Corticotrophin releasing hormone: its potential for a role in human myometrium.

Aside from its role as a hypothalamic stress hormone, corticotrophin releasing hormone (CRH) is also a placental hormone, at least in primates. Although the function of placentally derived CRH remains to be fully elucidated, elevated CRH levels have been associated with premature labour, suggesting that the hormone may be involved in regulating the duration of pregnancy. Indeed, pregnant human myometrium expresses functional CRH receptors (CRH R1 and CRH R2 subtypes) thought to signal predominantly via the second messenger cAMP. Thus, like other cAMP-producing hormones in the myometrium such as beta(2) agonists, CRH may play a part in maintaining uterine quiescence. However, several of the CRH receptor isoforms identified to date have a reduced ability to activate adenylate cyclase, raising the question as to whether they are linked to other signal transduction pathways. Here, we discuss critically the evidence for the peptide's role in regulating contractility, both directly at the myometrium and indirectly via the fetal membranes and decidua. The possibility of a role in myometrial growth modulation is also described. Experimental Physiology (2001) 86.2, 273-281.

Processing of procorticotropin-releasing hormone (pro-CRH): molecular forms of CRH in normal and preeclamptic pregnancy.

This study examined the different molecular forms of CRH in normal and preeclampsia maternal plasma and protease-blocked placental extracts using antibodies to different regions of the CRH precursor, pro-CRH. In the absence of protease inhibitors, chromatographed normal placental extracts contained four peaks of immunoreactivity corresponding to unprocessed approximately 19-kDa pro-CRH, its approximately 8-kDa intermediate metabolite, pro-CRH125-194, its approximately 2.8-kDa midportion fragment, pro-CRH125-151, and 4.75-kDa CRH1-41. However, if protease inhibitors were included in the extraction medium, only pro-CRH and pro-CRH125-194 were found. Pro-CRH processing was more extensive in protease-blocked preeclampsia placentas than in those from normal pregnancy, with three peaks corresponding to pro-CRH, proCRH125-194, and mature CRH1-41 peptide found. Using quantitative competitive PCR, the messenger ribonucleic acid levels of CRH precursor in preeclampsia placentas were 1.7-fold higher than those in normal placentas (37.83 +/- 3.48 vs. 21.83 +/- 2.59 attomoles/microg total ribonucleic acid, respectively; P < 0.005). Preeclampsia placentas contained significantly more CRH1-41 cross-reactivity (4.72 +/- 1.22 pmol/g) than normal term placentas (1.52 +/- 0.39 pmol/g; P < 0.048) extracted in medium containing protease inhibitors. The content of pro-CRH(125+/-151)-reactive species in these extracts followed the same pattern, with more immunoreactivity detected in preeclampsia placentas (4.23 +/- 1.39 pmol/g) than in those from normal term pregnancies (1.44 +/- 0.32 pmol/g; P < 0.01). Sequential plasma samples from 10 women with normal pregnancy and 5 women with preeclampsia were assayed for pro-CRH(125-151)- and CRH(1-41)-immunoreactive species In normal pregnancy, maternal plasma CRH(1-41) immunoreactivity rose with increasing gestational age, reaching 460 +/- 48 pmol/L at term. In women with preeclampsia, CRH(1-41) levels at each gestational age point were higher than those at the equivalent stage of normal pregnancy. In contrast, the levels of pro-CRH(125-151)-immunoreactive species remained barely detectable throughout normal and preeclamptic pregnancy. Both pro-CRH and CRH(1-41), but not pro-CRH(125-151), were shown to bind to the plasma CRH-binding protein. Our findings highlight the importance of protection of placental tissue from degrading enzymes during extraction and show that most of the CRH in the human placenta exists as unprocessed pro-CRH, with very little in the form of CRH(1-41) except in preeclampsia. Our studies using maternal plasma indicate that CRH(1-41) is the only one of the pro-CRH fragments studied to be maintained in significant amounts in the maternal circulation and also the only fragment studied for which a specific plasma binding protein exists.

Pharmacokinetics, cortisol release, and hemodynamics after intravenous and subcutaneous injection of human corticotropin-releasing factor in humans.

OBJECTIVE: Two clinical trials investigated the pharmacokinetics of human corticotropin-releasing factor (hCRF), resulting cortisol release, and associated hemodynamic changes. METHODS: In a 3 x 3 Latin square design, subjects were randomized to receive a single dose of 5 microg x kg(-1) hCRF as a 10-minute intravenous infusion, a 180-minute infusion, and a subcutaneous injection in separate study sessions 7 days apart. Twelve additional subjects obtained a subcutaneous dose of either 300, 600, or 1200 microg hCRF on 3 consecutive days. Noncompartmental and compartmental pharmacokinetic analysis was performed. Hemodynamic response was characterized with use of pharmacodynamic models. RESULTS: The volume of distribution at steady state was 9.81 +/- 3.0 and 15.61 +/- 2.9, and the clearance was 256 +/- 40 mL x min(-1) and 345 +/- 90 mL x min(-1) for the 10-minute and 180-minute intravenous infusion, respectively (P < .05). Corresponding elimination half-life was 45 +/- 7 minutes and 37 +/- 10 minutes. Two-compartment and 1-compartment models adequately described the 10-minute and 180-minute infusions, respectively. The bioavailability of hCRF after subcutaneous administration was 67% +/- 17%. Apparent clearance remained unchanged for different subcutaneous doses. Peak plasma cortisol concentrations were similar after subcutaneous and intravenous administration of hCRF. Repetitive administration of hCRF did not result in accumulation but produced a reduced plasma cortisol response. A sigmoidal model related plasma hCRF concentrations to increase in heart rate (maximum, 39 beats x min(-1)). The relationship between the modest decrease in diastolic blood pressure and plasma hCRF concentrations was linear. CONCLUSION: The pharmacokinetics of intravenously administered hCRF were nonlinear, but apparent clearance was constant for various subcutaneous doses. An excellent bioavailability and preserved bioactivity make the subcutaneous route an attractive choice. Repetitive administration of hCRF probably caused tolerance of the cortisol response.

Urocortin in pregnancy.

OBJECTIVES: The aim of this study was to investigate the possibility that urocortin is the ligand that displaces corticotropin-releasing hormone from its binding protein in the maternal circulation during pregnancy and, if so, to determine whether urocortin, like corticotropin-releasing hormone, is synthesized in substantial quantities in the placenta. STUDY DESIGN: A radioimmunoassay specific for urocortin was developed and used for measurement of the peptide in chorionic villi and fetal membranes (amnion and chorion) from normal and preeclamptic pregnancies. These tissues were also assayed for corticotropin-releasing hormone. Assays for urocortin were also carried out on normal term pregnant and nonpregnant myometrium and on plasma from nonpregnant individuals, and assays for both peptides were performed on sequential normal pregnancy plasma samples taken from mid gestation until term. RESULTS: Corticotropin-releasing hormone was present in normal term (1904 +/- 489 pg/g) and preeclamptic placentas (5897 +/- 1526 pg/g) and in normal term fetal membranes (645 +/- 155 pg/g, n = 6 in all cases). Urocortin was not detected in any of the tissues studied, nor was it found in the normal human plasma samples. Unlike the situation for corticotropin-releasing hormone, no pregnancy-related pattern was seen for urocortin in the plasma from pregnant women. CONCLUSIONS: Urocortin is not translated to any great extent in the pregnancy tissues investigated, nor is it present in the circulation of pregnant women in detectable amounts. Furthermore, it is unlikely that urocortin is responsible for the high maternal plasma levels of free corticotropin-releasing hormone circulating in the latter stages of pregnancy, but this does not preclude the possibility that another, as yet uncharacterized, corticotropin-releasing hormone-like peptide may be.

Elevated levels of N-terminal pro-opiomelanocortin peptides in fetal sheep plasma may contribute to fetal adrenal gland development and the pre-parturient cortisol surge.

Parturition in sheep is initiated by a rapid rise in fetal plasma cortisol. There is some controversy as to the exact nature of the drive for this pre-partum cortisol surge and it is thought that factors other than ACTH may act in concert to stimulate the development of the fetal adrenal gland. We have investigated the concentrations of ACTH and other peptides derived from pro-opiomelanocortin (POMC) in the circulation of fetal sheep during the final part of gestation, using specific 2-site immunoradiometric assays. The expected rise in fetal cortisol was seen with an 880% (p < 0.01) increase in concentration of this hormone between the initial measurement period (110-119 days gestation) and the final period (139-147 days). Fetal plasma ACTH increased less dramatically (137%; p < 0.03) during this time. The most surprising finding was the presence of very high relative concentrations of the N-terminal POMC peptide N-POMC(1-77) in the fetal circulation. Initially the concentration was 289 +/- 66 pmol/l compared to ACTH concentrations of 6.4 +/- 0.8 pmol/l. In the final week of gestation N-POMC(1-77) levels, although still high, had declined to 188 +/- 35 pmol/l (ACTH having increased to 13.7 +/- 2.2 pmol/l). Fetal plasma 3 yen-MSH was found to increase towards the end of gestation when the concentration of N-POMC(1-77) was declining, suggesting some cleavage of the latter. We postulate that the N-POMC(1-77) and its fragments, acting in concert with ACTH, play a vital role in stimulating the development of the fetal adrenal cortex and provide the additional drive to the adrenal gland required to stimulate parturition.

Biosynthesis of corticotrophin-releasing hormone (CRH) in mouse corticotrophic tumour cells expressing the human proCRH gene: intracellular storage and regulated secretion.

The mouse corticotrophic tumour cell line AtT-20 naturally synthesizes pro-opiomelanocortin (POMC) which is proteolytically processed to N-POMC(1-76), ACTH, beta-lipotrophin and beta-endorphin. The processed products are stored in secretory vesicles and released upon stimulation with specific secretagogues. AtT-20 cells which have been stably transfected with the human corticotrophin-releasing hormone (CRH) gene store and secrete immunoreactive CRH. The present results demonstrate that the CRH precursor is proteolytically processed in the transfected cells to yield the 41 amino acid neuropeptide CRH(1-41). On stimulation with the secretagogue noradrenaline, CRH(1-41) was released into the medium, while the precursor was not. Whilst treatment of wild-type AtT-20 cells with exogenous CRH(1-41) (1 nM) caused a fourfold stimulation of ACTH release above basal levels, the peptide had no effect on ACTH release from the stably transfected cells R1 and R4. These results suggest that the endogenous CRH produced by the transfected R1 and R4 cells may cause down-regulation of their CRH receptors, and thus exogenous CRH cannot cause further stimulation of ACTH release in these cells. We propose that the CRH precursor is correctly processed in the transfected AtT-20 cells (R1 and R4) and that the foreign prohormone is sorted into the secretory pathway.

Identification of high molecular weight peptides in bovine adrenomedullary chromaffin granules using monoclonal antibodies to a preproenkephalin A fusion peptide.

Two mouse monoclonal antibodies (PE-1 and PE-2) raised to a beta-galactosidase-preproenkephalin A(69-207) fusion peptide recognize pro-enkephalin A (pro-enk-A) peptides of 33-5 kDa isolated from bovine adrenal chromaffin granules. The preliminary characterization of the high molecular weight adrenomedullary pro-enk-A peptides recognized by PE-1 and PE-2 is described. The high molecular weight peptides were resolved after Sephadex G-50 chromatography and high-performance liquid chromatography (HPLC) into three components (peaks I, II and III). Immunoblot analysis showed each HPLC peak to be heterogeneous. Peak I contained PE-1-and PE-2-immunoreactive peptides of 33, 29, 24 and 22 kDa; peak II contained a peptide of 22 kDa recognized by PE-2, and peptides of 24 and 22 kDa recognized by PE-1; peak III contained a PE-2-immunoreactive peptide of 15 kDa and PE-1-immunoreactive peptide of 18 kDa. Using polyclonal antibodies to peptide F and methionineenkephalin-Arg6-Gly7-Leu8 (MetEnk-RGL), the 22 kDa band cross-reacted with both MetEnk-RGL and peptide F antibodies, whilst the 24 kDa band was shown to possess predominantly MetEnk-RGL immunoreactivity. The 15 kDa (PE-2-immunoreactive) band was recognized by the peptide F but not the MetEnk-RGL antibody, whereas the polyclonal antibodies did not recognize the 18 kDa (PE-1-immunoreactive) band. We propose that the immunological and size characteristics of some of these peptides (29, 24/22, 15 kDa) suggest their similarity to the peptides of predicted molecular mass 23.3, 18.2 and 12.6 kDa previously found in bovine adrenal medulla. The results also indicate the existence of high molecular weight pro-enk-A peptides shortened at the N-terminus. The use of an immunoradiometric assay designed to measure the proenk-A-derived 18.2 kDa peptide using PE-2 and an affinity purified and radioiodinated MetEnk-RGL IgG has supported these findings.

Phorbol esters, but not insulin, promote depletion of cytosolic protein kinase C in rat adipocytes.

The tumor-promoting phorbol esters have insulinomimetic effects in several tissues. Employing two different assay systems, we have compared the effects of phorbol ester and insulin on the activity and intracellular distribution of the Ca++ and phospholipid dependent protein kinase (protein kinase C) in isolated rat adipocytes. Phorbol ester leads to a prompt depletion of kinase activity from the cytosolic fraction and appearance of activity in membrane extracts; neither of these effects is mimicked by insulin. These results, taken together with other data, emphasize important divergences between the actions of these agonists and suggest that changes in protein kinase C activity or intracellular distribution are not a necessary concomitant of the cascade of insulin action.

Protein kinase C-stimulated phosphorylation in vitro of a Mr 80,000 protein phosphorylated in response to phorbol esters and growth factors in intact fibroblasts. Distinction from protein kinase C and prominence in brain.

In previous studies in intact 3T3-L1 fibroblasts and adipocytes, we demonstrated that the phosphorylation state of an acidic, multicomponent Mr 80,000 protein appeared to be a specific and useful marker for the activation state of protein kinase C (Blackshear, P.J., Witters, L.A., Girard, P.R., Kuo, J.F., and Quamo, S.N. (1985) J. Biol. Chem. 260, 13304-13315). In the present studies, we demonstrate that the Mr 80,000 protein from rat adipose tissue was a substrate for protein kinase C in vitro, and co-migrated on two-dimensional gels with the analogous protein from murine 3T3-L1 adipocytes labeled by exposure of intact cells to 32Pi and phorbol 12-myristate 13-acetate. Partial proteolytic maps of the two 32P-proteins were nearly identical, supporting the postulate that the sites phosphorylated by protein kinase C in vitro, and in response to phorbol 12-myristate 13-acetate in vivo, were similar or identical. Despite their similar apparent molecular weights, we were able to distinguish between the Mr 80,000 protein and protein kinase C by several physical criteria. The Mr 80,000 protein kinase C substrate was found in fractions of all rat tissues examined, but was most prominent in rat brain. Phorbol 12-myristate 13-acetate also stimulated phosphorylation of the Mr 80,000 protein in several types of cultured neuronal cells, suggesting a possible role for this protein in cholinergic neurotransmission. The Mr 80,000 protein appears to be a useful marker for protein kinase C activation in a variety of cell types.

gamma-Glutamyltransferase from Marthasterias glacialis: purification procedures and enzyme characterisation.

gamma-Glutamyl transferase has been purified 239-fold from the pyloric caeca of Marthasterias glacialis. It has a mol. wt. of 229,000. The pH optima for the hydrolysis and transfer reactions are 5.0 and 9.2. Novel purification procedures using hydroxyhexyl and aminohexyl derivatives of Sepharose 4B are described. The enzyme was bound by immobilised m-aminophenylboronic acid but not by immobilized concanavalin A.