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.
Margioris
A N
AN
Developmental Endocrinology Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Brockmann
G
G
Bohler
H C
HC
Grino
M
M
Vamvakopoulos
N
N
Chrousos
G P
GP
D000818:Animals; D015152:Blotting, Northern; D002850:Chromatography, Gel; D005260:Female; D013007:Growth Hormone-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics*; D066298:In Vitro Techniques; D009693:Nucleic Acid Hybridization; D010477:Perfusion / Q000379:methods; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D014018:Tissue Distribution
D012333:RNA, Messenger; D013007:Growth Hormone-Releasing Hormone