Correlation between serum 25-hydroxyvitamin D levels and quadriceps muscle strength in elderly cretans.

Vitamin D deficiency is associated with osteomalacic myopathy, and muscle weakness related to vitamin D deficiency has been implicated as a possible risk factor for falls in the elderly. This study investigated the possible correlation between serum 25-hydroxyvitamin D (25[OH]D) concentration and quadriceps muscle strength in ambulatory community dwelling Cretan men (n=13) and women (n=35) aged > or = 65 years. Quadriceps muscle strength was measured isometrically using Cybex 6000 apparatus. The mean serum 25(OH)D concentration was significantly higher in men than in women (76.00 versus 49.11 nmol/l, respectively). Serum 25(OH)D values were < 50 nmol/l in 15% of men and in 60% of women. Serum 25(OH)D concentration correlated positively with quadriceps muscle strength. In conclusion, vitamin D deficiency was common in the study participants despite the high levels of sunlight in Crete. Serum 25(OH)D levels were positively correlated with muscle strength.

Corticotropin-releasing factor (CRF) and the urocortins differentially regulate catecholamine secretion in human and rat adrenals, in a CRF receptor type-specific manner.

Corticotropin-releasing factor (CRF) affects catecholamine production both centrally and peripherally. The aim of the present work was to examine the presence of CRF, its related peptides, and their receptors in the medulla of human and rat adrenals and their direct effect on catecholamine synthesis and secretion. CRF, urocortin I (UCN1), urocortin II (UCN2), and CRF receptor type 1 (CRF1) and 2 (CRF2) were present in human and rat adrenal medulla as well as the PC12 pheochromocytoma cells by immunocytochemistry, immunofluorescence, and RT-PCR. Exposure of dispersed human and rat adrenal chromaffin cells to CRF1 receptor agonists induced catecholamine secretion in a dose-dependent manner, an effect peaking at 30 min, whereas CRF2 receptor agonists suppressed catecholamine secretion. The respective effects were blocked by CRF1 and CRF2 antagonists. CRF peptides affected catecholamine secretion via changes of subplasmaliminal actin filament polymerization. CRF peptides also affected catecholamine synthesis. In rat chromaffin and PC12 cells, CRF1 and CRF2 agonists induced catecholamine synthesis via tyrosine hydroxylase. However, in human chromaffin cells, activation of CRF1 receptors induced tyrosine hydroxylase, whereas activation of CRF2 suppressed it. In conclusion, it appears that a complex intraadrenal CRF-UCN/CRF-receptor system exists in both human and rat adrenals controlling catecholamine secretion and synthesis.

Adherence to the Mediterranean diet during pregnancy and offspring adiposity and cardiometabolic traits in childhood.

BACKGROUND: In adults, adherence to the Mediterranean diet has been inversely associated with cardiovascular risk, but the extent to which diet in pregnancy is associated with offspring adiposity is unclear. We aimed to investigate the association between adherence to Mediterranean diet in pregnancy and offspring cardiometabolic traits in two pregnancy cohorts. METHODS: We studied 997 mother-child pairs from Project Viva in Massachusetts, USA, and 569 pairs from the Rhea study in Crete, Greece. We estimated adherence to the Mediterranean diet with an a priori defined score (MDS) of nine foods and nutrients (0 to 9). We measured child weight, height, waist circumference, skin-fold thicknesses, blood pressure, and blood levels of lipids, c-reactive protein and adipokines in mid-childhood (median 7.7 years) in Viva, and in early childhood (median 4.2 years) in Rhea. We calculated cohort-specific effects and pooled effects estimates with random-effects models for cohort and child age. RESULTS: In Project Viva, the mean (SD, standard deviation) MDS was 2.7 (1.6); in Rhea it was 3.8 (1.7). In the pooled analysis, for each 3-point increment in the MDS, offspring BMI z-score was lower by 0.14 units (95% CI, -0.15 to -0.13), waist circumference by 0.39 cm (95% CI, -0.64 to -0.14), and the sum of skin-fold thicknesses by 0.63 mm (95% CI, -0.98 to -0.28). We also observed lower offspring systolic (-1.03 mmHg; 95% CI, -1.65 to -0.42) and diastolic blood pressure (-0.57 mmHg; 95% CI, -0.98 to -0.16). CONCLUSION: Greater adherence to Mediterranean diet during pregnancy may protect against excess offspring cardiometabolic risk.

Dehydroepiandrosterone sulfate and allopregnanolone directly stimulate catecholamine production via induction of tyrosine hydroxylase and secretion by affecting actin polymerization.

Adrenal cortical cells of zona reticularis produce the neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester dehydroepiandrosterone sulfate (DHEAS), and allopregnanolone (ALLO). An interaction between zona reticularis and adrenal medulla has been postulated based on their close proximity and their interwoven borders. The aim of this paper was to examine in vitro the possible paracrine effects of these steroids on catecholamine production from adrenomedullary chromaffin cells, using an established in vitro model of chromaffin cells, the PC12 rat pheochromocytoma cell line. We have found the following: 1) DHEA, DHEAS, and ALLO increased acutely (peak effect between 10-30 min) and dose-dependently (EC50 in the nanomolar range) catecholamine levels (norepinephrine and dopamine). 2) It appears that the acute effect of these steroids involved actin depolymerization/actin filament disassembly, a fast-response cellular system regulating trafficking of catecholamine vesicles. Specifically, 10(-6) m phallacidin, an actin filament stabilizer, completely prevented steroid-induced catecholamine secretion. 3) DHEAS and ALLO, but not DHEA, also affected catecholamine synthesis. Indeed, DHEAS and ALLO increased catecholamine levels at 24 h, an effect blocked by L-2-methyl-3-(-4-hydroxyphenyl)alanine and 3-(hydrazinomethyl)phenol hydrochloride, inhibitors of tyrosine hydroxylase and L-aromatic amino acid decarboxylase, respectively, suggesting that this effect involved catecholamine synthesis. The latter hypothesis was confirmed by finding that DHEAS and ALLO increased both the mRNA and protein levels of tyrosine hydroxylase. In conclusion, our findings suggest that neuroactive steroids exert a direct tonic effect on adrenal catecholamine synthesis and secretion. These data associate the adrenomedullary malfunction observed in old age and neuroactive steroids.

Opioids in neural and nonneural tissues.

The endogenous opioid peptides (EOP) are grouped in three families, each deriving from the posttranslational processing of a distinct precursor molecule and exhibiting high affinity for a specific opioid receptor. The genes of EOPs are expressed in a wide variety of sites, including many nerve, neurosecretory, and endocrine cells. In reviewing the vast literature on this subject, a few patterns begin to emerge. First, the distribution of EOPs in tissues appears to be a distinct characteristic of each family of opioids. Second, the EOP producing cells can be grouped into two broad categories: those expressing only one and those expressing multiple EOP genes. Most EOP-producing nerve and neurosecretory cells fall into the first category, that is, they express one EOP gene, whereas most nonneural cells fall into the second category, that is, they express multiple EOP genes. Third, it appears that there is a relationship between opioids, proliferation rate, and state of differentiation of cells, since it has been shown that (a) mitogenic factors may change the EOP profile of a cell, and that (b) opioids may inhibit the proliferation rate of normal or neoplastic cells. The physiologic implication of these observations is briefly discussed.

Fingolimod induces neurogenesis in adult mouse hippocampus and improves contextual fear memory.

Fingolimod (FTY720) was the first per os administered disease-modifying agent approved for the treatment of relapsing-remitting multiple sclerosis. It is thought that fingolimod modulates the immune response by activating sphingosine-1 phosphate receptor type 1 (S1P1) on lymphocytes following its in vivo phosphorylation. In addition to its immune-related effects, there is evidence that fingolimod exerts several other effects in the central nervous system, including regulation of the proliferation, survival and differentiation of various cell types and their precursors. In the present study, we have investigated the effect of fingolimod on the production of new neurons in the adult mouse hippocampus and the association of this effect with the ability for pattern separation, an established adult neurogenesis-dependent memory function. Immunofluorescence analysis after chronic administration of a physiologic dose of fingolimod (0.3 mg kg(-1)) revealed a significant increase in both the proliferation and the survival of neural progenitors in the area of dentate gyrus of hippocampus, compared with control animals. These effects were replicated in vitro, in cultures of murine hippocampal neural stem/precursor cells that express S1P1 receptor, suggesting cell-autonomous actions. The effects of fingolimod on neurogenesis were correlated to enhanced ability for context discrimination after fear conditioning. Since impairment of adult hippocampal neurogenesis and memory is a common feature of many neuropsychiatric conditions, fingolimod treatment may be beneficial in therapeutic armamentarium of these disorders.

Comparative study between normal rat chromaffin and PC12 rat pheochromocytoma cells: production and effects of corticotropin-releasing hormone.

The adrenal medulla of several species and some human pheochromocytomas contain CRH. The first aim of the present work was to find out whether normal rat adrenal chromaffin cells and the PC12 rat pheochromocytoma cell line produce CRH in vitro and what regulates its production. CRH was measured and characterized in the media of both types of chromaffin cells under basal conditions and after exposure to K+, nicotine, interleukin-1 beta, and nerve growth factor (NGF). The second aim was to examine the biological effect of exogenous CRH (and of its antagonist) on the production of catecholamines from these two types of cells. Our results are as follows: 1) Both types of chromaffin cells contained and secreted comparable amounts of immunoreactive-CRH under basal conditions and after K(+)-induced depolarization, nicotine, and interleukin-1 beta; 2) the physicochemical characteristics of the immunoreactive-CRH in the cells and the media were identical to the putative CRH peptide on both sieve chromatography and RP-HPLC; 3) synthetic CRH induced the production of catecholamines from both cell types in a dose- and time-dependent manner; this effect was abolished by the antagonist, alpha helical CRH; 4) exposure of PC12 cells to NGF (for 1 week) resulted in their neuronal differentiation and the stimulation of their production of CRH by 30 times and of dopamine by 10 times, compared with parallel controls; this effect of NGF was abolished by alpha helical CRH. In conclusion, our data suggest that the production of CRH by PC12 cells represents the preservation of a normal chromaffin cell characteristic rather than a tumor-induced ectopic phenomenon.

PC12 rat pheochromocytoma cells synthesize dynorphin. Its secretion is modulated by nicotine and nerve growth factor.

The PC12 is a cloned rat pheochromocytoma cell line that retains a number of chromaffin cell characteristics, such as the presence of nicotinic cholinergic receptors, the synthesis and secretion of catecholamines, and the expression of a number of neuropeptide genes. The PC12 cell line is a useful model for the study of neuronal development, since PC12 cells can be induced to differentiate toward sympathetic neurons after exposure to nerve growth factor (NGF). PC12 cells can also be induced to differentiate toward the opposite direction, i.e. toward mature chromaffin cells. Morphological and biochemical changes mark the differentiation of PC12 cells toward either direction. Among the substances proposed as biochemical markers of PC12 cell differentiation toward chromaffin cells is the endogenous opioid precursor proenkephalin and its posttranslational peptide products. Indeed, the proenkephalin gene is expressed in both adrenal chromaffin and PC12 cells. The secretion of enkephalins from PC12 cells increases by several-fold after differentiation toward chromaffin cells. On the other hand, prodynorphin (another endogenous opioid precursor) is not present in normal adrenal chromaffin cells, but it is synthesized by human pheochromocytomas. It, thus, appears that dedifferentiation of chromaffin cells induces expression of the prodynorphin gene. Consequently, the aim of this study was to determine whether the rat pheochromocytoma-derived PC12 cell line expresses the prodynorphin gene, if it secretes dynorphins, and if the NGF-induced differentiation of PC12 cells toward sympathetic neurons affects the secretion of the latter. We found the following. 1) PC12 cells synthesize prodynorphin and secrete its peptide products. 2) The size of the prodynorphin transcript and the mol wt of its dominant form of dynorphin appear to be similar or identical to those of prodynorphin in rat anterior pituitary. 3) PC12 dynorphin secretion is increased, in a dose-dependent manner, after nicotinic cholinergic stimulation, an effect blocked by the specific nicotine antagonist hexamethonium. Thus, it appears that after cholinergic stimulation, PC12 dynorphin is cosecreted with catecholamines, a phenomenon described for a number of neuropeptides, including the proenkephalin-derived opioids. 4) The NGF-mediated differentiation of PC12 cells into sympathetic neurons exerted a stimulatory effect on basal, nicotine-induced, and depolarization-dependent dynorphin secretion. However, NGF did not shift the nicotine dose-response curve of dynorphin secretion. In conclusion, it appears that while changes in the secretion of proenkephalin-derived peptides may serve as a marker of PC12 cell differentiation toward chromaffin cells, an increase in the secretion of prodynorphin-derived peptides may represent a marker of NGF-induced differentiation of PC12 cells toward sympathetic neurons.

Opioids suppress basal and nicotine-induced catecholamine secretion via a stabilizing effect on actin filaments.

Catecholamine secretion and actin filament disassembly are closely coupled in chromaffin cells. Opioid suppression of catecholamine secretion is fast and transient, both characteristics of actin filament involvement. The aim of the present work was to test the hypothesis that opioids suppress catecholamine secretion via an inhibitory effect on actin filament disassembly. For this purpose we used the PC12 rat pheochromocytoma cell line. Norepinephrine and dopamine were measured by enzyme-linked immunosorbent assay or RIA. Polymerized actin was measured by rhodamine-phalloidin and visualized by confocal laser scanning microscopy. Opioids suppressed basal catecholamine secretion. The onset of this effect was fast and transient, peaking at 2 min, and was reversible by opioid antagonists. Synchronously, opioids suppressed actin filament disassembly; this was also reversible by opioid antagonists. Cytochalasin B prevented the inhibitory effect of opioids on catecholamine secretion. In addition, opioids suppressed the stimulatory effect of nicotine on catecholamine secretion and actin depolymerization. Changes in actin cytoskeleton in neuron-like PC12 cells make them resistant to both effects of opioids, i.e. on catecholamine secretion and actin disassembly. In conclusion, our data suggest that the suppressive effect of opioids on basal and nicotine-induced catecholamine secretion may result from an opioid-provoked stabilization of cortical actin. It also appears that basal catecholamine secretion is associated with opioid-sensitive machinery regulating the continuous formation of short-lived areas of cortical actin filament disassembly.

KAT45, a noradrenergic human pheochromocytoma cell line producing corticotropin-releasing hormone.

KAT45 cells were derived from a human pheochromocytoma, which also caused ectopic Cushing's syndrome, and developed into a cell line spontaneously after the continuous primary culture of the tumor cells. These human pheochromocytoma cells were compared with the extensively characterized PC12 rat pheochromocytoma cell line. KAT45 cells resembled PC12 cells in morphology, proliferation rate, response to cholinergic stimuli, and the development of dendrite-like projections after exposure to nerve growth factor. They produced norepinephrine and epinephrine in a ratio of 50:1, as opposed to production of dopamine by PC12 cells, in amounts 1 order of magnitude higher compared with PC12. Because of the ectopic Cushing's syndrome in our patient, her normal ACTH level, and the knowledge that PC12 cells and even normal rat chromaffin cells appear to produce CRH, we examined whether KAT45 cells also produced this neuropeptide. Indeed, KAT45 cells released authentic CRH and contained an apparently intact CRH transcript. Nicotine and KCl depolarization stimulated the secretion of CRH, whereas interleukin-1beta, glucocorticoids, and nerve growth factor stimulated its synthesis. In addition to the potential systemic effects of CRH, which in our patient produced ectopic Cushing's syndrome, CRH can exert paracrine effects within normal or tumoral adrenals. We used KAT45 cells as a model for the study of the local role of CRH. CRH affected several parameters of KAT45 cell metabolism, including their proliferation rate, synthesis of catecholamines, and production of POMC-derived peptides. KAT45 cells, in addition to the data they provided regarding the in vitro profile of a human CRH-producing pheochromocytoma, may prove to be a valuable auxiliary to the PC12 cell line.

Characterization of immunoreactive proopiomelanocortin-related peptides in rat testes.

alpha-Endorphin, beta-endorphin, gamma-endorphin, and N-terminal ACTH immunoreactivity were detectable in acid extracts of rat testes with concentrations of 0.07 +/- 0.01, 0.18 +/- 0.03, 0.06 +/- 0.01, and 0.33 +/- 0.08 (+/- SD) pmol/g wet wt, respectively. The forms of these immunoreactive peptides were characterized by reverse phase high performance liquid chromatography. Immunoreactive beta-endorphin was also analyzed by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results indicated that the major form of immunoreactive beta-endorphin present appears to be beta-endorphin-(1-31). No alpha-N-acetylated forms of beta-endorphin or beta-lipotropin-sized material were detected. Immunoreactive alpha- and gamma-endorphin appear to be present as alpha-endorphin and des-Tyr1-gamma-endorphin, respectively. Immunoreactive alpha MSH was present as its desacetylated form. No immunoreactive ACTH fractionating with ACTH-(1-39) or its glycosylated forms was detected. This peptide profile is most similar to that seen for proopiomelanocortin-derived peptides in the brain. The low concentrations of these peptides in rat testes suggest a paracrine function.

Demonstration of immunoreactive beta-endorphin- and gamma 3-melanocyte-stimulating hormone-related peptides in the ovaries of neonatal, cyclic, and pregnant mice.

Antisera against proopiomelanocortin (POMC)-derived peptides have previously been employed to demonstrate immunostainable materials in the male reproductive tract and in the corpus luteum of rat ovary. The present study was designed to determine how the distribution of such stainable materials varies in mouse ovary as a function of the reproductive status of the animal. Peptide-like activities were localized with the unlabeled antibody peroxidase-antiperoxidase (PAP) technique in ovaries removed from mice during fetal and neonatal development, during different stages of estrous cycle, and during pregnancy, with antisera against beta-endorphin, gamma 3MSH, and an extended N-terminal portion of POMC (16 K). beta-endorphin-like activity was also quantified in ovarian extracts by RIA. Immunostainable beta-endorphin, gamma 3MSH, and 16 K fragment-like activities were present in ovaries of pregnant and normally cycling (but not immature) mice. Intense staining was found predominantly in the corpora lutea. Less intense staining was observed in the interstitium and in the following parts of large follicles: parietal granulosa, corona radiata, and cumulus oophorus. When neonatal mice were injected with hCG, immunostainable beta-endorphin-like material in the ovarian interstitial area increased. Treatment with PMSG increased staining in both secondary follicles and the interstitium. Immunoassayable beta-endorphin-like activity was twice as high (per g wet wt) at pregnancy as during the cycle. We conclude that peptides similar or identical to POMC and/or its components are present in ovarian cells and that the concentration of such material appears to be regulated by gonadotropins.

Expression and localization of growth hormone-releasing hormone messenger ribonucleic acid in rat placenta: in vitro secretion and regulation of its peptide product.

The placenta is the source of many hypothalamic peptides. We now report that GH-releasing hormone (GHRH) mRNA was detectable in rat placenta. On Northern blot hybridization analysis, the size of the placental GHRH transcript was the same with the putative GHRH precursor mRNA. On in situ hybridization, the cells expressing the GHRH mRNA had the morphological characteristics of cytotrophoblasts. In addition, immunoreactive (IR) postranslational products of GHRH were present in rat placental extracts and in the effluent of in vitro perifused placentae. On gel filtration chromatography, the bulk of IR-GHRH present in placental extracts and perifusion effluent had the same size as the authentic hypothalamic GHRH (5.2K). A higher mol wt form of IR-GHRH of about 10K was also present and may represent the pro-GHRH predicted from the sequence of the GHRH cDNA. The mean basal release of IR-GHRH in the perifusion effluent from full thickness rat placental fragments was 337.3 +/- 38.5 (+/- SE; n = 48) pg/10 min fraction.g tissue. Depolarization by 56 mM KCl increased the concentration of the secreted immunoreactive peptide to 632.2 +/- 50.5. A 10-min exposure to 8-bromo-cAMP caused an immediate, monophasic, and dose-dependent increase in IR-GHRH secretion, which lasted between 20-30 min. The ensuing response to a KCl pulse was similar in size and pattern to that in the control channels. In contrast, a 10-min pulse of phorbol 12-myristate 13-acetate (a protein kinase-C-irreversible activator) induced a gradual, prolonged, and dose-dependent increase in basal GHRH secretion which lasted for at least 4 h. Additionally, phorbol 12-myristate 13-acetate enhanced KCl-induced GHRH secretion. In conclusion, our data suggest that the GHRH gene is expressed in the rat placenta. The placental GHRH transcript and its peptide products appear to have the same size as their hypothalamic counterparts, while the site of its placental GHRH synthesis is the cytotrophoblast. Finally, the secretion of placental GHRH seems to be regulated by both the adenyl cyclase and the protein kinase-C pathways.

Steroid hormones regulate the release of immunoreactive beta-endorphin from the Ishikawa human endometrial cell line.

Immunoreactive beta-endorphin (IR-beta END) is present in human endometrium. Several indirect lines of evidence suggest that endometrial beta END is under steroid hormone control, i.e. IR-beta END is detectable in the secretory, but not the proliferative, endometrium, and progesterone administration increases the concentration of IR-beta END in uterine secretions of ovariectomized gilts. To study the effect of steroid hormones on endometrial beta END, we first questioned whether Ishikawa human endometrial adenocarcinoma cells (which respond to steroid hormones) express the proopiomelanocortin (POMC) gene. Indeed, on Northern blot analysis, a RNA similar or identical in size to pituitary POMC mRNA was present in Ishikawa cell RNA extracts. IR-beta END was also present in Ishikawa cell extracts and culture medium, which coeluted with synthetic human beta END in a Sephadex G-50 column. Ishikawa cells released most of their IR-beta END into the culture medium. Estradiol decreased the release of IR-beta END from Ishikawa cells, an effect that was dependent upon dose and time. The maximal effect was observed after a 4-day exposure to 10 nM estradiol (44 +/- 6% of the control value; n = 6; P less than 0.001). This effect was almost completely counteracted by a 100-fold excess of the antiestrogen 4-hydroxytamoxifen. Progesterone and dihydrotestosterone did not have a statistically significant effect on IR-beta END release. Dexamethasone had effects similar to those of estradiol, i.e. decreased the release of IR-beta END in a time- and dose-dependent manner. The maximal effect was detected after a 4-day exposure to 10 nM dexamethasone (53 +/- 6% of the control value; n = 6; P less than 0.001). Interestingly, the antiprogestin-antiglucocorticoid RU486 exhibited agonistic properties, i.e. diminished the release of IR-beta END in a time- and dose-dependent fashion, possibly via the glucocorticoid receptor. Its maximal effect was reached after a 4-day exposure to 10 nM RU486 (55 +/- 6% of the control value; n = 6; P less than 0.001). In conclusion, our data demonstrate that the release of IR-beta END from Ishikawa cells in culture is inhibited by estradiol and dexamethasone, suggesting that endometrial beta END is under estrogen and glucocorticoid regulation, as is the case with hypothalamic and pituitary POMC-derived peptides. This is the first time that the in vitro release of a peripheral-extracranial POMC-derived peptide has been found to be under the direct control of estrogens and glucocorticoids.

Identification and characterization of opioid-binding sites present in the Ishikawa human endometrial adenocarcinoma cell line.

Normal epithelial cells of human endometrium, and Ishikawa human endometrial adenocarcinoma cells (an in vitro model for the study of steroid hormone effects on human endometrium) have been found to express and secrete opioid peptides deriving from proenkephalin, prodynorphin, and proopiomelanocortin. These opioids may act locally, affecting the uterine tissues. In the present study, we identified and characterized opioid-binding sites on the Ishikawa cell line, producing evidence for the mechanism of local opioid action. We used an acid shock before the receptor assay to dissociate any endogenously bound peptide. The acidification improved specific binding by 2- to 4.5-fold. Characterization of opioid binding using different radiolabeled opioids and effectors has shown the existence of a low concentration of delta-sites (Kd, 6.20 nmol/L; 4,890 sites/cell), no mu-sites, low affinity kappa 1-sites (Kd, 10.8 nmol/L; 276,000 sites/cell), kappa 2-sites with high affinity for ethylketocyclazocine (Kd, approximately 1 nmol/L) and low affinity for diprenorphine (Kd, approximately 8 nmol/L) at a concentration of 93,000 sites/cell, and high affinity kappa 3-sites (Kd, 3.6 nmol/L; 77,000 sites/cell). In conclusion, our report characterizes opioid sites in a particular and homogeneous cell type of human endometrium, i.e. in epithelial cells. The coexistence of opioid sites and their endogenous ligands in the Ishikawa cell line makes these cells a good model for the study of autocrine/paracrine interactions of opioids in nonneural tissues.

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.

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.

The corticotropin-releasing hormone (CRH) in normal and tumoral epithelial cells of human endometrium.

CRH is produced by several intrauterine sites, including placenta and desidua, during pregnancy. However, no data are available regarding the presence of CRH in the nonpregnant uterus. We now report that CRH is produced in the epithelial cells of normally cycling human uterus and in an endometrial epithelial cell-derived tumor. Specifically, we have found that: 1) Northern blot hybridization analysis of normal glandular endometrium as well as of Ishikawa human endometrial adenocarcinoma cells showed the presence of the CRH messenger RNA; the size of the transcript seemed to be identical to that present in human placenta and rat hypothalamus; 2) immunoreactive CRH (ir-CRH) was detectable in normal dispersed glandular endometrial cells as well as in the Ishikawa adenocarcinoma cells; 3) gel filtration chromatography of normal glandular endometrial and Ishikawa cell extracts and their culture media showed that most ir-CRH present had the mol wt of the authentic CRH peptide; in addition, a larger form of ir-CRH was also present in both normal and tumoral endometrial epithelial cell extracts; the latter most probably correspondents to CRH precursor molecules; and 4) immunofluorescence staining of CRH in normal glandular endometrial and Ishikawa cells revealed a cytoplasm rich in granules positive for ir-CRH. Our findings suggest that CRH may play an important role in the physiological events taking place within the uterine cavity, since CRH seems to be present in nonpregnant as well as pregnant uteri. Since CRH is expressed in normal endometrial epithelial cells and in an epithelial tumoral cell line, we propose the use of the Ishikawa cell line as a convenient model for the in vitro study of endometrial CRH.

Immunoreactive corticotropin-releasing hormone in human plasma during pregnancy, labor, and delivery.

We previously reported that immunoreactive corticotropin-releasing hormone (CRH) is present in human placenta and third trimester maternal plasma, and that such material is very similar to rat CRH and the predicted structure of human CRH. We suggested that maternal plasma immunoreactive CRH may be of placental origin. To further investigate this possibility, we measured plasma immunoreactive CRH in women during pregnancy, labor, and delivery and 1 and 2 h postpartum, and in nonpregnant women. Umbilical cord plasma and placental CRH concentrations were also measured. In the first trimester of pregnancy, the mean maternal plasma level was 5.9 +/- 1.0 pg (+/- SEM)/ml (n = 24), not significantly different from that in 10 nonpregnant women (5.8 +/- 0.8 pg/ml). Plasma CRH concentrations progressively increased during pregnancy (second trimester, 35.4 +/- 5.9 pg/ml (n = 39); early third trimester (28-34 weeks), 263 +/- 41 pg/ml (n = 14); late third trimester (35-40 weeks), 800 +/- 163 pg/ml (n = 20)]. There was a significant correlation between maternal plasma CRH levels and weeks of pregnancy. Plasma CRH concentrations were further elevated (2215 +/- 329 pg/ml; n = 9). During early labor, peaked at delivery (4409 +/- 591 pg/ml; n = 28), and declined rapidly after delivery [1 h postpartum, 1042 +/- (353 pg/ml (n = 13); 2 h postpartum, 346 +/- 91 pg/ml (n = 13)]. There was a significant correlation (r = 0.562; P less than 0.01) between matched maternal plasma and placental CRH concentrations. The mean umbilical cord plasma CRH level (50.6 +/- 6.1 pg/ml; n = 28) was much lower than that in the mother at the time of delivery. Umbilical venous plasma CRH levels were significantly greater than those in simultaneously obtained umbilical arterial plasma (70.8 +/- 11.3 and 41.8 +/- 4.9 pg/ml, respectively; n = 11). There was a significant correlation (r = 0.384; P less than 0.05) between maternal and fetal CRH concentrations. Gel filtration of plasma obtained from women during the third trimester, at delivery, and early postpartum and placental extracts revealed two major peaks of immunoreactive CRH: a high mol wt peak and one at the elution position of rat CRH. In contrast, only rat CRH-sized material was detected in plasma from nonpregnant women and umbilical cord plasma. Maternal plasma immunoreactive CRH-sized material stimulated ACTH release from anterior pituitary tissue in a dose-dependent manner and was equipotent with rat CRH.(ABSTRACT TRUNCATED AT 400 WORDS)