of nitric oxide synthase isoforms in the human placenta is not altered by labor

Nitric oxide has various biological activities including smooth muscle relaxation, anti-inflammatory activity, anti-coagulatory activity. As the human placenta is known to express nitric oxide synthases, this study investigated the possible effect of labor on the expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in human placental tissues at term. Both eNOS and iNOS mRNA expression in placental tissues in labor were significantly higher than those in the amnion, chorion laeve, decidua vera and myometrium. The eNOS mRNA and protein expressions in placental tissues in labor (n = 12) were 1.6023 +/- 0.1652 (eNOS/GAPDH, mean +/- SEM) and 12.8 +/- 1.3 arbitrary units (AU), respectively, which were similar to those not in labor (n = 10), 1.5806 +/- 0.2042 (eNOS/GAPDH) and 11.4 +/- 1.8 AU. The iNOS mRNA and protein expressions in the placental tissues in labor were 1.2831 +/- 0.2436 (iNOS/GAPDH) and 10.7 +/- 2.1 AU respectively, similar to those not in labor, 1.9254 +/- 0.8004 (iNOS/GAPDH) and 13.3 +/- 1.8 AU. The guanosine 3',5'-cyclic monophosphate (cGMP) concentration in the placental tissues in labor was 23.6 +/- 1.4 fmol/g wet tissue, similar to that not in labor, 26.1 +/- 2.0 fmol/g wet tissue. These findings suggest that nitric oxide production in the human placenta is maintained during labor.     

Proopiocortin-related peptide plasma levels throughout prepuberty and puberty

The plasma patterns of ACTH, beta-lipotropin (beta LPH) and beta-endorphin (beta EP), in addition to those of cortisol and dehydroepiandrosterone sulfate (DHAS), were studied in 139 prepubertal children (subdivided into different age groups) and 38 adolescents (subdivided according to Tanner's pubertal stages) aged 10-16 yr. The adult control group was composed of 23 females and 12 males aged 17-40 yr. No sex differences were found in ACTH, beta LPH, beta EP, and cortisol plasma levels. ACTH plasma levels were slightly lower in the 1- to 3-yr-old groups than in males at 4-5 yr and females at 8-9 yr. No further significant differences were observed in any of the age or pubertal groups, the concentrations being constantly in the adult range. beta LPH and beta EP plasma levels were lowest at 1-3 yr in both males (beta LPH: 2.1 +/- 0.25, beta EP: 1.85 +/- 0.59 fmol/ml, mean +/- SE) and females (beta LPH: 2.8 +/- 0.31; beta EP: 2.41 +/- 0.41 fmol/ml); plasma levels of both hormones increased progressively in both sexes until Puberty 1 stage of sexual maturation, at which time levels were 7.3 +/- 0.78 and 8.69 +/- 1.0 fmol/ml in males and 7.1 +/- 0.34 and 6.76 +/- 0.13 fmol/ml in females; these levels are similar to adult values. A highly significant linear correlation was found between both beta LPH and beta EP concentrations and the age of the subjects; this was not true for ACTH plasma levels. Cortisol plasma levels were similar in all groups. DHAS plasma levels increased progressively from 1-3 yr to the end of sexual maturation when adult values were reached. During prepuberty, DHAS levels were significantly correlated with both beta LPH and beta EP, but not ACTH. These data indicate that plasma beta LPH and beta EP concentrations, in contrast to ACTH levels, increase progressively throughout prepuberty and suggest that the processing of the parent proopiocortin molecule or secretion of the processed peptides from the anterior pituitary (or other sources) may change from early infancy to adulthood. Furthermore, the correlation between both beta LPH and beta EP with DHAS plasma levels in prepuberty suggests a role of proopiocortin-related peptides in adrenarche.     

Immunoreactive corticotropin-releasing hormone present in human plasma may be derived from both hypothalamic and extrahypothalamic sources

Immunoreactive CRH concentrations were determined in human plasma using an immunoaffinity chromatographic extraction procedure and sensitive RIA. Immunoreactive CRH was detectable in the plasma of all normal subjects (mean +/- SD, 6.2 +/- 2.4 pg/mL; n = 15). Basal (0800-1000 h) plasma immunoreactive CRH levels were significantly lower in patients with Cushing's syndrome due to adrenal (2.8 +/- 1.1 pg/mL; n = 4) or pituitary adenomas (2.9 +/- 0.8 pg/mL; n = 5), in patients with hypothalamic hypopituitarism (3.2 +/- 0.9 pg/mL; n = 5), and in glucocorticoid-treated patients (3.9 +/- 1.9 pg/mL, n = 8). Basal plasma CRH levels were also low in patients with acromegaly (2.8 +/- 0.8 pg/mL; n = 14) and insulin-treated diabetic patients whose pituitary-adrenal function was normal (3.6 +/- 1.0 pg/mL; n = 12). In normal subjects plasma CRH levels increased after insulin-induced hypoglycemia; this response was abolished by the prior administration of dexamethasone. In contrast, basal plasma CRH levels were not affected by prior administration of metyrapone or dexamethasone. No evidence for diurnal variation in plasma immunoreactive CRH was found in normal subjects. In addition, in normal subjects oral glucose administration elicited a significant increase in plasma CRH (basal, 7.3 +/- 0.9 pg/mL; peak 30 min after glucose, 16.7 +/- 5.8 pg/mL; n = 5; P less than 0.05) without concomitant changes in ACTH. Gel filtration of extracts of pooled plasma from normal subjects revealed a major component of immunoreactive CRH in the position of synthetic rat CRH. Immunoreactive CRH-sized material had the same retention time as authentic rat CRH in a reverse phase high pressure liquid chromatography system. The content of immunoreactive CRH in human placenta, pancreas, and adrenal gland was much larger than that in hypothalamus. These findings suggest that immunoreactive CRH is present in peripheral plasma; the increase in plasma immunoreactive CRH after insulin-induced hypoglycemia may reflect stimulation of hypothalamic CRH release; the increase in plasma immunoreactive CRH after glucose administration may reflect extrahypothalamic CRH release; and the lack of diurnal variation in plasma immunoreactive CRH together with the lack of suppression of CRH by dexamethasone suggest that basal plasma CRH is of extrahypothalamic origin.     

Effect of restriction of placental growth on the concentrations of insulin, glucose and placental lactogen in the plasma of sheep

The effect of restricting placental growth on maternal glucose, insulin and placental lactogen was investigated in 16 ewes carrying singleton lambs. Uterine caruncles were removed from seven ewes (caruncle ewes) before pregnancy, resulting in reduced placental size and retarded intra-uterine fetal growth. The concentration of insulin in maternal plasma was similar in both control and caruncle ewes. The concentration of glucose was significantly higher in the caruncle than in the control ewes (3.26 +/- 0.15 (S.E.M.) mmol/l, number of observations (n) = 9, vs 2.75 +/- 0.1, n = 9, P less than 0.02, and 3.27 +/- 0.16, n = 7, vs 2.46 +/- 0.11, n = 12, P less than 0.001, for the carotid artery and utero-ovarian vein respectively). The concentration of ovine placental lactogen (oPL) in the utero-ovarian vein was reduced in the caruncle compared with the control ewes (283 +/- 65 micrograms/l, n = 8, and 705 +/- 106 micrograms/l, n = 18, P less than 0.02, respectively). Restriction of placental growth by removal of endometrial caruncles similarly reduced the concentrations of oPL in maternal arterial plasma (231 +/- 54 micrograms/l, n = 9, and 621 +/- 96 micrograms/l, n = 18, P less than 0.002). Production of oPL by the placenta was also reduced by limiting placental growth to 30 +/- 11 micrograms/min, n = 8, compared with 133 +/- 43 micrograms/min, n = 15, P less than 0.05, for the controls. Production of oPL per gram of placenta in the caruncle group, although only 34% of the control value, was not reduced significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticotropin/lipotropin common precursor-like material in normal rat extrapituitary tissues.

A Mr 26,000 corticotropin (ACTH)-like material is present in glacial acetic acid extracts of all normal rat extrapituitary tissues. In the present study, beta-melanotropin (beta-MSH) immunoactivity was detected in glacial acetic acid extracts of normal rat extrapituitary tissues. beta-MSH immunoactivity was also present in all extracts (mean +/- SEM, fmol/mg of protein): brain, 71.0 +/- 16.3; stomach, 11.5 +/- 1.6; kidney, 8.9 +/- 0.8; colon, 8.2 +/- 1.1; small intestine, 6.5 +/- 1.1; liver, 4.3 +/- 0.5; and heart, 3.2 +/- 0.5. Except in brain extracts, beta-MSH and ACTH immunoactivities of tissue extracts were strongly correlated to each other (r = 0.79; n = 42). When tissue extracts (except brain) were passed through a Sephadex G-75 (superfine) column, ACTH and beta-MSH immunoactivities were eluted in a single peak corresponding to Mr 26,000. In contrast, for brain extracts, the MrS of major peaks of ACTH and beta-MSH immunoactivities were 4,500 and 8,000, respectively; a smaller peak of Mr 26,000 ACTH/beta-MSH-like material was also eluted. Specific anti-ACTH immunocolumns, which did not bind purified synthetic beta-MSH, adsorbed both ACTH and beta-MSH immunoactivities of all tissue extracts except those of brain. One-third of the beta-MSH immunoactivity in brain extracts adsorbed to the anti-ACTH immunocolumn, but two-thirds of beta-MSH immunoactivity passed through the column. We conclude that ACTH and beta-MSH immunoactivities are present in all normal rat extrapituitary tissues and exist in most tissues on the same molecule. This Mr 26,000 molecule is closely related to the pituitary ACTH/beta-lipotropin common precursor.     

Immunoreactive growth hormone-releasing hormone in rat placenta

It has been shown that immunoreactive and biologically active GH-releasing hormone (GHRH)-like material is present in rat placenta. To investigate the role of placental GHRH, we measured it in human and rat placenta of different gestational stages, using specific RIA systems. GHRH and somatostatin contents in median eminence, pituitary GH contents, and plasma GHRH levels were also quantified in rats. Immunoreactive GHRH was detectable in rat placenta [13 days of gestation, 1.4 +/- 0.4 (+/- SD); 16 days, 1.4 +/- 0.2; 20 days, 1.7 +/- 0.4 ng/g] but not in human placenta (less than 0.06 ng/g in both full-term and mid-term placenta). GHRH concentrations in rat placenta did not change significantly during pregnancy, but total contents increased progressively in relation to placental growth. GHRH and somatostatin contents in median eminence of pregnant rats were not different from those of control female rats. In contrast, rat pituitary GH contents in pregnant rats were significantly lower than those of control female rats. Immunoreactive GHRH was not detectable in plasma of either pregnant rats or nonpregnant rats. Molecular sieve chromatography revealed two peaks of immunoreactive GHRH in rat placental extracts: a major peak eluted in the position of synthetic rat GHRH and a minor peak in the higher molecular weight region. In contrast, a single peak in the position of rat GHRH was observed in rat median eminence extracts. Detection of immunoreactive GHRH in rat placenta but not in human may suggest that the mechanism of GHRH gene expression in placenta is species specific. Failure of detection of immunoreactive GHRH in rat maternal circulation suggests that placental GHRH may not affect the maternal hypothalamic pituitary axis. Presence of high molecular weight materials of immunoreactive GHRH in rat placenta but not in median eminence suggests that posttranslational processing of the GHRH precursor molecule may be different in the two organs. Placental GHRH may have a paracrine function or may be secreted into fetal circulation and contribute to fetal growth.     

Dramatic elevation of plasma metastin concentrations in human pregnancy: metastin as a novel placenta-derived hormone in humans

Metastin is a novel peptide that was recently isolated from human placenta as the endogenous ligand of an orphan heptahelical receptor, hOT7T175. Metastin has been shown to suppress the motility of hOT7T175-transfected melanoma cells; however, studies of the physiological function of metastin have begun only recently. To investigate the possibility that metastin is an endocrine peptide, we determined the immunoreactive (ir-) metastin concentration in human plasma using our newly developed, sensitive, and specific two-site enzyme immunoassay. The plasma concentrations of ir-metastin in males and females were 1.30 +/- 0.14 (n = 12) and 1.31 +/- 0.37 fmol/ml (n = 10), respectively. As metastin is known to be abundant in human placenta, the ir-metastin concentration in the maternal plasma was then determined. The ir-metastin concentrations were 1230 +/- 346 fmol/ml (n = 11) in the first trimester, 4590 +/- 555 (n = 16) in the second trimester, and 9590 +/- 1640 (n = 12) in the third trimester. On d 5 after delivery, the ir-metastin concentration returned to nearly the nonpregnant level (7.63 +/- 1.33 fmol/ml; n = 10), suggesting that ir-metastin increases in pregnancy and is derived mainly from the placenta. The plasma from both nonpregnant and pregnant women showed a single ir-metastin peak at the same retention time as authentic metastin on reverse phase HPLC analysis, indicating that the major portion of the circulating metastin, as determined by our two-site enzyme immunoassay, represents endogenous metastin. Histochemical studies of human placenta localized metastin mRNA and immunoreactivity to the syncytiotrophoblasts. The present study provides evidence for metastin as a novel placenta-derived hormone in humans.     

Functional opioid receptor sites in human placentas

We characterized the presence of opioid peptide receptor sites in plasma membranes and cells from human midterm and term placentas. Incubations with [3H]ethylketo-cyclazocine (EKC) at increasing doses revealed the presence of high affinity, low capacity, opioid peptide receptor-specific binding of the kappa-type. Scatchard analysis of the binding data showed, in the plasma membranes, linear plots at both stages of pregnancy with similar mean equilibrium association constants of 1.31 +/- 0.29 (+/- SE) X 10(9) mol/L-1 (n = 4) at midterm and 0.52 +/- 0.63 X 10(9) mol/L-1 at term (n = 4). In placental cells (n = 4) from term gestations, the binding plots were curvilinear; the first component had a Ka of 5.51 +/- 0.50 X 10(9) mol/L-1, and the second component had a Ka of 1.33 +/- 0.81 X 10(8) mol/L-1 (P less than 0.01). When standardized per mg tissue protein, the number of binding sites in plasma membranes increased from 13.8 +/- 9.8 fmol at midterm to 50.0 +/- 18.6 fmol at term (P less than 0.05). For term placental cells, the concentration of binding sites was 81.2 +/- 36.0 fmol for the high affinity sites and 713 +/- 390 fmol for the lower affinity sites. Specificity for the kappa-type of OPR was found based on the inability of mu- or delta-opioid peptides, as well as LHRH and TRH, to compete for [3H]EKC binding. Term placental cells incubated with various doses of opioid peptides had a 50% increase in placental lactogen production. The increase was significantly higher than controls only with kappa-agonists (P less than 0.05), maximal with 10(-9) mol/L EKC, and completely inhibited by 5 X 10(-6) mol/L naloxone. These results expand on previous data demonstrating the presence of opioid peptide receptor in placental plasma membranes and suggest a role for opioid peptides in regulating secretion of placental lactogen by placental cells.     

Beta-Adrenergic receptors and catecholamine-sensitive adenylate cyclase of the human placenta

Beta-Adrnergic receptor and beta-adrenergic sensitive adenylate cyclase were demonstrated in membrane fractions of human placenta. Placental membranes from normal term pregnancies bound the beta-adrenergic antagonist (-)[3H]dihydroalprenolol to a single saturable class of sites (Kd = 2.31 +/- 0.23 nM; n = 9; maximal capacity, 112 +/- 9 fmol/mg). Competition for binding was stereoselective for (-)isomers of propranolol, and beta-adrenergic agonists displayed competition for the placental receptor in the order (-)isoproterenol greater than (-)epinephrine greater than (-)norepinephrine, typical of a beta 2 type receptor. Beta-Adrenergic receptor was present in placental tissue as early as 10 weeks gestational age, and binding capacity decreased slightly with advancing gestation. [3H]Dihydroalprenolol binding was coupled to epinephrine-stimulated adenylate cyclase activity throughout gestation. The subcellular distribution of both beta-adrenergic receptors and epinephrine-stimulated adenylate cyclase suggest their localization primarily in nonbrush border membrane fractions, presumably from plasma membranes more closely related to the fetal rather than to the maternal circulation. Epinephrine-sensitive adenylate cyclase was not present in purified brush border preparations which were directly exposed to maternal blood in the intervillous space.     

Effects of low dose ovine corticotropin-releasing hormone in humans: endocrine relationships and beta-endorphin/beta-lipotropin responses

The effects of low doses (0.03 and 0.1 microgram/kg) of ovine CRH (oCRH) on plasma beta-endorphin/beta-lipotropin (beta End/beta LPH), ACTH, and corticosteroid levels were studied in normal men. The 0.03 microgram/kg oCRH dose produced a reproducible response, with a rapid increase in plasma oCRH to peak levels between 45 and 95 fmol/mL and an appropriate doubling of plasma peptide and corticosteroid concentrations. The relationship between the corticosteroid rise and the rapid beta End/beta LPH and ACTH declines suggested negative feedback by corticosteroids on the release of these pituitary products. Plasma oCRH levels were proportionate to those reported in studies using much higher oCRH doses, and produced plasma oCRH levels in the reported range for the hypophyseal portal circulation. Molecular sieving of the beta End-immunoreactive materials in basal and post-oCRH (0.1 microgram/kg) plasma samples revealed an average basal beta End to beta LPH ratio of 1:1.5; 15 min after oCRH stimulation the average ratio was 4:1. We conclude that a low (threshold) dose of oCRH can reliably stimulate POMC peptide secretion and may preferentially release beta End from the anterior pituitary.     

Trophoblastic cells of the hydatidiform mole contain a beta 1-subtype adrenergic receptor

Both beta 1- and beta 2-adrenergic receptors have been previously described in normal human placental homogenates; the cells upon whose surface membranes these receptors reside have not been identified. In order to show that a beta 1-adrenergic receptor is present on trophoblastic cells, the cells which mediate maternal-fetal transport and produce placental hormones, beta-adrenergic receptors were demonstrated in membrane fractions of human hydatidiform mole. Microscopic sections of the mole samples used demonstrated edematous villi lined by trophoblastic cells with minimal nontrophoblastic (stromal or vascular) contamination compared with placenta. (--)-[3H]Dihydroalprenolol [(--)-[3H]DHA] binding to molar membranes was reversible and saturable to a single class of sites (Kd = 0.97 +/- 0.12 nM; n = 7; maximum binding capacity, 72.9 +/- 6.4 fmol/mg protein). (--)-[3H]DHA binding was associated with catecholamine-stimulated adenylate cyclase activity. Agonist competition for the molar beta-adrenergic receptor showed the order of potency to be (--)isoproterenol much greater than norepinephrine = epinephrine, characteristic of a beta 1-adrenergic receptor subtype. Competition for (--)-[3H]DHA binding to trophoblastic membranes by the beta-adrenergic receptor subtype-specific agents metoprolol (beta 1 selective) and zinterol (beta 2 selective) was also characteristic of a homogeneous subtype of beta 1-adrenergic receptors. Because beta 1-adrenergic receptors alone were seen on trophoblast cells, the beta 2-adrenergic receptor in placenta must reside on nontrophoblastic elements (stromal or vascular endothelium). No differences in beta-adrenergic receptor binding were seen related with ploidy (2 or 3 N), the presence or absence of a fetus, or the progression of the mole to choriocarcinoma. Two choriocarcinoma cell lines, BeWo and JEG-3, however, showed no specific (--)-[3H]DHA binding. Human trophoblast contains beta 1-adrenergic receptors coupled to catecholamine-sensitive adenylate cyclase, supporting a role for catecholamines in the regulation of placental metabolism.     

Difference of plasma leptin levels in venous and arterial cord blood: relation to neonatal and placental weight

To investigate the physiological significance of umbilical plasma leptin in the fetal growth and its possible origin of production during pregnancy, plasma leptin concentrations in venous and arterial cord bloods were measured in 42 neonates (male = 23, female = 19, gestational age 36-41 weeks, birth weight 2600-4000 g). Leptin concentrations in umbilical veins (5.65 +/- 0.53 ng/mL, n = 42) and umbilical arteries (0.56 +/- 0.07 ng/mL, n = 42) were significantly (P < 0.001) lower than those in maternal peripheral veins (22.36 +/- 1.06 ng/mL, n = 42). Mean plasma leptin concentration (+/- SEM) in venous cord blood was significantly (P < 0.001) higher than that of arterial cord blood. There was significantly positive correlation (r = 0.447, P < 0.01) between umbilical venous leptin levels and neonatal body mass index (BMI). A positive correlation (r = 0.435, P < 0.01) was also found between umbilical arterial leptin and neonate BMI. There was no positive correlation between umbilical leptin levels in venous cord blood and placental weight. We thus conclude that umbilical plasma leptin may play an essential role in the fetal growth and metabolism during pregnancy and placenta is one of the major sources of leptin production, but the level of leptin in umbilico-placental circulation is independent of the weight of placenta.