Superoxide (O2−) produced by NADPH oxidase regulates Na absorption and renal hemodynamics. Increased NaCl in the thick ascending limb (TAL) stimulates O2− generation. However, we do not know whether physiological changes in NaCl concentration augment O2− generation, nor do we know the mediator(s) involved. In other cells, Rac1, a regulatory subunit of NADPH oxidase, is activated by elevated NaCl. We hypothesized that increasing luminal NaCl within the physiological range activates Rac1 and NADPH oxidase and, thereby, increases O2− production. We increased NaCl from 10 to 57 mM in medullary TAL suspensions and used lucigenin to measure O2− generation and Western blot to measure Rac1 activity. Increasing NaCl stimulated O2− generation from 1.41 ± 0.16 to 2.71 ± 0.30 nmol O2−·min−1·mg protein−1 ( n = 6, P < 0.05). This increase was blocked by the Na-K-2Cl cotransporter inhibitor furosemide and the NADPH oxidase inhibitor apocynin. To examine the role of Rac1 in NaCl-induced O2− production, we measured Rac1 translocation by Western blot. When we added NaCl, Rac1 in the particulate fraction increased from 6.8 ± 0.8 to 11.7 ± 2.4% of total Rac1 ( n = 7, P < 0.05). Then we measured O2− generation in the presence and absence of the Rac1 inhibitor. In the absence of the Rac1 inhibitor, NaCl increased O2− generation from 1.07 ± 0.24 to 2.02 ± 0.49 nmol O2−·min−1·mg protein−1, and this increase was completely blocked by the inhibitor. Similarly, in vivo treatment of TALs with adenovirus expressing dominant-negative Rac1 decreased NaCl-induced O2− generation by 60% compared with control (0.33 ± 0.04 vs. 0.81 ± 0.17 nmol O2−·min−1·mg protein−1, n = 6, P < 0.05). We concluded that physiological increases in NaCl stimulate TAL O2− generation by activating Rac1.
Putrescine, a source of γ-aminobutyric acid in the adrenal gland of the rat