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 n
m
) 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/μg (
p
< 0.01). Upon adding the AT
2
antagonist PD123319 (1 μ
m
), 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 μ
m
) resulted in Ang II stimulating NO by 0.9 ± 0.1 fluorescence units/min. Akt inhibitor (5 μ
m
) 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.