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.