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Cellular and Subcellular Localization of the Type 2 Vasopressin Receptor in Kidney
Author(s) -
Fenton Robert Andrew,
Brønd Lone,
Praetorius Jeppe,
Nielsen Søren
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1444-b
Subject(s) - aquaporin 2 , vasopressin , immunolabeling , nephron , intercalated cell , kidney , subcellular localization , biology , vasopressin receptor , receptor , chemistry , microbiology and biotechnology , medicine , endocrinology , biochemistry , immunohistochemistry , cytoplasm , water channel , antagonist , mechanical engineering , engineering , immunology , inlet
Arginine vasopressin (AVP) is essential in body fluid homeostasis. The antidiuretic effects of AVP are initialized by binding of AVP to the type 2 vasopressin receptor (V2R), resulting in the exocytic insertion of aquaporin‐2 water channels into the apical plasma membrane. Furthermore, V2R has been proposed to play a role in the TAL in the regulation of sodium balance. However, difficulties in obtaining antibodies, defining the exact localization and studying the regulation of the V2R have prevented progress in the understanding of V2R. We have generated and characterized a polyclonal antibody targeted against the 19 NH 2 ‐terminal amino acids of the rat V2R. HEK293 cells overexpressing the rat or mouse V2R showed strong intracellular immunolabeling, whereas no labeling was observed in non‐transfected cells. In vitro translation of rat V2R cDNA followed by immunoblotting revealed an approximately 60kDa protein product that was not apparent in control reactions. Immunoblots of rat kidney showed an approximately 60kDa band in all regions that was not apparent after peptide pre‐absorption. Immunohistochemistry of rat and mouse kidney revealed abundant labeling of the collecting‐duct throughout the kidney. In addition, double labeling confocal immunofluorescence microscopy (using DCT and CNT specific marker calbindin and CD principal cell specific marker AQP2) showed that staining was restricted to collecting‐duct and connecting tubule principal cells and IMCD cells. There was a complete absence of labeling in vascular structures and other renal tubules, including PT and TAL. At the subcellular level, labeling was predominantly intracellular, although some staining was apparent in basolateral membrane domains.