Extracellular ATP Stimulates NO Production in Rat Thick Ascending Limb

Abstract

NO produced by NO synthase (NOS) 3 acts as an autacoid to regulate NaCl absorption in the thick ascending limb. ATP induces NO production by NOS 3 in endothelial cells. We hypothesized that extracellular ATP activates NOS in thick ascending limbs through P2 receptors. To test this, we measured intracellular NO production using the NO-selective fluorescent dye DAF-2 in suspensions of rat medullary thick ascending limbs. We found that ATP increased DAF-2 fluorescence in a concentration-dependent manner, reaching saturation at &200 μmol/L with an EC ₅₀ of 37 μmol/L. The increase was blunted by 74% by the nonselective NOS inhibitor l -ω-nitro-arginine-methyl-ester (2 mmol/L; 60±7 versus 16±6 arbitrary fluorescence units; P n =5). In the presence of the P2 receptor antagonist suramin (300 μmol/L), ATP-induced NO production was reduced by 64% (101±11 versus 37±5 arbitrary fluorescence units; P n =5). Blocking ATP hydrolysis with a 5′-ectonucleotidase inhibitor, ARL67156 (30 μmol/L) enhanced the response to ATP and shifted the EC ₅₀ to 0.8 μmol/L. In the presence of ARL67156, the EC ₅₀ of the P2X-selective agonist β,γ-methylene-adenosine 5′-triphosphate was 4.8 μmol/L and the EC ₅₀ for the P2Y–selective agonist UTP was 40.4 μmol/L. The maximal responses for both agonists were similar. Taken together, these data indicate that ATP stimulates NO production in the thick ascending limb primarily through P2X receptor activation and that ATP hydrolysis may regulate NO production.