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.

Fig. 1.

A: effect of l-arginine (Arg) on net O₂⁻ production by thick ascending limbs in the presence and absence of luminal flow. B: effect of Arg on flow-induced change in net O₂⁻ production. *P < 0.002; n = 5 vs. control (Con). Tubules were perfused at 20 nl/min.

Fig. 2.

A: effect of the nitric oxide (NO) synthase inhibitor N^G-nitro-l-arginine methyl ester (l-NAME) on Arg-induced inhibition of net O₂⁻ production by thick ascending limbs in the absence and presence of luminal flow. B: effect of l-NAME on Arg's effect on flow-stimulated net O₂⁻ production. *P < 0.002; n = 5 vs. Arg alone. Tubules were perfused at 20 nl/min.

Fig. 3.

A: effect of the soluble guanylate cyclase inhibitor LY-83583 (LY) on the effect of Arg on net O₂⁻ production by thick ascending limbs in the absence and presence of luminal flow. B: effect of LY on Arg's effect on flow-stimulated net O₂⁻ production. *P < 0.01; n = 5 vs. Arg alone. C: effect of Arg in the presence of LY on net O₂⁻ production by thick ascending limbs in the absence and presence of luminal flow. D: effect of Arg on the flow-induced change in net O₂⁻ production in the presence of LY. *P < 0.006; n = 5 vs. LY alone. Tubules were perfused at 20 nl/min.

Fig. 4.

A: effect of dibutyryl (db)-cGMP on net O₂⁻ production by thick ascending limbs in the absence and presence of luminal flow. B: effect of db-cGMP on flow-stimulated net O₂⁻ production. *P < 0.03; n = 5 vs. Con. Tubules were perfused at 20 nl/min.

Fig. 5.

A: effect of KT-5823 (KT), a PKG inhibitor, on the effect of Arg on net O₂⁻ production by thick ascending limbs in the absence and presence of luminal flow. B: effect of KT on Arg's effect on net O₂⁻ production induced by flow. *P < 0.03; n = 5 vs. Arg alone. C: effect of KT, a PKG inhibitor, on the effect of db-cGMP on net O₂⁻ production by thick ascending limbs in the absence and presence of luminal flow. D: effect of KT on db-cGMP's effect on net O₂⁻ production induced by flow. *P < 0.01; n = 5 vs. db-cGMP alone. Tubules were perfused at 20 nl/min.

Fig. 6.

A: effect of Arg in the presence of the phosphodiesterase II inhibitor BAY 60–7550 (BAY) on net O₂⁻ production by thick ascending limbs in the absence and presence of luminal flow. B: effect of Arg in the presence of BAY on flow-induced change in net O₂⁻ production by thick ascending limbs. P > 0.05; n = 5 vs. Arg alone. Tubules were perfused at 20 nl/min.