NDLI: cAMP Stimulates Apical Exocytosis of the Renal $Na^{+}-K^{+}-2Cl^{−}$ Cotransporter NKCC2 in the Thick Ascending Limb

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cAMP Stimulates Apical Exocytosis of the Renal $Na^{+}-K^{+}-2Cl^{−}$ Cotransporter NKCC2 in the Thick Ascending Limb

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Ares, Gustavo R. Ortiz, Pablo A. Caceres, Paulo S.
Description	Author Affiliation: Caceres PS ( Hypertension and Vascular Research Division, Henry Ford Hospital, Wayne State University, Detroit, Michigan 48202, USA.)
Abstract	The apical renal $Na^{+}-K^{+}-2Cl^{−}$ cotransporter NKCC2 mediates NaCl absorption by the thick ascending limb (TAL) of Henle's loop. cAMP stimulates NKCC2 by enhancing steady-state apical membrane levels of this protein; however, the trafficking and signaling mechanisms by which this occurs have not been studied. Here, we report that stimulation of endogenous cAMP levels with either forskolin/3-isobutyl-1-methylxanthine (IBMX) or the V2 receptor agonist $[deamino-Cys^{1},d-Arg^{8}]vasopressin$ increases steady-state surface NKCC2 and that the protein kinase A (PKA) inhibitor H-89 blocks this effect. Confocal imaging of apical surface NKCC2 in isolated perfused TALs confirmed a stimulatory effect of cAMP on apical trafficking that was blocked by PKA inhibition. Selective stimulation of PKA with the agonist $N^{6}-benzoyl-cAMP$ (500 μm) stimulated steady-state surface NKCC2, whereas the Epac-selective agonist 8-p-chlorophenylthio-2′-O-methyl-cAMP (100 and 250 μm) had no effect. To explore the trafficking mechanism by which cAMP increases apical NKCC2, we measured cumulative apical membrane exocytosis and NKCC2 exocytic insertion in TALs. By monitoring apical FM1–43 fluorescence, we observed rapid stimulation of apical exocytosis (2 min) by forskolin/IBMX. We also found constitutive exocytic insertion of NKCC2 in TALs over time, which was increased by 3-fold in the presence of forskolin/IBMX. PKA inhibition blunted cAMP-stimulated exocytic insertion but did not affect the rate of constitutive exocytosis. We conclude that cAMP stimulates steady-state apical surface NKCC2 by stimulating exocytic insertion and that this process is highly dependent on PKA but not Epac.
ISSN	00219258
e-ISSN	1083351X
Journal	Journal of Biological Chemistry
Issue Number	37
Volume Number	284
Language	English
Publisher	American Society for Biochemistry and Molecular Biology (United States)
Publisher Date	2009-09-11
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Cyclic AMP-Dependent Protein Kinases Metabolism Cyclic AMP Guanine Nucleotide Exchange Factors Sodium-Potassium-Chloride Symporters Sodium-Potassium-Exchanging ATPase 1-Methyl-3-isobutylxanthine Pharmacology Animals Cell Membrane Colforsin Enzyme Inhibitors Exocytosis Isoquinolines Rats, Sprague-Dawley Solute Carrier Family 12, Member 1 Sulfonamides Research Support, N.I.H., Extramural Biochemistry Molecular Biology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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