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(Hypertension. 2000;35:319.)
© 2000 American Heart Association, Inc.

Scientific Contributions

eNOS Mediates L-Arginine–Induced Inhibition of Thick Ascending Limb Chloride Flux

Craig F. Plato; Edward G. Shesely; Jeffrey L. Garvin

From the Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Mich.

Correspondence to Jeffrey L. Garvin, PhD, Henry Ford Hospital, Hypertension and Vascular Research Division, 2799 W Grand Blvd, Detroit, MI 48202.

Abstract—We recently reported that the rat thick ascending limb (THAL) possesses an active isoform of nitric oxide synthase (NOS) that is substrate-limited in vitro. NO produced by THAL NOS inhibits chloride flux. Protein and transcript for each of the primary NOS isoforms-endothelial (eNOS), inducible (iNOS), and neuronal (nNOS)-have been demonstrated in THALs. However, the NOS isoform that mediates NO-induced inhibition of chloride flux is unknown. We hypothesized that NO produced from eNOS in the THAL inhibits NaCl transport. THALs from male eNOS, iNOS, and nNOS knockout mice and C57BL/6J wild-type controls were perfused in vitro and the response of transepithelial chloride flux (J_Cl) to L-arginine (L-Arg), the substrate for NOS, and spermine NONOate (SPM), an NO donor was measured. We first tested whether isolated mouse THALs could synthesize NO and whether this NO inhibits transport. Addition of 0.5 mmol/L L-Arg to the bath decreased J_Cl from 105.8±17.5 to 79.2±15.8 pmol/mm per minute (P<0.01) in C57BL/6J wild-type mice, whereas addition of D-Arginine had no effects on J_Cl. In contrast, addition of 0.5 mmol/L L-Arg to the bath did not alter J_Cl of THALs from eNOS knockout mice. When 10 µmol/L SPM was added to the bath of eNOS knockout THALs, J_Cl decreased from 89.1±8.6 to 74.8±7.5 pmol/mm/min (P<0.05). Thus the lack of responsiveness of eNOS knockout THALs to L-Arg was not due to an inability to respond to NO. We next evaluated the role of iNOS and nNOS in the response to L-Arg. Addition of 0.5 mmol/L L-Arg to the bath decreased J_Cl in THALs from iNOS and nNOS knockout mice by 37.7±6.4% (P<0.05) and 31.8±8.3% (P<0.01), respectively. We conclude that eNOS is the active isoform of NOS in the THAL under basal conditions. Mouse THAL eNOS responds to exogenous L-Arg by increasing NO production, which, in turn, inhibits J_Cl.

Key Words: nitric oxide • kidney • mice

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