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