Abstract
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.
Highlights
Duction by both enzymes in the kidney [7,8,9,10]
We first investigated whether endothelin-1 increases Nitric oxide (NO) via NOS3 activation by measuring changes in DAF2 fluorescence in thick ascending limbs isolated from wild-type and NOS3Ϫ/Ϫ mice
We found that 1 nmol/liter endothelin-1 increased NO production by 2.81 Ϯ 0.32 fluorescence units/ min, whereas vehicle had no effect
Summary
Duction by both enzymes in the kidney [7,8,9,10]. Inhibition of endothelin-induced NOS activation can cause salt-sensitive hypertension [6]. We hypothesized that endothelin-1 stimulates thick ascending limb NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser1177. We first investigated whether endothelin-1 increases NO via NOS3 activation by measuring changes in DAF2 fluorescence in thick ascending limbs isolated from wild-type and NOS3Ϫ/Ϫ mice.
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