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Interaction of O 2 − and NO in the Thick Ascending Limb

2002; Lippincott Williams & Wilkins; Volume: 39; Issue: 2 Linguagem: Inglês

10.1161/hy0202.103287

1524-4563

Pablo A. Ortiz, Jeffrey L. Garvin,

Electron Spin Resonance Studies

Abstract— Nitric oxide (NO) is an important regulator of NaCl absorption by the thick ascending limb of the loop of Henle (THAL). The free radical superoxide (O 2 − ) reacts with NO, decreasing its bioavailability. O 2 − is produced by mitochondria and various oxidases, some of which are present in the THAL. However, the ability of the THAL to produce O 2 − and its interaction with NO have not been studied. We hypothesized that NO bioavailability is decreased by O 2 − . THALs were isolated and perfused and NO production was measured with an NO-selective microelectrode. Addition of L-Arg (250 μmol/L), but not D-arginine, to the bath increased NO release by 34.8±11.8 pA (n=7). The response to L-Arg was completely abolished by the NO synthase inhibitor L-NAME (n=7). Scavenging THAL O 2 − with the superoxide dismutase (SOD) mimetic Tempol (50 μmol/L) increased L-Arg-induced NO release. At all concentrations of L-Arg tested (50, 100, 250, 500, and 750 μmol/L), further addition of Tempol to the bath significantly increased NO release by THALs. Addition of SOD (300 U/mL) to the bath increased L-Arg-induced NO levels by 82% (n=5; P <0.02). Pretreatment of THALs with the SOD inhibitor diethyl-dithiocarbamate (250 μmol/L) blunted L-Arg-induced NO release by 63% compared with untreated tubules (n=5; P <0.05). Finally, we tested the effect of Tempol on NO-induced inhibition of THAL chloride transport. Addition of L-Arg decreased THAL Cl − absorption by 35%. Subsequent addition of Tempol (50 μmol/L) to the bath further decreased Cl − absorption by 35% (n=6; P <0.05). We conclude that NO bioavailability in the THAL is decreased by O 2 − . In addition, we believe our studies are the first to show that endogenous O 2 − may act as a physiological regulator of nephron NaCl transport.