A high-salt diet dissociates NO synthase-3 expression and NO production by the thick ascending limb

NO produced by endothelial NO synthase (NOS3) decreases sodium transport by the thick ascending limb (THAL). We found previously that 7 days of high salt (HS) increased THAL-NOS3 expression but not NO production. NOS3 phosphorylation regulates enzyme activity. We hypothesized that HS acutely increases NOS3 expression and NO production, and, over time, changes in NOS3 phosphorylation dissociate NO production from expression. NOS3 expression increased by 71+/-13%, 127+/-24%, and 69+/-16% at days 1, 3, and 7 of HS, respectively. At days 14 and 28, expression was back to normal salt. After 1 day of HS, NO production in response to 250 micromol/L L-arginine was elevated by 146% and, by day 3, returned to normal salt. Similar increases were found in response to endothelin-1. Inhibitors of NOS1/2 did not blunt the salt-induced increase in NO. Phosphorylation at Thr495, an inhibitory site, decreased by 39+/-8% at day 1 of HS and then increased by 116+/-18% at day 3. Phosphorylation at Ser633 and Ser1177 (stimulatory sites) decreased by &25% at day 1 and remained depressed at day 3. Superoxide production increased by 71% at day 1, decreased by 57% at day 3, and decreased by 55% at day 7. The NOS inhibitor L-NG-nitroarginine methyl ester did not alter superoxide levels at any time point. The addition of reduced nicotinamide-adenine dinucleotide phosphate and tetrahydrobiopterin had no effect on NO release after 3 days of HS. We conclude the following: (1) HS transiently increases NO production and NOS3 expression; (2) NOS3 expression and NO production are dissociated by HS; and (3) changes in phosphorylation explain how THAL NOS3 activity and expression are dissociated by HS.