Endogenous flow - induced nitric oxide reduces superoxide - stimulated Na / H 1 exchange activity via PKG in thick ascending limbs

19 Luminal flow stimulates endogenous NO and O2 production by renal thick 20 ascending limbs (TALs). The delicate balance between these two factors regulates Na 21 transport in TALs; NO enhances natriuresis whereas O2 augments Na absorption. 22 Endogenous, flow-stimulated O2 enhances Na/H exchange (NHE). Flow-stimulated NO 23 reduces flow-induced O2 , a process mediated by cGMP-dependent protein kinase 24 (PKG). However, whether flow-stimulated, endogenously-produced NO diminishes O2 25 stimulated NHE activity and the signaling pathway involved are unknown. We 26 hypothesized that flow-induced NO reduces the stimulation of NHE activity caused by 27 flow-induced O2 via PKG in TALs. Intracellular pH recovery after an acid-load was 28 measured as an indicator of NHE activity in isolated, perfused rat TALs. L-arginine, the 29 NO synthase substrate, decreased NHE activity by 34 ± 5% (n = 5; P < 0.04). The O2 30 scavenger tempol decreased NHE activity by 46 ± 8% (n = 6; P < 0.004) in the absence 31 of NO. In the presence of L-arginine, the inhibitory effect of tempol on NHE activity was 32 reduced to -19 ± 6% (n = 6; P < 0.03). The soluble guanylate cyclase inhibitor LY-83583 33 blocked the effect of L-arginine thus restoring tempol’s effect on NHE activity to -42 ± 34 4% (n = 6; P < 0.0005). The PKG inhibitor KT-5823 also inhibited L-arginine’s effect on 35 tempol-reduced NHE activity (-43 ± 5%; n = 5; P < 0.03). We conclude that flow-induced 36 NO reduces the stimulatory effect of endogenous, flow-induced O2 on NHE activity in 37 TALs via an increase in cGMP and PKG activation. 38 39