Nitric oxide and atrial natriuretic factor stimulate cGMP-dependent membrane insertion of aquaporin 2 in renal epithelial cells
Richard Bouley, … , Dennis A. Ausiello, Dennis Brown
Richard Bouley, … , Dennis A. Ausiello, Dennis Brown
Published November 1, 2000
Citation Information: J Clin Invest. 2000;106(9):1115-1126. https://doi.org/10.1172/JCI9594.
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Article

Nitric oxide and atrial natriuretic factor stimulate cGMP-dependent membrane insertion of aquaporin 2 in renal epithelial cells

Abstract

In collecting duct principal cells, aquaporin 2 (AQP2) is shuttled from intracellular vesicles to the plasma membrane upon vasopressin (VP) stimulation. VP activates adenylyl cyclase, increases intracellular cAMP, activating protein kinase A (PKA) to phosphorylate AQP2 on the COOH-terminal residue, serine 256. Using rat kidney slices and LLC-PK1 cells stably expressing AQP2 (LLC-AQP2 cells), we now show that AQP2 trafficking can be stimulated by cAMP-independent pathways. In these systems, the nitric oxide (NO) donors sodium nitroprusside (SNP) and NONOate and the NO synthase substrate L-arginine mimicked the effect of VP, stimulating relocation of AQP2 from cytoplasmic vesicles to the plasma membrane. Unlike VP, these other agents did not increase intracellular cAMP. However, SNP increased intracellular cGMP, and exogenous cGMP stimulated AQP2-membrane insertion. Atrial natriuretic factor, which signals via cGMP, also stimulated AQP2 translocation. The VP and SNP effects were blocked by the kinase inhibitor H89. SNP did not stimulate membrane insertion of AQP2 in LLC-PK1 cells expressing the phosphorylation-deficient mutant 256SerAla-AQP2, indicating that phosphorylation of Ser256 is required for signaling. Both PKA and cGMP-dependent protein kinase G phosphorylated AQP2 on this COOH-terminal residue in vitro. These results demonstrate a novel, cAMP-independent and cGMP-dependent pathway for AQP2 membrane insertion in renal epithelial cells.

Authors

Richard Bouley, Sylvie Breton, Tian-xiao Sun, Margaret McLaughlin, Ndona N. Nsumu, Herbert Y. Lin, Dennis A. Ausiello, Dennis Brown

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Figure 5

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Immunogold electron microscopy showing apical plasma membrane insertion ...
Immunogold electron microscopy showing apical plasma membrane insertion of AQP2 induced by VP/forskolin and SNP treatment of kidney slices. AQP2 was localized in nonpermeabilized tissues with an Ab against an external epitope of AQP2. (a) SNP treatment for 15 minutes. Abundant gold particles, representing AQP2 antigenic sites, are located on the apical plasma membrane of a principal cell (left). An adjacent intercalated cell (right) is unlabeled. (b) Apical plasma membrane localization of gold labeling for AQP2 in a principal cell after VP/forskolin treatment. The adjacent intercalated cell is unlabeled. (c) Control tissue incubated for 15 minutes in the absence of agonist. Very few gold particles are seen on the apical membrane, indicating that most of the AQP2 is inside the cell in this condition, supporting the confocal data shown in Figures 1 and 3. In all figures, most of the gold particles are on the external surface of the apical membrane, consistent with the use of an Ab raised against an external epitope of AQP2. The position of the cell junction between the principal cell (left) and the intercalated cell (right) is indicated with an arrow in each figure. Bar, 1 μm.