Ang II Type 1 Receptor Signaling Research Articles

Angiotensin II Induces Oxidative Stress and Endothelial Dysfunction in Mouse Ophthalmic Arteries via Involvement of AT1 Receptors and NOX2.

Angiotensin II (Ang II) has been implicated in the pathophysiology of various age-dependent ocular diseases. The purpose of this study was to test the hypothesis that Ang II induces endothelial dysfunction in mouse ophthalmic arteries and to identify the underlying mechanisms. Ophthalmic arteries were exposed to Ang II in vivo and in vitro to determine vascular function by video microscopy. Moreover, the formation of reactive oxygen species (ROS) was quantified and the expression of prooxidant redox genes and proteins was determined. The endothelium-dependent artery responses were blunted after both in vivo and in vitro exposure to Ang II. The Ang II type 1 receptor (AT1R) blocker, candesartan, and the ROS scavenger, Tiron, prevented Ang II-induced endothelial dysfunction. ROS levels and NOX2 expression were increased following Ang II incubation. Remarkably, Ang II failed to induce endothelial dysfunction in ophthalmic arteries from NOX2-deficient mice. Following Ang II incubation, endothelium-dependent vasodilation was mainly mediated by cytochrome P450 oxygenase (CYP450) metabolites, while the contribution of nitric oxide synthase (NOS) and 12/15-lipoxygenase (12/15-LOX) pathways became negligible. These findings provide evidence that Ang II induces endothelial dysfunction in mouse ophthalmic arteries via AT1R activation and NOX2-dependent ROS formation. From a clinical point of view, the blockade of AT1R signaling and/or NOX2 may be helpful to retain or restore endothelial function in ocular blood vessels in certain ocular diseases.

Cardioprotective Angiotensin-(1-7) Peptide Acts as a Natural-Biased Ligand at the Angiotensin II Type 1 Receptor.

Hyperactivity of the renin-angiotensin-aldosterone system through the angiotensin II (Ang II)/Ang II type 1 receptor (AT1-R) axis constitutes a hallmark of hypertension. Recent findings indicate that only a subset of AT1-R signaling pathways is cardiodeleterious, and their selective inhibition by biased ligands promotes therapeutic benefit. To date, only synthetic biased ligands have been described, and whether natural renin-angiotensin-aldosterone system peptides exhibit functional selectivity at AT1-R remains unknown. In this study, we systematically determined efficacy and potency of Ang II, Ang III, Ang IV, and Ang-(1-7) in AT1-R-expressing HEK293T cells on the activation of cardiodeleterious G-proteins and cardioprotective β-arrestin2. Ang III and Ang IV fully activate similar G-proteins than Ang II, the prototypical AT1-R agonist, despite weaker potency of Ang IV. Interestingly, Ang-(1-7) that binds AT1-R fails to promote G-protein activation but behaves as a competitive antagonist for Ang II/Gi and Ang II/Gq pathways. Conversely, all renin-angiotensin-aldosterone system peptides act as agonists on the AT1-R/β-arrestin2 axis but display biased activities relative to Ang II as indicated by their differences in potency and AT1-R/β-arrestin2 intracellular routing. Importantly, we reveal Ang-(1-7) a known Mas receptor-specific ligand, as an AT1-R-biased agonist, selectively promoting β-arrestin activation while blocking the detrimental Ang II/AT1-R/Gq axis. This original pharmacological profile of Ang-(1-7) at AT1-R, similar to that of synthetic AT1-R-biased agonists, could, in part, contribute to its cardiovascular benefits. Accordingly, in vivo, Ang-(1-7) counteracts the phenylephrine-induced aorta contraction, which was blunted in AT1-R knockout mice. Collectively, these data suggest that Ang-(1-7) natural-biased agonism at AT1-R could fine-tune the physiology of the renin-angiotensin-aldosterone system.

Angiotensin II Stimulation of DPP4 Activity Regulates Megalin in the Proximal Tubules.

Proteinuria is a marker of incipient kidney injury in many disorders, including obesity. Previously, we demonstrated that megalin, a receptor endocytotic protein in the proximal tubule, is downregulated in obese mice, which was prevented by inhibition of dipeptidyl protease 4 (DPP4). Obesity is thought to be associated with upregulation of intra-renal angiotensin II (Ang II) signaling via the Ang II Type 1 receptor (AT1R) and Ang II suppresses megalin expression in proximal tubule cells in vitro. Therefore, we tested the hypothesis that Ang II will suppress megalin protein via activation of DPP4. We used Ang II (200 ng/kg/min) infusion in mice and Ang II (10−8 M) treatment of T35OK-AT1R proximal tubule cells to test our hypothesis. Ang II-infused mouse kidneys displayed increases in DPP4 activity and decreases in megalin. In proximal tubule cells, Ang II stimulated DPP4 activity concurrent with suppression of megalin. MK0626, a DPP4 inhibitor, partially restored megalin expression similar to U0126, a mitogen activated protein kinase (MAPK)/extracellular regulated kinase (ERK) kinase kinase (MEK) 1/2 inhibitor and AG1478, an epidermal growth factor receptor (EGFR) inhibitor. Similarly, Ang II-induced ERK phosphorylation was suppressed with MK0626 and Ang II-induced DPP4 activity was suppressed by U0126. Therefore, our study reveals a cross talk between AT1R signaling and DPP4 activation in the regulation of megalin and underscores the significance of targeting DPP4 in the prevention of obesity related kidney injury progression.

Assessing the Relationship of Angiotensin II Type 1 Receptors with Erythropoietin in a Human Model of Endogenous Angiotensin II Type 1 Receptor Antagonism

Hypothesis/Introduction: Angiotensin II (Ang II) has been shown to control erythropoietin (EPO) synthesis as Ang II type 1 receptor (AT1R) blockers block Ang-II-induced EPO oversecretion. To further explore the involvement of AT1R in processes controlling EPO levels, plasma EPO and mononuclear cell NADPH oxidase 4 (NOX4) - a NOX family member involved in oxygen sensing, which is a process central to controlling EPO levels - were assessed in Bartter's/Gitelman's syndrome (BS/GS) patients, a human model of endogenous AT1R antagonism and healthy subjects. Heme oxygenase (HO)-1, antioxidant and anti-inflammatory factor related to NOX4 activation, and the relationship of EPO and NOX4 to HO-1 were also assessed. Materials and Methods: EPO was measured by chemiluminescent immunoassay, HO-1 by sandwich immunoassay and NOX4 protein expression by Western blot. Results: EPO was increased in BS/GS patients compared to healthy subjects (7.64 ± 2.47 vs. 5.23 ± 1.07 U/l; p = 0.025), whereas NOX4 did not differ between BS/GS and healthy subjects (1.76 ± 0.61 vs. 1.65 ± 0.54 densitometric units; p = n.s.), and HO-1 was increased in BS/GS patients compared to healthy subjects (9.58 ± 3.07 vs. 5.49 ± 1.04 ng/ml; p = 0.003). NOX4 positively correlated with HO-1 only in BS/GS patients; no correlation was found between EPO and either NOX4 or HO-1 in those two groups. Conclusions: The effect of the renin-angiotensin system on EPO cannot be solely mediated by Ang II via AT1R signaling, but rather, EPO levels are also determined by a complex interrelated set of signals that involve AT2R, nitric oxide levels, NOX4 and HO-1 activity.

Pleiotropic Effects of Angiotensin II Receptor Signaling in Cardiovascular Homeostasis and Aging.

Most of the pathophysiological actions of angiotensin II (Ang II) are mediated through the Ang II type 1 (AT1) receptor, a member of the seven-transmembrane G protein-coupled receptor family. Essentially, AT1 receptor signaling is beneficial for organismal survival and procreation, because it is crucial for normal organ development, and blood pressure and electrolyte homeostasis. On the other hand, AT1 receptor signaling has detrimental effects, such as promoting various aging-related diseases that include cardiovascular diseases, diabetes, chronic kidney disease, dementia, osteoporosis, and cancer. Pharmacological or genetic blockade of AT1 receptor signaling in rodents has been shown to prevent the progression of aging-related phenotypes and promote longevity. In this way, AT1 receptor signaling exerts antagonistic and pleiotropic effects according to the ages and pathophysiological conditions. Here we review the pleiotropic effects of AT1 receptor signaling in cardiovascular homeostasis and aging.

The Physiology and Pathophysiology of a Novel Angiotensin Receptor-binding Protein ATRAP/Agtrap

The Ang II type 1 receptor (AT1R)-associated protein (ATRAP/Agtrap) is a molecule specifically interacting with the carboxyl- terminal domain of AT1R. The results of in vitro studies showed that ATRAP suppresses Ang II-mediated pathological responses in cardiovascular cells by promoting AT1R internalization. With respect to the tissue distribution and regulation of ATRAP expression in vivo, ATRAP is broadly expressed in many tissues as is AT1R. Accumulating evidence indicates that a tissue-specific regulatory balancing of ATRAP and AT1R expression may be involved in the modulation of AT1R signaling at local tissue sites and also in the pathophysiology of hypertension and its associated end-organ injury. Furthermore, the activation of ATRAP in transgenic-models inhibited inflammatory vascular remodeling and cardiac hypertrophy in response to Ang II stimulation. These results suggest the clinical potential benefit of an ATRAP activation strategy in the treatment of hypertension and related organ injury.

β-Arrestin-biased AT1R stimulation promotes cell survival during acute cardiac injury

Pharmacological blockade of the ANG II type 1 receptor (AT1R) is a common therapy for treatment of congestive heart failure and hypertension. Increasing evidence suggests that selective engagement of β-arrestin-mediated AT1R signaling, referred to as biased signaling, promotes cardioprotective signaling. Here, we tested the hypothesis that a β-arrestin-biased AT1R ligand TRV120023 would confer cardioprotection in response to acute cardiac injury compared with the traditional AT1R blocker (ARB), losartan. TRV120023 promotes cardiac contractility, assessed by pressure-volume loop analyses, while blocking the effects of endogenous ANG II. Compared with losartan, TRV120023 significantly activates MAPK and Akt signaling pathways. These hemodynamic and biochemical effects were lost in β-arrestin-2 knockout (KO) mice. In response to cardiac injury induced by ischemia reperfusion injury or mechanical stretch, pretreatment with TRV120023 significantly diminishes cell death compared with losartan, which did not appear to be cardioprotective. This cytoprotective effect was lost in β-arrestin-2 KO mice. The β-arrestin-biased AT1R ligand, TRV120023, has cardioprotective and functional properties in vivo, which are distinct from losartan. Our data suggest that this novel class of drugs may provide an advantage over conventional ARBs by supporting cardiac function and reducing cellular injury during acute cardiac injury.

Prepubertal angiotensin blockade exerts long-term therapeutic effect through sustained ATRAP activation in salt-sensitive hypertensive rats

We previously showed that the molecule interacting with Ang II type 1 receptor (AT1R), ATRAP, promotes AT1R internalization and attenuates AT1R-mediated pathological responses. In this study we examined whether the regulation of renal ATRAP expression is related to the development of salt-induced hypertension and renal injury as well as to the beneficial effects of AT1R blockade. Dahl Iwai salt-sensitive hypertensive and Dahl Iwai salt-resistant rats were divided into six groups for the administration of vehicle or olmesartan either continuously from 3 to 16 weeks, or transiently from weaning to puberty (3-10 weeks), and fed high salt diet from 6 to 16 weeks. In Dahl Iwai salt-sensitive rats, not only continuous, but also prepubertal olmesartan treatment improved hypertension at 15 weeks. Renal ATRAP expression was suppressed in vehicle-treated Dahl Iwai salt-sensitive rats, concomitant with up-regulation of renal oxidative stress, inflammation and fibrosis-related markers such as p22phox, TGF-β, fibronectin, monocyte chemotactic protein-1 and type 1 collagen. However, prepubertal as well as continuous olmesartan treatment recovered the suppressed renal ATRAP expression and inhibited the renal activation of p22phox, TGF-β, fibronectin, MCP-1 and type 1 collagen. In Dahl Iwai salt-resistant rats, such suppression of renal ATRAP expression or induction of renal pathological responses by salt loading was not observed. These results indicate that prepubertal transient blockade of AT1R signaling exerts a long-term therapeutic effect on salt-induced hypertension and renal injury in Dahl Iwai salt-sensitive rats, partly through a sustained enhancement of renal ATRAP expression, thereby suggesting ATRAP a novel molecular target in salt-induced hypertension and renal injury.

Intrarenal suppression of angiotensin II type 1 receptor binding molecule in angiotensin II-infused mice

ATRAP [ANG II type 1 receptor (AT1R)-associated protein] is a molecule which directly interacts with AT1R and inhibits AT1R signaling. The aim of this study was to examine the effects of continuous ANG II infusion on the intrarenal expression and distribution of ATRAP and to determine the role of AT1R signaling in mediating these effects. C57BL/6 male mice were subjected to vehicle or ANG II infusions at doses of 200, 1,000, or 2,500 ng·kg(-1)·min(-1) for 14 days. ANG II infusion caused significant suppression of ATRAP expression in the kidney but did not affect ATRAP expression in the testis or liver. Although only the highest ANG II dose (2,500 ng·kg(-1)·min(-1)) provoked renal pathological responses, such as an increase in the mRNA expression of angiotensinogen and the α-subunit of the epithelial sodium channel, ANG II-induced decreases in ATRAP were observed even at the lowest dose (200 ng·kg(-1)·min(-1)), particularly in the outer medulla of the kidney, based on immunohistochemical staining and Western blot analysis. The decrease in renal ATRAP expression by ANG II infusion was prevented by treatment with the AT1R-specific blocker olmesartan. In addition, the ANG II-mediated decrease in renal ATRAP expression through AT1R signaling occurred without an ANG II-induced decrease in plasma membrane AT1R expression in the kidney. On the other hand, a transgenic model increase in renal ATRAP expression beyond baseline was accompanied by a constitutive reduction of renal plasma membrane AT1R expression and by the promotion of renal AT1R internalization as well as the decreased induction of angiotensinogen gene expression in response to ANG II. These results suggest that the plasma membrane AT1R level in the kidney is modulated by intrarenal ATRAP expression under physiological and pathophysiological conditions in vivo.

The renin-angiotensin system mediates hyperoxia-induced collagen production in human lung fibroblasts

A high concentration of oxygen can cause lung injury and lead to pulmonary fibrosis. Angiotensin (Ang) II induces human lung fibroblast proliferation and stimulates collagen synthesis. However, the role of the renin-angiotensin system (RAS) in the pathogenesis of hyperoxia-induced collagen production is unclear. The aims of this study were to investigate the effects of hyperoxia on the components of the RAS and collagen expression in human lung fibroblasts (MRC-5). Hyperoxia increased total collagen, collagen type I, and α-smooth muscle actin (α-SMA) mRNA and protein expression. RAS components and Ang II production were also significantly increased after hyperoxic exposure. Hyperoxia induced Ang II type 1 receptor (AT1R) expression but did not alter AT2R expression, furthermore, silencing of AT1R signaling with small interfering RNA suppressed hyperoxia-induced phosphorylated-ERK (p-ERK) 1/2, α-SMA, and collagen type I expression. Ang II increased p-ERK 1/2 and collagen type I expression, and these increases were inhibited by the AT1R inhibitor, losartan, but not by the AT2R inhibitor, PD123319 under both normoxic and hyperoxic conditions. These data suggest Ang II-mediated signaling transduction via AT1R is involved in hyperoxia-induced collagen synthesis in human lung fibroblasts.

Regulation of angiotensin converting enzyme II by angiotensin peptides in human cardiofibroblasts

Numerous studies have suggested that angiotensin peptides modulate the expression of angiotensin converting enzyme II (ACE2) in the cardiovascular system, but the molecular mechanisms remain poorly understood. In the present study, human cardiac fibroblasts (HCF) were used to test the regulatory effects of angiotensin II (Ang II) and angiotensin-(1-7) [Ang-(1-7)] on ACE2 expression. The results show that Ang II upregulates ACE2 expression. This action is modulated through activation of Ang II type 1 receptor (AT1R). Ang II-mediated ACE2 upregulation was blocked by antagonists of AT1R and ERK-MAPK signaling pathways. Additionally, Ang-(1-7) increased ACE2 expression, and this upregulation was inhibited by Ang-(1-7) Mas receptor blockade. Our results further reveal that the activation of p-ERK1/2 proteins plays a critical role in upregulating ACE2 in Ang-(1-7)-stimulated HCF cells. This effect occurs independently of the Ang II-AT1R signaling pathway. In conclusion, we propose that Ang II-upregulated ACE2 may increase Ang-(1-7) formation from Ang II, and that ACE2 expression is further enhanced by Ang-(1-7) in a positive feedback loop.

Abstract 1363: Novel Role of Early Endosome Antigen 1 in Reactive Oxygen Species-dependent AT1 Receptor-mediated Signaling Linked to Hypertrophy in Vascular Smooth Muscle Cells

Angiotensin II (Ang II) stimulates hypertrophy in vascular smooth muscle cells (VSMCs) primarily through the G protein-coupled receptor (GPCR) Ang II type 1 receptor (AT1R) at the cell membrane and after its internalization. Major outputs of AT1R leading to vascular hypertrophy are triggered by reactive oxygen species (ROS)-dependent transactivation of the EGF receptor (EGFR), followed by activation of p38MAPK, Akt, p70S6K, and ERK1/2. However, underlying regulatory mechanisms are largely unknown. The small GTPase Rab5 and its downstream effector early endosome antigen 1 (EEA1) regulate endosomal internalization of GPCR. We hypothesized that EEA1 could be a scaffold facilitating the multiple outputs of AT1R signaling including those involved in hypertrophy. We found that Ang II (0.1 μ M, 5 min) and H2O2 (100 μ M, 5 min) induce formation of a complex composed of EEA1, Rab5, AT1R and EGFR that is inhibited by PEG-catalase (100 U/ml) and PEG-superoxide dismutase (SOD, 200 U/ml), inferring the redox sensitivity of the process. To explore this possibility further, we showed that Ang II and H2O2 stimulate EEA1 threonine phosphorylation (3.0- and 2.0-fold, respectively) co-temporaneously with Ang II- and H2O2-induced complex formation. Inhibition of p38MAPK by inhibitors (SB202190, PD169316; 1 μ M) or treatment with PEG-catalase and PEG-SOD prevents EEA1 phosphorylation and its recruitment to the complex. To gain further insight, we used EEA1 siRNA to knock down EEA1 (80%). EEA1 siRNA significantly inhibits (79% control) Ang II-stimulated [3H]leucine incorporation in VSMC and is associated with decrease in Akt phosphorylation and its downstream p70S6K (80% and 50%, respectively) without affecting ROS-independent Erk1/2 phosphorylation. EEA1 siRNA does not affect Ang II-induced EGFR phosphorylation. Conclusion: ROS- and p38MAPK-dependent Ang II-induced EEA1 phosphorylation is involved in formation of a signaling complex (AT1R, EGFR, EEA1, Rab5) that is related to Ang II stimulated, AT1R- and EGFR-mediated vascular hypertrophy. Akt-dependent phosphorylation of the p70S6K kinase is also EEA1-dependent. These findings provide insight into a novel role for EEA1 in Ang II signaling in VSMC hypertrophy.

Mechanisms of angiotensin II signaling on cytoskeleton of podocytes

Podocytes are significant in establishing the glomerular filtration barrier. Sustained rennin-angiotensin system (RAS) activation is crucial in the pathogenesis of podocyte injury and causes proteinuria. This study demonstrates that angiotensin II (Ang II) caused a reactive oxygen species (ROS)-dependent rearrangement of cortical F-actin and a migratory phenotype switch in cultured mouse podocytes with stable Ang II type 1 receptor (AT1R) expression. Activated small GTPase Rac-1 and phosphorylated ezrin/radixin/moesin (ERM) proteins provoked Ang II-induced F-actin cytoskeletal remodeling. This work also shows increased expression of Rac-1 and phosphorylated ERM proteins in cultured podocytes, and in glomeruli of podocyte-specific AT1R transgenic rats (Neph-hAT1 TGRs). The free radical scavenger DMTU eliminated Ang II-induced cell migration, ERM protein phosphorylation and cortical F-actin remodeling, indicating that ROS mediates the influence of Rac-1 on podocyte AT1R signaling. Heparin, a potent G-coupled protein kinase 2 inhibitor, was found to abolish ERM protein phosphorylation and cortical F-actin ring formation in Ang II-treated podocytes, indicating that phosphorylated ERM proteins are the cytoskeletal effector in AT1R signaling. Moreover, Ang II stimulation triggered down-regulation of alpha actinin-4 and reduced focal adhesion expression in podocytes. Signaling inhibitor assay of Ang II-treated podocytes reveals that Rac-1, RhoA, and F-actin reorganization were involved in expressional regulation of alpha actinin-4 in AT1R signaling. With persistent RAS activation, the Ang II-induced phenotype shifts from being dynamically stable to adaptively migratory, which may eventually exhaust podocytes with a high actin cytoskeletal turnover, causing podocyte depletion and focal segmental glomerulosclerosis.

Angiotensin II Type 1 Receptor Gene Regulation

Angiotensin II (Ang II), the major bioactive peptide of the renin–angiotensin system, was originally described as a potent vasoconstrictor. It is now recognized as a multifunctional hormone influencing many cellular processes, including cell growth, apoptosis, migration, inflammation, and fibrosis.1,2 Under pathological conditions, the vasoconstrictor, mitogenic, proinflammatory, and profibrotic actions of Ang II contribute to altered vascular tone, structural remodeling, and endothelial dysfunction.3–6 Thus, Ang II plays a key role in the pathogenesis of cardiovascular diseases. The biological responses of Ang II are mediated by its interaction with 2 distinct high-affinity G protein–coupled receptors now designated Ang II type 1 receptor (AT1R) and Ang II type 2 receptor.7 Most of the known physiological and pathophysiological effects of Ang II are mediated via the AT1R.1–7 This receptor subtype is expressed in cardiovascular-relevant cell types including vascular smooth muscle cells (VSMCs), endothelial cells, cardiac myocytes, cardiac fibroblasts, and renal mesangial cells.7 The multiple actions of Ang II, mediated through the AT1R, are a result of complex intracellular signaling pathways including stimulation of the phospholipase C/inositol 1,4,5-trisphosphate/diacylglycerol cascade, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases, tyrosine kinases, and RhoA/Rho kinase.1,2,4,6,7 In addition, AT1Rs mediate many of their pathophysiological effects by stimulating reactive oxygen species generation via an reduced nicotinamide-adenine dinucleotide/reduced nicotinamide-adenine dinucleotide phosphate oxidase-dependent mechanism.2,8 Reactive oxygen species, in turn, influences downstream signaling molecules, including transcription factors, tyrosine kinases/phosphatases, Ca2+ channels, and MAPKs.2,8 The expression level of the AT1R defines the biological efficacy of Ang II; hence, overexpression of the AT1R is one potential mechanism by which Ang II can contribute to cardiovascular disease. Importantly, aberrant expression of the AT1R has been shown to have pathophysiological relevance in cell culture, animal studies, …

Caveolin-1 Is Essential for Activation of Rac1 and NAD(P)H Oxidase After Angiotensin II Type 1 Receptor Stimulation in Vascular Smooth Muscle Cells

Angiotensin II (Ang II) is a potent mediator of vascular hypertrophy in vascular smooth muscle cells (VSMCs). These effects are mediated through the Ang II type 1 receptor (AT1R) and require its trafficking through caveolin-1 (Cav1)-enriched lipid rafts and reactive oxygen species (ROS) derived from Rac1-dependent NAD(P)H oxidase. The specific role(s) of Cav1 in AT1R signaling is incompletely understood. Knockdown of Cav1 protein by small interfering RNA (siRNA) inhibits Ang II-stimulated Rac1 activation and membrane translocation, H2O2 production, ROS-dependent epidermal growth factor receptor (EGF-R) transactivation, and subsequent phosphorylation of Akt without affecting ROS-independent extracellular signal-regulated kinase 1/2 phosphorylation. Ang II stimulates tyrosine phosphorylation of Sos-1, a Rac-guanine nucleotide exchange factor, which is inhibited by Cav1 siRNA, demonstrating involvement of Cav1 in Rac1 activation. Detergent-free fractionation showed that EGF-Rs are found basally in Cav1-enriched lipid raft membranes and associate with Cav1. Ang II stimulates AT1R movement into these microdomains contemporaneously with the egress of EGF-R. Both aspects of this bidirectional receptor trafficking are inhibited by Cav1 siRNA. Moreover, Cav1 siRNA inhibits Ang II-induced vascular hypertrophy. Cav1 plays an essential role in AT1R targeting into Cav1-enriched lipid rafts and Rac1 activation, which are required for proper organization of ROS-dependent Ang II signaling linked to VSMC hypertrophy.

Microtubules regulate angiotensin II type 1 receptor and Rac1 localization in caveolae/lipid rafts: role in redox signaling.

Microtubules are important in signal transduction temporal-spatial organization. Full expression of angiotensin II (Ang II) signaling in vascular smooth muscle cells (VSMCs) is dependent on the reactive oxygen species (ROS) derived from nicotinamide-adenine dinucleotide phosphate (NAD(P)H) oxidase and the dynamic association of the Ang II type 1 receptor (AT1R) with caveolae/lipid rafts. Translocation of the small GTPase Rac1 to the plasma membrane is an essential step for activation of NAD(P)H oxidase; however, its precise localization in the plasma membrane after agonist stimulation and how it is targeted are unknown. We hypothesized that microtubules are involved in regulating multiphasic Ang II signaling events in VSMC. We show that Ang II promotes Rac1 and AT1R trafficking into caveolae/lipid rafts, which is blocked by disruption of microtubules with nocodazole. As a consequence, nocodazole significantly inhibits Ang II-stimulated H2O2 production, its downstream ROS-dependent epidermal growth factor receptor transactivation, Akt phosphorylation, and vascular hypertrophy without affecting Rac1 activation or ROS-independent extracellular signal-regulated kinase 1/2 phosphorylation. These results suggest that proper Rac1 and AT1R trafficking into caveolae/lipid rafts requires the integrity of microtubules and provide insight into the essential role of microtubules for the spatial-temporal organization of ROS-dependent and caveolae/lipid rafts-dependent AT(1)R signaling linked to vascular hypertrophy.