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This Article Full Text Full Text (PDF) All Versions of this Article: 296/1/F87    most recent 90509.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Raychowdhury, M. K. Articles by Cantiello, H. F. Search for Related Content PubMed PubMed Citation Articles by Raychowdhury, M. K. Articles by Cantiello, H. F.

Vasopressin receptor-mediated functional signaling pathway in primary cilia of renal epithelial cells

¹Nephrology Division and Electrophysiology Core, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown; ²Program in Membrane Biology, Massachusetts General Hospital, Boston, Massachusetts; ³Department of Physiology, University of Alberta, Edmonton, Alberta, Canada; and ⁴Laboratorio de Canales Iónicos, ININCA, UBA-CONICET, Buenos Aires, Argentina

Submitted 26 August 2008 ; accepted in final form 16 October 2008

The primary cilium of renal epithelial cells is a nonmotile sensory organelle, implicated in mechanosensory transduction signals. Recent studies from our laboratory indicate that renal epithelial primary cilia display abundant channel activity; however, the presence and functional role of specific membrane receptors in this organelle are heretofore unknown. Here, we determined a functional signaling pathway associated with the type 2 vasopressin receptor (V2R) in primary cilia of renal epithelial cells. Besides their normal localization on basolateral membrane, V2R was expressed in primary cilia of LLC-PK₁ renal epithelial cells. The presence of V2R in primary cilia was determined by spontaneous fluorescence of a V2R-gfp chimera and confirmed by immunocytochemical analysis of wild-type LLC-PK₁ cells stained with anti-V2R antibodies and in LLC-PK₁ cells overexpressing the V2R-Flag, with anti-Flag antibody. Ciliary V2R colocalized with adenylyl cyclase (AC) type V/VI in all cell types tested. Functional coupling of the receptors with AC was confirmed by measurement of cAMP production in isolated cilia and by testing AVP-induced cation-selective channel activity either in reconstituted lipid bilayers or subjected to membrane-attached patch clamping. Addition of either 10 µM AVP (trans) or forskolin (cis) in the presence but not the absence of ATP (1 mM, cis) stimulated cation-selective channel activity in ciliary membranes. This channel activity was reduced by addition of the PKA inhibitor PKI. The data provide the first demonstration for the presence of V2R in primary cilia of renal epithelial cells, and a functional cAMP-signaling pathway, which targets ciliary channel function and may help control the sensory function of the primary cilium.

apical vasopressin; cystic kidney disease; cAMP production; polycystin-2; ciliary function