title: In vivo processing and release into the circulation of GFP fusion protein in arginine vasopressin enhanced GFP transgenic rats: response to osmotic stimulation.
abstract: Arginine vasopressin (AVP) is a neurohypophysial hormone synthesized as a part of a prepropeptide precursor containing the signal peptide, AVP hormone, AVP-associated neurophysin II and copeptin in the hypothalamic neurosecretory neurons. A transgenic (Tg) rat line expressing the AVP-eGFP fusion gene has been generated. To establish the AVP-eGFP Tg rat as a unique model for an analysis of AVP dynamics in vivo, we first examined the in vivo molecular dynamics of the AVP-eGFP fusion gene, and then the release of GFP in response to physiological stimuli. Double immunoelectron microscopy demonstrated that GFP was specifically localized in neurosecretory vesicles of AVP neurons in this Tg rat. After stimulation of the posterior pituitary with high potassium we demonstrated the exocytosis of AVP neurosecretory vesicles containing GFP at the ultrastructural level. Biochemical analyses indicated that the AVP-eGFP fusion gene is subjected to in vivo post-translational modifications like the native AVP gene, and is packaged into neurosecretory vesicles as a fusion protein: copeptin1-14 -GFP. Moreover, GFP release into the circulating blood appeared to be augmented after osmotic stimulation, like native AVP. Thus, here we show for the first time the in vivo molecular processing of the AVP-eGFP fusion gene and stimulated secretion after osmotic stimulation in rats. Because GFP behaved like native AVP in the hypothalamo-pituitary axis, and in particular was released into the circulation in response to a physiological stimulus, the AVP-eGFP Tg rat model appears to be a powerful tool for analyzing neuroendocrine systems at the organismal level.
authors.affiliation: Ushimado Marine Institute, Graduate School of Natural Science and Technology, Okayama University, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Satoh
authors.affiliation: Ushimado Marine Institute, Graduate School of Natural Science and Technology, Okayama University, Japan.
authors.forename: Takumi
authors.initials: T
authors.lastname: Oti
authors.affiliation: Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Katoh
authors.affiliation: Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Ueta
authors.affiliation: Department of Physiology, Anatomy and Genetics, Le Gros Clark Building, University of Oxford, UK.
authors.forename: John F
authors.initials: JF
authors.lastname: Morris
authors.affiliation: Ushimado Marine Institute, Graduate School of Natural Science and Technology, Okayama University, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sakamoto
authors.affiliation: Ushimado Marine Institute, Graduate School of Natural Science and Technology, Okayama University, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Sakamoto
mesh_terms: D000818:Animals; D001127:Arginine Vasopressin / Q000378:metabolism*; D005089:Exocytosis; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D016253:Microscopy, Immunoelectron; D009994:Osmolar Concentration; D011499:Protein Processing, Post-Translational; D051381:Rats; D055647:Rats, Transgenic; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D001127:Arginine Vasopressin; D049452:Green Fluorescent Proteins
keywords: arginine vasopressin; eGFP; exocytosis; molecular processing; neurosecretion