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Nitric oxide reduces flow-induced superoxide production via cGMP-dependent protein kinase in thick ascending limbs

¹Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit; and ²Department of Physiology, Wayne State University, Detroit, Michigan

Submitted 25 November 2008 ; accepted in final form 21 February 2009

We have shown that increased luminal flow induces O₂^– and nitric oxide (NO) production in thick ascending limbs (TALs). However, the interaction of flow-stimulated NO and O₂^– in TALs is unclear. We hypothesized that NO inhibits flow-induced O₂^– production in TALs via cGMP-dependent protein kinase (PKG). We measured flow-stimulated O₂^– 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 O₂^– 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 µM) also enhanced flow-induced net O₂^– 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 O₂^– 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 O₂^– from 39 ± 9 to 7 ± 3 AU/s (P < 0.03). The PKG inhibitor KT-5823 (5 µM) partially restored flow-induced net O₂^– 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 O₂^– production. We conclude that 1) NO reduces flow-stimulated O₂^– production, 2) this occurs primarily via the cGMP/PKG pathway, and 3) O₂^– scavenging by NO plays a minor role.

reactive oxygen species; soluble guanylate cyclase; phosphodiesterase; scavenging