eNOS mediates L-Arginine-induced inhibition of thick ascending limb chloride flux

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 transcri pt for each of the primary NOS isoforms-endothelial (eNOS), inducible (iNOS ), and neuronal (nNOS)-have been demonstrated in THALs. However, the NOS is oform that mediates NO-induced inhibition of chloride flux is unknown. We h ypothesized 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 chlorid e flux (J(Cl)) to L-arginine (L-Arg), the substrate for NOS, and spermine N ONOate (SPM), an NO donor was measured. We first tested whether isolated mo use THALs could synthesize NO and whether this NO inhibits transport. Addit ion 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 a ddition 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 m ice. When 10 mu mol/L SPM was added to the bath of eNOS knockout THALs, J(C l) decreased from 89.1+/-8.6 to 74.8+/-7.5 pmol/mm/min (P<.05). Thus the la ck of responsiveness of eNOS knockout THALs to L-Arg was not due to an inab ility 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 exo genous L-Arg by increasing NO production, which, in turn, inhibits;I,,.