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

Abstract

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

Keywords: luminal flow; protein kinases; reactive oxygen species; sodium/hydrogen exchange.

Fig. 1.

Effect of the nitric oxide synthase substrate l-arginine (Arg; 0.5 mM) on luminal Na/H exchange (NHE) activity as measured by intracellular pH (pH_i) recovery rate after an acid load in flow-stimulated thick ascending limbs. *P < 0.04; n = 5.

Fig. 2.

Effect of tempol (100 μM) on NHE activity as measured by pH_i recovery rate after an acid load in flow-stimulated thick ascending limbs. *P < 0.004; n = 6.

Fig. 3.

Effect of tempol (100 μM) on NHE activity as measured by pH_i recovery rate after an acid load in flow-stimulated thick ascending limbs in the presence of l-arginine (0.5 mM). *P < 0.03; n = 6.

Fig. 4.

Effect of l-arginine (0.5 mM) in the presence of the guanylate cyclase inhibitor LY-83583 (LY; 10 μM) on tempol's (100 μM) effect on NHE activity as measured by pH_i recovery rate after an acid load in flow-stimulated thick ascending limbs. A: effect of tempol alone. Con = control; *P < 0.04; n = 6. B: effect of tempol in the presence of l-arginine and LY-83583. *P < 0.0005; n = 6.

Fig. 5.

Effect of tempol (100 μM) on NHE activity as measured by pH_i recovery rate after an acid load in flow-stimulated thick ascending limbs in the presence of dibutyryl-cGMP (db-cGMP; 0.5 mM); n = 6.

Fig. 6.

Effect of l-arginine (0.5 mM) in the presence of the PKG inhibitor KT-5823 (KT; 5 μM) on tempol's (100 μM) effect on NHE activity as measured by pH_i recovery rate after an acid load in flow-stimulated thick ascending limbs. *P < 0.03; n = 5.