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 pree clampsia maternal plasma and protease-blocked placental extracts using anti bodies to different regions of the CRH precursor, pro-CRH. In the absence o f protease inhibitors, chromatographed normal placental extracts contained four peaks of immunoreactivity corresponding to unprocessed approximately 1 90kDa 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 extracti on medium, only pro-CRH and pro-CRH125-194 were found. Pro-CRH processing w as more extensive in protease-blocked preeclampsia placentas than in those from normal pregnancy, with three peaks corresponding to pro-CRH, pro-CRH12 5-194, and mature CRH1-41 peptide found. Using quantitative competitive PCR , the messenger ribonucleic acid levels of CRH precursor in preeclampsia pl acentas were 1.7-foId higher than those in normal placentas (37.83 +/- 3.48 us. 21.83 +/- 2.59 attomoles/mu g 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 +/- 0.3 9 pmol/g; P < 0.048) extracted in medium containing protease inhibitors. Th e content of pro-CRH125-151-reactive species in these extracts followed the same pattern, with more immunoreactivity detected in preeclampsia placenta s (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 nor mal pregnancy and 5 women with preeclampsia were assayed for pro-CRH125-152 - and CRH1-41-immunoreactive species. In normal pregnancy, maternal plasma CRH1-41 immunoreactivity rose with increasing gestational age, reaching 460 +/- 48 pmol/L at term. In women with preeclampsia, CRH1-41 levels at each gestational age point were higher than those at the equivalent stage of nor mal pregnancy. In contrast, the levels of pro-CRH125-151-immunoreactive spe cies remained barely detectable throughout normal and preeclamptic pregnanc y. Both pro-CRH and CRH1-41, but not pro-CRH125-151, were shown to bind to the plasma CRH-binding protein. Our findings highlight the importance of protection of placental tissue fro m 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 CRH1-41 except in preeclampsia. Our studies using maternal plasma indica te that CRH1-41 is the only one of the pro-CRH fragments studied to be main tained in significant amounts in the maternal circulation and also the only fragment studied for which a specific plasma binding protein exists.