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 2

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Quantification of the effect of VP plus forskolin and SNP on AQP2 redist...
Quantification of the effect of VP plus forskolin and SNP on AQP2 redistribution in collecting duct (inner stripe) principal cells. Total principal cell area (excluding the nucleus) and the area occupied by AQP2 fluorescence were determined using IP Lab Spectrum software on confocal microscope images as in Figure 1. Results are expressed as the percentage of total cell area occupied by AQP2 fluorescence. VP plus forskolin and SNP both induce a similar and marked reduction in the area of AQP2 fluorescence, indicating that these agonists cause the AQP2 staining to become concentrated in a smaller region of the cell, i. e., the apical pole. AP < 0.05 compared with the control value. FK, forskolin.