title: Nitric oxide reduces flow-induced superoxide production via cGMP-dependent protein kinase in thick ascending limbs.
abstract: We have shown that increased luminal flow induces O(2)(-) and nitric oxide (NO) production in thick ascending limbs (TALs). However, the interaction of flow-stimulated NO and O(2)(-) in TALs is unclear. We hypothesized that NO inhibits flow-induced O(2)(-) production in TALs via cGMP-dependent protein kinase (PKG). We measured flow-stimulated O(2)(-) production in rat TALs using dihydroethidium in the absence and presence of L-arginine (0.3 mM), the substrate for NO synthase. The addition of L-arginine reduced flow-induced net O(2)(-) production from 68 +/- 9 to 17 +/- 4 AU/s (P < 0.002). The addition of the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 5 mM) in the presence of L-arginine stimulated production (L-arginine: 15 +/- 4 AU/s vs. L-arginine + L-NAME: 63 +/- 7 AU/s; P < 0.002). The guanylate cyclase inhibitor LY-83583 (10 microM) also enhanced flow-induced net O(2)(-) production in the presence of L-arginine (L-arginine: 7 +/- 4 AU/s vs. L-arginine + LY-83583: 53 +/- 7 AU/s; P < 0.01). In the presence of LY-83583, L-arginine only reduced flow-induced net O(2)(-) by 36% (LY-83583: 80 +/- 7 AU/s vs. LY-83583 + L-arginine: 51 +/- 3 AU/s; P < 0.006). The cGMP analog dibutyryl (db)-cGMP reduced flow-induced net O(2)(-) from 39 +/- 9 to 7 +/- 3 AU/s (P < 0.03). The PKG inhibitor KT-5823 (5 microM) partially restored flow-induced net O(2)(-) in the presence of L-arginine (L-arginine: 4 +/- 4 AU/s vs. L-arginine + KT-5823: 32 +/- 9 AU/s; P < 0.03) and db-cGMP (db-cGMP: 9 +/- 7 AU/s vs. db-cGMP + KT-5823: 54 +/- 5 AU/s; P < 0.01). Phosphodiesterase II inhibition had no effect on arginine-inhibited O(2)(-) production. We conclude that 1) NO reduces flow-stimulated O(2)(-) production, 2) this occurs primarily via the cGMP/PKG pathway, and 3) O(2)(-) scavenging by NO plays a minor role.
authors.affiliation: Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, MI 48202, USA.
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Hong
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Garvin
mesh_terms: D000818:Animals; D001120:Arginine / Q000378:metabolism / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000378:metabolism; D008138:Loop of Henle / Q000098:blood supply* / Q000201:enzymology*; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012079:Renal Circulation / Q000502:physiology*; D000071756:Soluble Guanylyl Cyclase; D013481:Superoxides / Q000378:metabolism*
chemical_list: D002227:Carbazoles; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D018160:Receptors, Cytoplasmic and Nuclear; D013481:Superoxides; C073601:KT 5823; D009569:Nitric Oxide; D001120:Arginine; D017869:Cyclic GMP-Dependent Protein Kinases; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester