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Extracellular ATP‐induced Akt activation stimulates NO production by NOS3
Author(s) -
Silva Guillermo B,
Garvin Jeffrey L
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.796.14
Subject(s) - protein kinase b , chemistry , extracellular , purinergic receptor , phosphorylation , microbiology and biotechnology , medicine , endocrinology , biochemistry , biology
ATP simulates NO production in thick ascending limbs by activating purinergic receptors (P2R). However the signaling cascade involved is unknown. We hypothesized that ATP enhances NOS3 activity by stimulating PI3 Kinase and Akt. We measured NO production by thick ascending limb suspensions using the NO‐sensitive dye DAF‐2 DA and Akt activity by fluorescence resonance energy transfer (FRET). ATP (100 μM) increased NO production by 32±8 arbitrary units (AU). In the presence of selective NOS1 and NOS2 inhibitors, 7‐NI (10 μM) and 1400W (100nM), ATP stimulated NO production by 22±9 AU and 31±9 AU, respectively. In contrast, ATP only stimulated NO production by 6±5 AU in NOS3 ‐/‐ mice ( p <0.04 vs. wild‐type). In the presence of the PI3 kinase inhibitor LY294003 (10 μM), ATP did not increase NO production (5±2 vs . 28±4 AU; p <0.02). With the PDK1 inhibitor (5 μM), ATP did not induce NO production (6±3 vs . 47±8 AU; p <0.02). ATP alone increased Akt activity by 120±12 FRET units (FU) and only by 11±17 FU in the presence suramin, a P2R antagonist (300 μM; p <0.01). In the presence of the Akt‐selective inhibitor (5 μM), ATP‐induced NO production was blocked by 90±4% ( p <0.04). In vivo transduction of thick ascending limbs with a dominant‐negative Akt decreased ATP‐induced NO production by 60±6% ( p <0.05). We concluded that ATP stimulates NO production in the thick ascending limb by activating the PI3 kinase/ PDK1/Akt signaling pathway.