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Processing of Procorticotropin-Releasing Hormone (Pro-CRH): Molecular Forms of CRH in Normal and Preeclamptic Pregnancy¹

Nuffield Department of Obstetrics and Gynecology, University of Oxford, John Radcliffe Hospital (I.A., B.P.G., A.V.P., E.A.L.), Headington, Oxford, United Kingdom OX3 9DU; the Molecular Medicine Unit, Department of Medicine, University of Manchester (M.G.C., J.R., E.M.), Manchester, United Kingdom M13 9PT

Address all correspondence and requests for reprints to: Dr. E. A. Linton, Nuffield Department of Obstetrics and Gynecology, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom OX3 9DU.

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-CRH_125–194, its approximately 2.8-kDa midportion fragment, pro-CRH_125–151, and 4.75-kDa CRH_1–41. However, if protease inhibitors were included in the extraction medium, only pro-CRH and pro-CRH_125–194 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, pro-CRH_125–194, and mature CRH_1–41 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/µg total ribonucleic acid, respectively; P < 0.005). Preeclampsia placentas contained significantly more CRH_1–41 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.

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