Published Online
on December 27, 2005

A more recent version of this article appeared on March 1, 2006

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Submitted on September 29, 2005
Revised on October 23, 2005

Extracellular ATP Stimulates NO Production in Rat Thick Ascending Limb

Guillermo Silva; William H. Beierwaltes; and Jeffrey L. Garvin^*

From the Division of Hypertension and Vascular Research (G.S., W.H.B., J.L.G.), Henry Ford Hospital and Department of Physiology (W.H.B., J.L.G.), School of Medicine, Wayne State University, Detroit, Mich.

^* To whom correspondence should be addressed. E-mail: jgarvin1{at}hfhs.org.

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 EC₅₀ of 37 µmol/L. The increase was blunted by 74% by the nonselective NOS inhibitor L--nitro-arginine-methyl-ester (2 mmol/L; 60±7 versus 16±6 arbitrary fluorescence units; P<0.02; 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<0.002; n=5). Blocking ATP hydrolysis with a 5'-ectonucleotidase inhibitor, ARL67156 (30 µmol/L) enhanced the response to ATP and shifted the EC₅₀ to 0.8 µmol/L. In the presence of ARL67156, the EC₅₀ of the P2X-selective agonist ,-methylene-adenosine 5'-triphosphate was 4.8 µmol/L and the EC₅₀ 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.

Key words: nitric oxide • kidney • receptors, purinergic • signal transduction

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