title: Stimulation of AQP2 membrane insertion in renal epithelial cells in vitro and in vivo by the cGMP phosphodiesterase inhibitor sildenafil citrate (Viagra).
abstract: Vasopressin-stimulated insertion of the aquaporin 2 (AQP2) water channel into the plasma membrane of kidney collecting duct principal cells is a key event in the urinary concentrating mechanism. The paradigm for vasopressin-receptor signaling involves cAMP-mediated protein kinase A activation, which results in the functionally critical phosphorylation of AQP2 on amino acid serine 256. We previously showed that a parallel cGMP-mediated signaling pathway also leads to AQP2 membrane insertion in AQP2-transfected LLC-PK1 (LLC-AQP2) cells and in outer medullary collecting duct principal cells in situ (Bouley R, Breton S, Sun T, McLaughlin M, Nsumu NN, Lin HY, Ausiello DA, and Brown D. J Clin Invest 106: 1115-1126, 2000). In the present report, we show by immunofluorescence microscopy, and Western blotting of plasma membrane fractions, that 45-min exposure of LLC-AQP2 cells to the cGMP phosphodiesterase type 5 (PDE5) inhibitors sildenafil citrate (Viagra) or 4-{[3',4'-methylene-dioxybenzyl]amino}-6-methoxyquinazoline elevates intracellular cGMP levels and results in the plasma membrane accumulation of AQP2; i.e., they mimic the vasopressin effect. Importantly, our data also show that acute exposure to PDE5 inhibitors for 60 min induces apical accumulation of AQP2 in kidney medullary collecting duct principal cells both in tissue slices incubated in vitro as well as in vivo after intravenous injection of Viagra into rats. These data suggest that AQP2 membrane insertion can be induced independently of vasopressin-receptor activation by activating a parallel cGMP-mediated signal transduction pathway with cGMP PDE inhibitors. These results provide proof-of-principle that pharmacological activation of vasopressin-independent, cGMP signaling pathways could aid in the treatment of those forms of nephrogenic diabetes insipidus that are due to vasopressin-2 receptor dysfunction.
authors.affiliation: Program in Membrane Biology and Renal Unit, Rm. 8207, Massachusetts General Hospital East, 149 13th St.. Charlestown, MA 02129, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Bouley
authors.forename: Nuria
authors.initials: N
authors.lastname: Pastor-Soler
authors.forename: Ori
authors.initials: O
authors.lastname: Cohen
authors.forename: Margaret
authors.initials: M
authors.lastname: McLaughlin
authors.forename: Sylvie
authors.initials: S
authors.lastname: Breton
authors.forename: Dennis
authors.initials: D
authors.lastname: Brown
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D051399:Aquaporin 2; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D018500:Diabetes Insipidus, Nephrogenic / Q000188:drug therapy* / Q000378:metabolism / Q000503:physiopathology; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology*; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology* / Q000502:physiology; D018374:LLC-PK1 Cells; D008297:Male; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology*; D011687:Purines; D051381:Rats; D011910:Rats, Brattleboro; D017207:Rats, Sprague-Dawley; D017483:Receptors, Vasopressin / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D000068677:Sildenafil Citrate; D013450:Sulfones; D013552:Swine; D014162:Transfection
chemical_list: C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D020346:Aquaporins; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D017483:Receptors, Vasopressin; D013450:Sulfones; D000068677:Sildenafil Citrate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517384:Pde5a protein, rat; D006152:Cyclic GMP