Phosphorylation of Neuronal and Endothelial Nitric Oxide Synthase in the Kidney with High and Low Salt Diets

Abstract

Background: Renal nitric oxide (NO) synthesis increases with increasing salt intake, however, the mechanisms underlying this are poorly understood. We hypothesized that activating or inhibitory phosphorylation of neuronal and endothelial nitric oxide synthase (nNOS, eNOS) regulates renal NO production in response to altered dietary salt. Methods:Sprague-Dawley rats were fed low, normal or high salt diets for 12 h or 2 weeks, and kidney NOS phosphorylation was analyzed by Western blot using phosphopeptide antibodies against the sites nNOS-Ser¹⁴¹², nNOS-Ser⁸⁴⁷, eNOS-Ser¹¹⁷⁶ and eNOS-Thr⁴⁹⁴. Results:At 12 h, total nNOS increased 1.4-fold (p < 0.01) in the high salt group and decreased by 26% (p < 0.05) in the low salt group. Changes in expression of phospho-nNOS at 12 h were accounted for by the changes in total nNOS. No change in total or phospho-eNOS was seen at 12 h. At 2 weeks, in the low salt group expression of total nNOS increased 1.8-fold (p < 0.05) whereas expression of nNOS phosphorylated at the inhibitory site Ser⁸⁴⁷ increased 4.3-fold (p < 0.01). Total eNOS was increased 3-fold in the low salt group (p < 0.01), with parallel increases in eNOS phosphorylated at both activating and inhibitory sites (p < 0.05). In the 2-week high salt group no changes in NOS expression or phosphorylation were seen, despite the observed increased excretion of urinary NO metabolites. Conclusion:In summary, changes in phospho-nNOS and phospho-eNOS expression occurred in parallel with changes in total expression, thus, the overall activating and inhibitory effects of nNOS and eNOS phosphorylation at the sites studied were not changed by altered dietary salt.