Abstract P230: Uncoupling of NOS3 is Involved in Diminished Flow-induced No Production in Dahl Salt-sensitive Rat Thick Ascending Limbs

Abstract

Increased luminal flow enhances nitric oxide (NO) production in thick ascending limbs (TALs). NO produced by NO synthase 3 (NOS3) inhibits Na transport. However, its effect on transport is reduced in Dahl salt-sensitive (SS) vs salt-resistant rats (SR). In Sprague-Dawley rat TALs, angiotensin II can acutely cause NOS3 to uncouple to produce superoxide (O 2 - ) thereby reducing NO production. We hypothesized that flow-induced NO production is decreased in SS TALs and that this is due to NOS3 uncoupling. We measured flow-induced NO in isolated perfused TALs using the fluorescent dye DAF-FM and performed Western blots of renal medullary lysates. Flow-induced NO production was reduced 69% in TALs from SS (11±2 arbitrary units (AU)/min, n=6) vs SR (35±6 AU/min, n=8, p < 0.008). This difference between strains was not due to altered NOS3 expression (NOS3/GAPDH ratio of 0.91 ± 0.08 for SS vs 1.09 ± 0.08 for SR; n = 5 for each). The difference in flow-induced NO between strains was slightly reduced in the presence of the superoxide (O 2 - ) scavenger tempol (19±2 vs 30±5 AU/min for SS and SR, respectively; n=9 for each strain, p < 0.04), suggesting that scavenging of NO by O 2 - plays only a minor role in the difference in flow-induced NO production between SS and SR thick ascending limbs. We next investigated whether NOS3 uncoupling could account for the difference between strains by using the fluorescent dye dihydroethidium to measure flow-induced O 2 - before and after treatment with the NOS inhibitor L-NAME. Blocking NOS3 reduced O 2 - production in SS TALs by 21±7%, from 38±5 to 30±5 AU/min (n=6, p < 0.05) whereas it had no effect in SR TALs (26±6 vs 28±3, n=5). We conclude that the diminished flow-induced NO in SS TALs is not due to differences in NOS3 expression nor acute flow-induced O 2 - , but rather in large part due to uncoupling of NOS3. Impaired flow-induced NO production in TALs could contribute to the Na retention associated with salt-sensitive hypertension.