title: 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 micromol/L with an EC50 of 37 micromol/L. The increase was blunted by 74% by the nonselective NOS inhibitor L-omega-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 micromol/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 micromol/L) enhanced the response to ATP and shifted the EC(50) to 0.8 micromol/L. In the presence of ARL67156, the EC50 of the P2X-selective agonist beta,gamma-methylene-adenosine 5'-triphosphate was 4.8 micromol/L and the EC50 for the P2Y-selective agonist UTP was 40.4 micromol/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.
authors.affiliation: Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, MI 48202, USA.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Silva
authors.forename: William H
authors.initials: WH
authors.lastname: Beierwaltes
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Garvin
mesh_terms: D000255:Adenosine Triphosphate / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism*; D019793:Fluorescein; D005456:Fluorescent Dyes; D006868:Hydrolysis / Q000187:drug effects; D008138:Loop of Henle / Q000378:metabolism*; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D058910:Purinergic P2 Receptor Agonists; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000378:metabolism*; D013498:Suramin / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000008:administration & dosage / Q000494:pharmacology
chemical_list: C111205:4,5-diaminofluorescein; C092431:6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D009569:Nitric Oxide; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D013498:Suramin; D000255:Adenosine Triphosphate; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D019793:Fluorescein; D014544:Uridine Triphosphate; D019331:NG-Nitroarginine Methyl Ester