HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Videos All Versions of this Article: 295/6/C1476    most recent 00344.2008v1 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 Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Nunes, P. Articles by Brown, D. Search for Related Content PubMed PubMed Citation Articles by Nunes, P. Articles by Brown, D.

METHODS IN CELL PHYSIOLOGY

A fluorimetry-based ssYFP secretion assay to monitor vasopressin-induced exocytosis in LLC-PK₁ cells expressing aquaporin-2

MGH Center for Systems Biology, Program in Membrane Biology, Nephrology Division, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts

Submitted 2 July 2008 ; accepted in final form 14 September 2008

Vasopressin (VP)-induced exocytosis was dissected in native and aquaporin-2 (AQP2)-expressing renal LLC-PK₁ cells by a fluorimetric exocytosis assay based on soluble secreted yellow fluorescent protein (ssYFP). YFP was targeted to the secretory pathway by addition of an 18-amino acid signal peptide from hen egg white lysozyme. Immunofluorescence labeling, together with analysis of Alexa 555-dextran internalization, revealed that ssYFP is exclusively located in the secretory pathway. Immunofluorescence and immunogold electron microscopy showed significant colocalization of ssYFP and AQP2. Fluorimetry and Western blot analysis demonstrated similar constitutive ssYFP secretion in native LLC-PK₁ and AQP2-expressing cells. In AQP2-expressing cells, a twofold increase in ssYFP secretion was observed within 15 min of VP stimulation. This transient burst of ssYFP secretion was abolished by the PKA inhibitor H-89 and was not observed in native cells. The endocytotic inhibitor methyl-β-cyclodextrin, which also promotes membrane accumulation of AQP2, had no effect on ssYFP secretion. Although cells expressing phosphorylation-deficient AQP2-S256A showed significantly lower baseline levels of constitutive secretion, VP induced a significant increase in exocytosis. Our data indicate that 1) this assay can monitor exocytosis in cultured epithelial cells, 2) VP has an acute stimulatory effect on ssYFP secretion in AQP2-expressing, but not native, cells, and 3) phosphorylation of AQP2 at S256 may be involved in the regulation of constitutive AQP2 exocytosis and play only a minor role in the VP-induced burst. These results support the idea that, in addition to its role in reducing AQP2 endocytosis, VP increases AQP2 exocytosis.

recycling; antidiuretic hormone; phosphorylation; epithelial cells; kidney

This article has been cited by other articles:

				U. Hasler Controlled aquaporin-2 expression in the hypertonic environment Am J Physiol Cell Physiol, April 1, 2009; 296(4): C641 - C653. [Abstract] [Full Text] [PDF]

				H. B. Moeller, N. MacAulay, M. A. Knepper, and R. A. Fenton Role of multiple phosphorylation sites in the COOH-terminal tail of aquaporin-2 for water transport: evidence against channel gating Am J Physiol Renal Physiol, March 1, 2009; 296(3): F649 - F657. [Abstract] [Full Text] [PDF]

				J. Peti-Peterdi and R. H. Chow A novel tool to visualize the cell secretory pathway. Focus on "A fluorimetry-based ssYFP secretion assay to monitor vasopressin-induced exocytosis in LLC-PK1 cells expressing aquaporin-2" Am J Physiol Cell Physiol, December 1, 2008; 295(6): C1473 - C1473. [Full Text] [PDF]