Angiotensin II stimulates thick ascending limb NO production via AT(2) receptors and Akt1-dependent nitric-oxide synthase 3 (NOS3) activation - PubMed (original) (raw)

Angiotensin II stimulates thick ascending limb NO production via AT(2) receptors and Akt1-dependent nitric-oxide synthase 3 (NOS3) activation

Marcela Herrera et al. J Biol Chem. 2010.

Abstract

Angiotensin II (Ang II) acutely stimulates thick ascending limb (TAL) NO via an unknown mechanism. In endothelial cells, activation of Ang II type 2 receptor (AT(2)) stimulates NO. Akt1 activates NOS3 by direct phosphorylation. We hypothesized that Ang II stimulates TAL NO production via AT(2)-mediated Akt1 activation, which phosphorylates NOS3 at serine 1177. We measured NO production by fluorescence microscopy. In isolated TALs, Ang II (100 nm) increased NO production by 1.1 +/- 0.2 fluorescence units/min (p < 0.01). Ang II increased cGMP accumulation by 4.9 +/- 1.3 fmol/microg (p < 0.01). Upon adding the AT(2) antagonist PD123319 (1 microm), Ang II failed to stimulate NO (0.1 +/- 0.1 fluorescence units/min; p < 0.001 versus Ang II); adding the AT(1) antagonist losartan (1 microm) resulted in Ang II stimulating NO by 0.9 +/- 0.1 fluorescence units/min. Akt inhibitor (5 microm) blocked Ang II-stimulated NO (-0.1 +/- 0.2 fluorescence units/min versus inhibitor alone). Phospho-Akt1 increased by 72% after 5 min (p < 0.006), returning to basal after 10 min. Phospho-Akt2 did not change after 5 min but increased by 115 and 163% after 10 and 15 min (p < 0.02). Phospho-Akt3 did not change. An AT(2) agonist increased pAkt1 by 78% (p < 0.02), PI3K inhibition blocked this effect. In TALs transduced with dominant negative Akt1, Ang II failed to stimulate NO (0.1 +/- 0.2 fluorescence units/min versus 1.2 +/- 0.2 for controls; p < 0.001). Ang II increased phospho-NOS3 at serine 1177 by 130% (p < 0.01) and 150% after 5 and 10 min (p < 0.02). Ang II increased phosphoNOS3 at serine 633 by 50% after 5 min (p < 0.01). Akt inhibition prevented NOS3 phosphorylation. We concluded that Ang II enhances TAL NO production via activation of AT(2) and Akt1-dependent phosphorylation of NOS3 at serines 1177 and 633.

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Figures

FIGURE 1.

FIGURE 1.

Effect of 100 nm angiotensin II on NO production by isolated thick ascending limbs. A, representative experiment for an angiotensin II- and a vehicle-stimulated tubule, respectively. B, mean data. p < 0.004 versus vehicle (n = 5).

FIGURE 2.

FIGURE 2.

Effect of the angiotensin type 1 (AT1) blocker losartan and the angiotensin type 2 (AT2) blocker PD 123319 on angiotensin II-induced NO production by isolated thick ascending limbs. p < 0.001 versus vehicle or losartan (n = 5–6).

FIGURE 3.

FIGURE 3.

Effect of 5 μm Akt inhibitor (inh) VIII on angiotensin II-stimulated NO production by isolated thick ascending limbs. p < 0.017 versus vehicle (n = 5).

FIGURE 4.

FIGURE 4.

Time-dependent effect of 100 nm angiotensin II on phosphorylation of Akt1 (A), Akt2 (B), and Akt3 (C) (n = 6 for A and C and n = 7 for B). Right, representative Western blots for each panel.

FIGURE 5.

FIGURE 5.

A, effect of the Akt inhibitor VIII (Akt inh, 5 μ

m

) on basal and Ang II-stimulated phosphorylation of Akt1 at serine 473 (n = 4). B, effect of the AT2 receptor agonist (AT2 ago) GCP-42112A on Akt1 phosphorylation. Effect of the PI3K inhibitor wortmannin (wortm, 150 n

m

) on basal and AT2-stimulated Akt1 phosphorylation (n = 6). Right, representative Western blot for each panel.

FIGURE 6.

FIGURE 6.

Effect of 100 nm angiotensin II on NO production by thick ascending limbs transduced in vivo with an adenovirus expressing a scrambled DNA sequence (scr-DNA) or dn-Akt1. p < 0.003 versus scrambled DNA (n = 5–6).

FIGURE 7.

FIGURE 7.

Top, time-dependent effect of 100 n

m

Ang II on NOS3 phosphorylation at serines 1177 (A) and 633 (B) in the absence (dark bars) or presence of 5 μ

m

Akt inhibitor VIII (light bars) (n = 6). Bottom, representative Western blot for each panel.

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