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Expression and Coupling Characteristics of the CRH and Orexin Type 2 Receptors in Human Fetal Adrenals

The Sir Quinton Hazel Research Center for Molecular Medicine, Department of Biological Sciences, University of Warwick (E.K., H.S.R., D.K.G., E.W.H.), Coventry, United Kingdom CV4 7AL; Center for Reproductive Sciences, University of California (R.B.J.), San Francisco, California 94143

Address all correspondence and requests for reprints to: Prof. E. W. Hillhouse, Department of Biological Sciences, University of Warwick, Coventry, United Kingdom CV4 7AL. E-mail: eh{at}dna.bio.warwick.ac.uk

Abstract

Hormones produced by the fetal adrenal regulate fetal growth, steroidogenic activity, and intrauterine homeostasis, which are essential for the maintenance 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 adrenal cells. Moreover, in a rodent model administration of orexin A induced corticosterone production. To examine this relationship in more detail we measured the expression of the different subtypes of CRH and orexin receptors and their specific coupling to G protein -subunits upon activation with CRH and orexin A, respectively. Using RT-PCR and fluorescent in situ hybridization analysis, we demonstrated the presence of CRH receptors 1 and 2, and orexin type 2 receptor mRNA. None of the other CRH receptor variants or orexin type 1 receptor were detected. Immunofluorescent analysis and Western blotting confirmed the protein expression of both receptors, which also bind fluo-CRH and fluo-orexin with high affinity. Immunoblotting analysis confirmed the expression of prepro-orexin and orexin A in fetal adrenals. Using photoaffinity labeling, we determined which G proteins are 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 insights into the components of the signal transduction machinery in human fetal adrenals and demonstrate for the first time the presence of functional orexin receptors outside of the CNS in humans.

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