Endothelin-1 Inhibits Thick Ascending Limb Transport via Akt-stimulated Nitric Oxide Production

Endothelin-1 inhibits sodium reabsorption in the thick ascending limb (THAL) via stimulation of nitric oxide (NO) production. The mechanism whereby endothelin-1 stimulates THAL NO is unknown. We hypothesized that endothelin-1 stimulates THAL NO production by activating phosphatidylinositol 3-kinase (PI3K), stimulating Akt activity, and phosphorylating NOS3 at Ser¹¹⁷⁷. This enhances NO production and inhibits sodium transport. We measured 1) NO production by fluorescence microscopy using DAF2-DA, 2) Akt activity using a fluorescence resonance energy transfer-based Akt reporter, 3) phosphorylated NOS3 and Akt by Western blotting, and 4) NKCC2 activity by fluorescence microscopy. In isolated THAL, endothelin-1 (1 nmol/liter) increased NO production from 0.23 ± 0.24 to 2.81 ± 0.32 fluorescence units/min (p < 0.001; n = 5) but failed to stimulate NO production in THALs isolated from NOS3^–/– mice. Wortmannin (150 nmol/liter), a PI3K inhibitor, reduced endothelin-1-stimulated NO by 83% (0.49 ± 0.13 versus 3.31 ± 0.49 fluorescence units/min for endothelin-1 alone; p < 0.006; n = 5). Endothelin-1 stimulated Akt activity by 0.16 ± 0.02 arbitrary units as measured by fluorescence resonance energy transfer (p < 0.001; n = 5) and increased phosphorylation of Akt at Ser⁴⁷³ by 56 ± 11% (p < 0.002; n = 7). Dominant-negative Akt blocked endothelin-1-induced NO by 60 ± 8% (p < 0.001 versus control; n = 6), and an Akt inhibitor had a similar effect. Endothelin-1 increased phosphorylation of NOS3 at Ser¹¹⁷⁷ by 89 ± 24% (p < 0.01; n = 7) but had no effect on Ser⁶³³. Endothelin-1 inhibited NKCC2 activity, an effect that was blocked by dominant-negative Akt and NOS inhibition. We conclude that endothelin-1 stimulates THAL NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser¹¹⁷⁷. This enhances NO production and inhibits sodium transport.Nitric oxide (NO) augments salt and water excretion by the kidney (1–6). NO produced by both NOS1 and NOS3 (neuronal and endothelial NOS²) contributes to this effect (7–9). Endothelin-1 appears to be one factor that stimulates NO production by both enzymes in the kidney (7–10). Inhibition of endothelin-induced NOS activation can cause salt-sensitive hypertension (6). The thick ascending limb reabsorbs ∼30% of the filtered NaCl, and improper regulation of sodium reabsorption by this segment has been implicated in salt-sensitive hypertension (11, 12). Thus, studying the effects of endothelin-1 on the thick ascending limb is physiologically significant.Endothelin-1 inhibits thick ascending limb NaCl reabsorption via stimulation of NO (9). NO has been shown to inhibit apical Na⁺-K⁺-2Cl^– co-transport (NKCC2) (13), the main route for sodium entry in this segment and the first step in NaCl absorption (14, 15). The thick ascending limb expresses all three NOS isoforms. The actions of endothelin-1 are likely due to NOS3 activation because 1) this isoform is responsible for regulating thick ascending limb NaCl reabsorption (8), and 2) endothelin-1 stimulates NOS3 expression in the thick ascending limb (16). However, whether endothelin-1 acutely stimulates NO production via NOS3 activation in the thick ascending limb is uncertain.NOS3 can be activated by several signaling pathways, including those that involve Ca²⁺/calmodulin and phosphatidylinositol 3-kinase (PI3K). In endothelial cells, both pathways are important. However, in the thick ascending limb, only the latter has been shown to activate NOS3 (17, 18). Thus, the signaling cascades that activate NOS3 in the thick ascending limb and endothelial cells likely differ (19). The mechanisms by which endothelin-1 stimulates NOS3 and inhibits sodium transport in this segment are unknown. We hypothesized that endothelin-1 stimulates thick ascending limb NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser¹¹⁷⁷. This enhances NO production and inhibits sodium transport.