Angiotensin II Stimulates Thick Ascending Limb NO Production via AT2 Receptors and Akt1-dependent Nitric-oxide Synthase 3 (NOS3) Activation

Abstract

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. The thick ascending limb (TAL)2 of the loop of Henle is the diluting segment of the renal nephron. It reabsorbs ϳ30% of filtered NaCl and generates the osmotic gradient necessary for water reabsorption by the collecting duct. Enhanced Na ϩ reabsorption by this segment has been implicated in the development of hypertension (1, 2). The TAL cells produce NO. Endogenously produced NO inhibits Na ϩ reabsorption by the TAL (3, 4). Inappropriate NO bioavailability can lead to enhanced TAL Na ϩ reabsorption, Na ϩ retention, and hypertension. Thus, understanding the signaling cascade leading to NO production by TALs could help understand the pathophysiology of hypertension and identify new targets for its treatment.In the TAL, NO is stimulated by a number of factors including angiotensin II (Ang II) (5). Ang II can activate two different receptors: type 1 (AT 1 ) or type 2 (AT 2 ). In the vasculature, activation of AT 1 leads to vasoconstriction, resulting in increased renal vascular resistance (6). In contrast, activation of AT 2 in endothelial cells stimulates NO release (7). Similarly, in the kidney, activation of AT 1 mediates the salt-retaining and prohypertensive actions of Ang II (8 -12), whereas activation of AT 2 leads to natriuresis via activation of the NO/cGMP signaling pathway (13, 14). To our knowledge, the receptor that mediates the stimulatory actions of Ang II on NO production by the TAL has not yet been identified.In the TAL, NO is produced by nitric-oxide synthase 3 (NOS3 or eNOS) (15, 16). NOS3 can be activated by several signaling pathways, including those involving Ca 2ϩ /calmodulin (17) and Akt-dependent phosphorylation of NOS3 (18,19). In endothelial cells, both pathways are important. However, in epithelial cells, activation of Akt appears to be the main mechanism (20). Three Akt isoforms have been identified: Akt1, -2, and -3. In endothelial cells, Akt1 directly phosphorylates NOS3 at serine 1177, heightening enzyme activity and NO production (18,(21)(22)(23)(24)(25). In the TAL, activation of Akt has been shown to participate in the activation of NOS3 by different stimuli (16,20,26,27,28). However, the mechanism by which Ang II stimulates NO in the TAL has not been investigated to our knowledge. We hypothesized that Ang II stimulates TAL NO production via activation of AT 2 and Akt1, phosphorylating NOS3 at serine 1177 and enhancing NO production. EXPERIMENTAL PROCEDURES

Angiotensin II Stimulates Thick Ascending Limb Superoxide Production via Protein Kinase Cα-dependent NADPH Oxidase Activation

“…production in human platelets, and this effect was blunted by an AT 1 receptor antagonist. Although we recently reported that Ang II acts on AT 2 receptors to activate other signaling events in the TAL (34), in the present study the AT 2 antagonist PD123319 had no effect on Ang II-stimulated O 2 . production, suggesting that AT 2 receptors do not play a role in AT 1 -stimulated O 2 .…”

Dietary salt intake modulates differential splicing of the Na-K-2Cl cotransporter NKCC2

“…In view of the results obtained during the high-and low-salt diets, we supposed that angiotensin II might be involved in regulating the differential splicing of NKCC2. Both the AT 1 and AT 2 receptors have been shown to be expressed in the TAL and to be relevant for TAL function (14,15,24,30). Our experiments showed that chronic angiotensin II infusion led to a shift in the expression of NKCC2 from the A isoform to the B isoform similar to that which occurred for dietary salt restriction, and this regulation was most obvious in the renal cortex.…”

Antihypertensive therapy increases tetrahydrobiopterin levels and NO/cGMP signaling in small arteries of angiotensin II-infused hypertensive rats

“…Phosphorylation at Ser1177 of NOS3 is regulated by Akt kinase activation (13,28,36), while ANG II increases Akt phosphorylation at Ser473 in renal thick ascending limb (15). Thus we analyzed total and phosphorylated Akt at Ser473 in small mesenteric arteries from NORM and ANG rats (n ϭ 7; Fig.…”