Expression and coupling characteristics of the CRH and orexin type 2 receptors in human fetal adrenals

Hormones produced by the fetal adrenal regulate fetal growth, steroidogenic activity, and intrauterine homeostasis, which are essential for the mainte nance of pregnancy and the preparation of the fetus for extrauterine life. There is a functional interaction between CRH and the fetal adrenal, as CRH increases dehydroepiandrosterone sulfate production in cultured fetal adre nal cells. Moreover, in a rodent model administration of orexin A induced c orticosterone production. To examine this relationship in more detail we me asured the expression of the different subtypes of CRH and orexin receptors and their specific coupling to G protein alpha -subunits upon activation w ith CRH and orexin A, respectively. Using RT-PCR and fluorescent in situ hy bridization analysis, we demonstrated the presence of CRH receptors 1 alpha and 2 alpha, and orexin type 2 receptor mRNA. None of the other CRH recept or variants or orexin type 1 receptor were detected. Immunofluorescent anal ysis and Western blotting confirmed the protein expression of both receptor s, which also bind fluo-CRH and fluo-orexin with high affinity. Immunoblott ing analysis confirmed the expression of prepro-orexin and orexin A in feta l adrenals. Using photoaffinity labeling, we determined which G proteins ar e coupled to the CRH and orexin receptors in fetal adrenals when challenged with CRH or orexin. Treatment of fetal adrenal membranes with CRH (100 nM) increased the labeling of G(o) and, to a lesser extent, G(s), but not G(i) and G(q), whereas treatment with orexin A (100 nm) increased the labeling of G(s) and G(i), but not G(o) and G(q). These findings provide new insight s into the components of the signal transduction machinery in human fetal a drenals and demonstrate for the first time the presence of functional orexi n receptors outside of the CNS in humans.