Akt1 mediates purinergic-dependent NOS3 activation in thick ascending limbs

Abstract

Silva GB, Garvin JL. Akt1 mediates purinergic-dependent NOS3 activation in thick ascending limbs. Am J Physiol Renal Physiol 297: F646 -F652, 2009. First published July 1, 2009 doi:10.1152/ajprenal.00270.2009.-Extracellular ATP regulates many physiological processes via release of nitric oxide (NO). ATP stimulates NO in thick ascending limbs (TALs), but the signaling cascade involved in the cells of this nephron segment, as well as many other types of cells, is poorly understood. We hypothesized that ATP enhances NO synthase (NOS) activity by stimulating PI3 kinase and Akt. We measured 1) NO in TALs using the NO-sensitive dye DAF-2 DA and 2) Akt activity by fluorescence resonance energy transfer and phosphorylation of Akt isoforms. ATP (100 M) stimulated NO in wild-type mice [26 Ϯ 4 arbitrary units (AU)], but not in NOS3 Ϫ/Ϫ mice (2 Ϯ 2 AU; P Ͻ 0.04). In the presence of the NOS1-and NOS2-selective inhibitors 7-NI and 1400W, ATP stimulated NO by 30 Ϯ 2 and 33 Ϯ 3 AU, respectively (not significant vs. control). In the presence of the PI3 kinase inhibitor LY294002, ATP-increased NO was reduced by 85% (5 Ϯ 2 vs. 28 Ϯ 4 AU; P Ͻ 0.02). ATP alone increased Akt activity and this effect was significantly blocked by suramin, a P2 receptor antagonist. In the presence of an Akt-selective inhibitor, ATP-induced NO was blocked by 90 Ϯ 4%. ATP significantly stimulated Akt1 phosphorylation at Ser 473 by 91 Ϯ 13%, whereas Akt2 phosphorylation remained unchanged and Akt3 phosphorylation decreased. In vivo transduction of TALs with a dominantnegative Akt1 significantly decreased ATP-induced NO by 88 Ϯ 6%. We concluded that ATP increases NOS3-derived NO via Akt1 activation in the TAL. purinergic signaling; adenosine triphosphate; Na-K-2Cl cotransporter IN THE KIDNEY, ADENOSINE triphosphate (ATP) is released from several types of cells. Macula densa cells release ATP in response to an increase in luminal NaCl (19), while mesangial cells release ATP in response to elevated glucose (35). The cells that constitute the thick ascending limb release ATP in response to decreases in osmolality (34) and increases in luminal flow (16). The ATP released from these and other cells has pleotrophic effects. In vivo studies showed that extracellular ATP inhibits fluid absorption (1). In the collecting duct extracellular ATP also decreases Na (32) and water (18) reabsorption. Increases in ATP in the renal interstitium correlate with decreases in renal blood flow (26), most likely related to afferent arterial autoregulation (14). Moreover, we recently demonstrated that extracellular ATP stimulates nitric oxide (NO) by thick ascending limbs via activation of purinergic 2X (P2X) receptors (33). Indeed, ATP has been shown to stimulate NO production in a variety of cells, including platelets (20), endothelial cells (37), and cells of the central nervous system (31). However, the mechanisms by which ATP stimulates NO production are poorly understood.Activation of P2X receptors may lead to stimulation of several signaling cascades. These include glycoge...

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

“…The fact that phosphoserine 1177 remained elevated at 10 min, whereas phosphoserine 633 was already back to basal levels, may indicate that phosphorylation at serine 633 is required during early stages of the signaling event. The fact that Ang II enhances phosphorylation of NOS3 at positive regulatory sites in conjunction with our previous data (16,28), indicate that NOS3 is the NOS isoform activated by Ang II.…”

“…In addition, luminal flow activates the PI3K/Akt path- way, stimulating NO production in isolated TALs (27). More recently, it has been reported that ATP stimulates TAL NO production upon activation of Akt1 (28). From these studies and the current work, we concluded that Akt1 is the primary mechanism by which Ang II activates NOS3 to stimulate NO production in the TAL.…”

“…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.…”

Purinergic signalling in the kidney in health and disease

“…AQP1, AQP2, AQP3, AQP4 Aquaporin water channel isoforms (reproduced from [189] with permission from Springer) it was argued that the response was mediated primarily by P2X receptors. Further studies from the same group have defined NOS3 (eNOS) as the target for P2 receptor activation [338], in a process requiring activation of Akt1 (serine threonine kinase; aka PKB).…”

Angiotensin II stimulates superoxide production in the thick ascending limb by activating NOX4

“…Separate TAL suspensions from the adenovirus-injected left kidney and noninjected right kidney were prepared as described previously (79). Total RNA was isolated from the suspensions using an RNeasy Plus mini kit (Qiagen, Valencia, CA) and subsequently used for RT-PCR analysis using a OneStep RT-PCR kit (Qiagen) according to the manufacturer's instructions.…”