title: A fluorimetry-based ssYFP secretion assay to monitor vasopressin-induced exocytosis in LLC-PK1 cells expressing aquaporin-2.
abstract: Vasopressin (VP)-induced exocytosis was dissected in native and aquaporin-2 (AQP2)-expressing renal LLC-PK(1) 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-PK(1) 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-beta-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.
authors.affiliation: Massachusetts General Hospital, Boston, MA 02114, USA. pnunes@fas.harvard.edu
authors.forename: Paula
authors.initials: P
authors.lastname: Nunes
authors.forename: Udo
authors.initials: U
authors.lastname: Hasler
authors.forename: Mary
authors.initials: M
authors.lastname: McKee
authors.forename: Hua A J
authors.initials: HA
authors.lastname: Lu
authors.forename: Richard
authors.initials: R
authors.lastname: Bouley
authors.forename: Dennis
authors.initials: D
authors.lastname: Brown
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D005089:Exocytosis / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D005470:Fluorometry; D015870:Gene Expression; D007150:Immunohistochemistry; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000378:metabolism*; D055571:Secretory Pathway / Q000502:physiology*; D013552:Swine; D014667:Vasopressins / Q000378:metabolism*
chemical_list: D051399:Aquaporin 2; D008164:Luminescent Proteins; D014667:Vasopressins
references.citation: J Clin Invest. 2000 Nov;106(9):1115-26
references.pmid: 11067864
references.citation: J Biol Chem. 2002 Mar 22;277(12):10379-86
references.pmid: 11782489
references.citation: PLoS Comput Biol. 2007 Mar 23;3(3):e57
references.pmid: 17381238
references.citation: J Biol Chem. 2004 Nov 5;279(45):46969-80
references.pmid: 15319442
references.citation: Am J Physiol Renal Physiol. 2003 May;284(5):F893-901
references.pmid: 12676734
references.citation: J Biol Chem. 2008 Sep 26;283(39):26643-61
references.pmid: 18664568
references.citation: J Biol Chem. 2000 Nov 24;275(47):36839-46
references.pmid: 10973964
references.citation: Am J Physiol Cell Physiol. 2007 Sep;293(3):C1129-38
references.pmid: 17626240
references.citation: Am J Physiol Renal Physiol. 2000 Mar;278(3):F388-94
references.pmid: 10710543
references.citation: J Cell Biol. 2008 Aug 11;182(3):587-601
references.pmid: 18678705
references.citation: J Biol Chem. 2003 Jan 10;278(2):1101-7
references.pmid: 12374804
references.citation: Endocrinology. 2005 Dec;146(12):5063-70
references.pmid: 16150901
references.citation: Curr Opin Neurobiol. 1997 Oct;7(5):631-9
references.pmid: 9384543
references.citation: Curr Opin Nephrol Hypertens. 2008 Sep;17(5):491-8
references.pmid: 18695390
references.citation: Neuron. 2007 Nov 21;56(4):657-69
references.pmid: 18031683
references.citation: Semin Nephrol. 2008 May;28(3):266-78
references.pmid: 18519087
references.citation: Am J Physiol Renal Physiol. 2005 Jun;288(6):F1103-12
references.pmid: 15644488
references.citation: Am J Physiol Renal Physiol. 2001 Dec;281(6):F1092-101
references.pmid: 11704560
references.citation: Annu Rev Cell Dev Biol. 2006;22:439-55
references.pmid: 16824007
references.citation: Am J Physiol Cell Physiol. 2003 Oct;285(4):C750-62
references.pmid: 12801889
references.citation: Am J Physiol Renal Physiol. 2002 Jun;282(6):F998-1011
references.pmid: 11997316
references.citation: J Cell Biol. 2005 Sep 26;170(7):1113-25
references.pmid: 16186257
references.citation: Biochem J. 1998 Apr 15;331 ( Pt 2):669-75
references.pmid: 9531511
references.citation: Physiol Rev. 2002 Jan;82(1):205-44
references.pmid: 11773613
references.citation: Am J Physiol Endocrinol Metab. 2006 Jun;290(6):E1276-86
references.pmid: 16418206
references.citation: Nephrol Dial Transplant. 2000;15 Suppl 6:21-2
references.pmid: 11143977
references.citation: Am J Physiol Renal Physiol. 2006 Oct;291(4):F882-90
references.pmid: 16684923
references.citation: Eur J Cell Biol. 1997 Oct;74(2):133-42
references.pmid: 9352218
references.citation: Traffic. 2007 Feb;8(2):110-23
references.pmid: 17156409
references.citation: J Cell Sci. 2000 Jun;113 ( Pt 11):1985-92
references.pmid: 10806109
references.citation: J Membr Biol. 1998 Jan 15;161(2):141-9
references.pmid: 9435270
references.citation: J Cell Biol. 1998 Dec 14;143(6):1485-503
references.pmid: 9852146
references.citation: Am J Physiol Renal Physiol. 2005 May;288(5):F930-8
references.pmid: 15625084
references.citation: Nat Rev Genet. 2007 Jul;8(7):533-43
references.pmid: 17572691
references.citation: Histochem Cell Biol. 2005 Jul;124(1):1-12
references.pmid: 16049696
references.citation: Am J Physiol Renal Physiol. 2000 Jan;278(1):F29-42
references.pmid: 10644653
references.citation: Am J Physiol Renal Physiol. 2004 Feb;286(2):F233-43
references.pmid: 14519593
references.citation: Plant Cell Physiol. 2000 Sep;41(9):993-1001
references.pmid: 11100771
references.citation: Experientia. 1994 Nov 30;50(11-12):1012-20
references.pmid: 7988659
references.citation: Mol Cell Biol. 1985 Dec;5(12):3610-6
references.pmid: 3915782
references.citation: Nat Rev Mol Cell Biol. 2004 Feb;5(2):121-32
references.pmid: 15040445
references.citation: Am J Physiol Renal Physiol. 2006 Jul;291(1):F246-53
references.pmid: 16449354
references.citation: Proc Natl Acad Sci U S A. 2006 May 2;103(18):7159-64
references.pmid: 16641100
references.citation: Am J Physiol. 1997 Jun;272(6 Pt 2):F817-22
references.pmid: 9227644
references.citation: J Biol Chem. 2007 Sep 28;282(39):28721-28732
references.pmid: 17636261
references.citation: J Am Soc Nephrol. 2005 Jun;16(6):1571-82
references.pmid: 15843469
references.citation: J Am Soc Nephrol. 1999 Jul;10(7):1416-29
references.pmid: 10405197
references.citation: J Am Soc Nephrol. 2005 Oct;16(10):2881-9
references.pmid: 16093449
references.citation: J Microsc. 2006 Dec;224(Pt 3):213-32
references.pmid: 17210054
references.citation: Mol Cell Proteomics. 2005 Aug;4(8):1095-106
references.pmid: 15905145
references.citation: Am J Physiol Renal Physiol. 2006 Jan;290(1):F177-87
references.pmid: 15985652
references.citation: EMBO J. 1996 Feb 1;15(3):510-9
references.pmid: 8599934
references.citation: Biol Cell. 2006 Apr;98(4):215-32
references.pmid: 16563128
references.citation: Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7212-6
references.pmid: 7543677
references.citation: J Biol Chem. 1997 Jun 6;272(23):14800-4
references.pmid: 9169447
references.citation: Proc Natl Acad Sci U S A. 2007 Oct 16;104(42):16696-701
references.pmid: 17940053
references.citation: Am J Physiol Renal Physiol. 2000 Feb;278(2):F317-26
references.pmid: 10662736
references.citation: Traffic. 2005 Dec;6(12):1142-56
references.pmid: 16262725
references.citation: EMBO Rep. 2003 Jan;4(1):88-93
references.pmid: 12524527
references.citation: Proc Natl Acad Sci U S A. 2004 Sep 7;101(36):13368-73
references.pmid: 15326289
references.citation: Nature. 1997 Sep 4;389(6646):81-5
references.pmid: 9288971
references.citation: Am J Physiol. 1993 Aug;265(2 Pt 2):F214-24
references.pmid: 8396343