NADPH oxidase-derived superoxide anion mediates angiotensin II-induced pressor effect via activation of p38 mitogen-activated protein kinase in the rostral ventrolateral medulla

The rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, is a central site via which angiotensin II (Ang II) elicits its pressor effect. We tested the hypothesis that NADPH oxidase-derived superoxide anion (O2*-) in the RVLM mediates Ang II-induced pressor response via activation of mitogen-activated protein kinase (MAPK) signaling pathways. Bilateral microinjection of Ang II into the RVLM resulted in an angiotensin subtype 1 (AT1) receptor-dependent phosphorylation of p38 MAPK and extracellular signal-regulated protein kinase (ERK)1/2, but not stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), in the ventrolateral medulla. The Ang II-induced p38 MAPK or ERK1/2 phosphorylation was attenuated by application into the RVLM of a NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), an antisense oligonucleotide that targets against p22phox or p47phox subunit of NADPH oxidase mRNA, or the superoxide dismutase mimetic tempol. DPI or antisense p22phox or p47phox oligonucleotide treatment also attenuated the AT1 receptor-dependent increase in O2*- production in the ventrolateral medulla elicited by Ang II at the RVLM. Functionally, Ang II-elicited pressor response in the RVLM was attenuated by DPI, tempol, or a p38 MAPK inhibitor, SB203580. The AT1 receptor-mediated enhancement of the frequency of glutamate-sensitive spontaneous excitatory postsynaptic currents induced by Ang II in RVLM neurons was also abolished by SB203580. These results suggest that NADPH oxidase-derived O2*- underlies the activation of p38 MAPK or ERK1/2 by Ang II in the ventrolateral medulla. Furthermore, the p38 MAPK signaling pathway may mediate Ang II-induced pressor response via enhancement of presynaptic release of glutamate to RVLM neurons.     

Downregulation of angiotensin subtype 1 receptor in rostral ventrolateral medulla during endotoxemia

We reported recently that an upregulation of the inducible nitric oxide synthase (iNOS) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, is a crucial determinant for the elicitation of cardiovascular depression during experimental endotoxemia. The current study evaluated the hypothesis that a downregulation of the molecular synthesis and functional expression of angiotensin subtype 1 receptor (AT1R) in the RVLM is consequential to this upregulated iNOS. In adult Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of Escherichia coli lipopolysaccharide (15 mg/kg) elicited a reduction, followed by an augmentation and a secondary decrease in sympathetic vasomotor outflow, together with progressive hypotension and bradycardia. There was also a progressive increase in iNOS mRNA and protein level in the ventrolateral medulla. This was followed by a significant downregulation of both mRNA and protein levels of AT1R in the ventrolateral medulla, alongside reduced efficacy of angiotensin II (50 pmol) to induce an increase in systemic arterial pressure, heart rate, or sympathetic vasomotor outflow on unilateral microinjection into the RVLM. Pretreatment with microinjection of a selective iNOS inhibitor, S-methylisothiourea (250 pmol) bilaterally into the RVLM significantly reversed the reduction in both synthesis and activity of AT1R. We conclude that a downregulation of molecular synthesis and functional expression of AT1R in the ventrolateral medulla is consequential to the overproduction of NO through upregulation of iNOS in the RVLM and may underlie the cardiovascular depression that takes place during experimental endotoxemia.     

Sympathoinhibition caused by orally administered telmisartan through inhibition of the AT(1) receptor in the rostral ventrolateral medulla of hypertensive rats

In patients and animals with hypertension, sympathetic nervous system (SNS) activation is present. We have demonstrated that angiotensin II type 1 receptor (AT(1)R)-induced oxidative stress in the rostral ventrolateral medulla (RVLM), a vasomotor center in the brainstem, causes SNS activation in hypertensive rats. The aim of the present study was to determine whether orally administered AT(1)R blockers (ARBs) inhibit SNS activation through an anti-oxidant effect via inhibition of AT(1)R in the RVLM of hypertensive rats and, if so, whether the benefits are class effects of ARBs. Stroke-prone spontaneously hypertensive rats (SHRSPs), a hypertensive model with sympathoexcitation, were divided into four groups: SHRSPs treated with telmisartan (TLM), candesartan (CAN), or hydralazine (HYD) and a vehicle group (VEH). Although systolic blood pressure was reduced in the TLM, CAN and HYD groups to the same level, heart rate, SNS activation and oxidative stress in the RVLM were significantly lower in the TLM group only. The pressor effect caused by the microinjection of angiotensin II into the RVLM and the depressor effect caused by the microinjection of tempol, a superoxide dismutase mimetic, into the RVLM were both significantly smaller in TLM, but not in CAN or HYD. These results suggest that orally administered TLM inhibits SNS activation through an anti-oxidant effect via inhibition of AT(1)R in the RVLM of SHRSPs; these results are also independent of depressor effects and are not class effects of ARBs.     

ERK and p38 MAPK, but not NF-kappaB, are critically involved in reactive oxygen species-mediated induction of IL-6 by angiotensin II in cardiac fibroblasts

We recently reported that angiotensin II (Ang II) induced IL-6 mRNA expression in cardiac fibroblasts, which played an important role in Ang II-induced cardiac hypertrophy in paracrine fashion. The present study investigated the regulatory mechanism of Ang II-induced IL-6 gene expression, focusing especially on reactive oxygen species (ROS)-mediated signaling in cardiac fibroblasts. Ang II increased intracellular ROS in cardiac fibroblasts, and the increase was completely inhibited by the AT-1 blocker candesartan and the NADH/NADPH oxidase inhibitor diphenyleneiodonium (DPI). We first confirmed that antioxidant N-acetylcysteine, superoxide scavenger Tiron, and DPI suppressed Ang II-induced IL-6 expression. Because we observed that exogenous H(2)O(2) also increased IL-6 mRNA, the signaling pathways downstream of Ang II and exogenous H(2)O(2) were compared. Ang II, as well as exogenous H(2)O(2), activated ERK, p38 MAPK, and JNK, which were significantly inhibited by N-acetylcysteine and DPI. In contrast with exogenous H(2)O(2), however, Ang II did not influence phosphorylation and degradation of IkappaB-alpha/beta or nuclear translocation of p65, nor did it increase NF-kappaB promoter activity. PD98059 and SB203580 inhibited Ang II-induced IL-6 expression. Truncation and mutational analysis of the IL-6 gene promoter showed that CRE was an important cis-element in Ang II-induced IL-6 gene expression. NF-kappaB-binding site was important for the basal expression of IL-6, but was not activated by Ang II. Ang II phosphorylated CREB through the ERK and p38 MAPK pathway in a ROS-sensitive manner. Collectively, these data indicated that Ang II stimulated ROS production via the AT1 receptor and NADH/NADPH oxidase, and that these ROS mediated activation of MAPKs, which culminated in IL-6 gene expression through a CRE-dependent, but not NF-kappaB-dependent, pathway in cardiac fibroblasts.     

AT1 receptors in the RVLM mediate pressor responses to emotional stress in rabbits

In this study, we examined the role of angiotensin type 1 (AT1) receptors in the rostral ventrolateral medulla (RVLM) in mediating the pressor action of emotional stress in conscious rabbits. Rabbits were chronically instrumented with guide cannulas for bilateral microinjections into the RVLM and an electrode for measuring renal sympathetic nerve activity (RSNA). Airjet stress evoked increases in arterial pressure, heart rate, and RSNA, which reached a maximum (+9+/-1 mm Hg, +20+/-5 beats/min, and +93+/-17%, respectively) in the first 2 minutes of stress exposure. Then RSNA rapidly returned to prestress values, while arterial pressure and heart rate remained close to the maximal level until the conclusion of the 7-minute airjet exposure. Microinjections of the nonselective angiotensin receptor antagonist sarile (0.5 nmol, n=8) or AT1 receptor antagonists losartan (2 nmol, n=6) or candesartan (0.2 nmol, n=6) into the RVLM did not alter resting cardiovascular parameters. By contrast, the antagonists attenuated the sustained phase (4 to 7 minutes) of the pressor stress response by 55% to 89%. However, only sarile decreased the onset of this response. The antagonists affected neither the stress-induced tachycardia nor the pressor response to glutamate microinjections. Microinfusion of angiotensin II (4 pmol/min, n=8) into the RVLM did not change the pressor response to airjet stress but attenuated tachycardic response by 47%. Microinjections of vehicle did not alter the cardiovascular stress response. Sarile, losartan, and angiotensin II did not affect the sympathoexcitatory response to baroreceptor unloading. These results suggest that AT1 receptors in the RVLM are important in mediating the pressor effects of emotional stress in conscious rabbits.     

Effects of angiotensin type 2 receptor overexpression in the rostral ventrolateral medulla on blood pressure and urine excretion in normal rats

Central angiotensin II plays a critical role in the regulation of cardiovascular function and autonomic activity, in part, via angiotensin type 1 receptors in the rostral ventrolateral medulla (RVLM). Increasing evidence indicates that angiotensin II can also act on angiotensin type 2 receptors (AT(2)Rs) to exert antagonistic effects. In the current study we determined the effects of overexpression of AT(2)R in the RVLM on sodium and water excretion and on blood pressure in conscious rats. The overexpression of AT(2)R was induced by bilateral microinjection of the AT(2)R adenovirus (Ad5-SYN-AT2R-IRES-EGFP, 2.5 x 10(6) infection units in 0.5 microL; Ad5-SYN-EGFP as the control, 2.5 x 10(6) infection units in 0.5 microL) into the RVLM of rats. Immunofluorescence staining showed that microinjection of AT(2)R adenovirus into the RVLM evoked local overexpression. Significant overexpression of AT(2)R in the RVLM began at 24 hours and was sustained up to 12 days after microinjection. Overexpression of AT(2)R in the RVLM significantly decreased the nocturnal arterial blood pressure and increased the 24-hour urine excretion at days 2, 3, and 4 after gene delivery compared with the control rats. These alterations were abolished by the microinfusion of captopril into the RVLM and were enhanced by angiotensin II infusion. Overexpression of AT(2)R in the RVLM also significantly decreased the urine concentration of noradrenaline and 24-hour noradrenaline excretion (1.1+/-0.5 microg in control rats and 2.4+/-0.5 microg in AT(2)R rats; P<0.05). These results suggest that overexpression of AT(2)R in the RVLM induced a diuresis that may be mediated, in part, by sympathoinhibition.     

AT(1) receptors mediate excitatory inputs to rostral ventrolateral medulla pressor neurons from hypothalamus

Angiotensin II type 1 (AT(1)) receptors are located on pressor neurons in the rostral ventrolateral medulla, and their activation results in an increase in arterial pressure. However, the normal role of these AT(1) receptors in cardiovascular regulation is unknown. In this study, we tested the hypothesis that these receptors mediate synaptic excitation of rostral ventrolateral medullary pressor neurons in response to activation of the hypothalamic paraventricular nucleus. In anesthetized rats, microinjections of the gamma-aminobutyric acid receptor antagonist bicuculline were made into the paraventricular nucleus; this injection causes activation of the nucleus as a consequence of disinhibition. The pressor and sympathoexcitatory responses evoked by paraventricular nucleus activation were significantly reduced (by approximately 40% to 50%) after microinjection of the specific AT(1) receptor antagonists losartan or L-158,809 into the rostral ventrolateral medulla on the ipsilateral, but not contralateral, side. These responses were reduced to a similar degree after microinjections of the neuroinhibitory compound muscimol into the ipsilateral, but not contralateral, rostral ventrolateral medulla. However, bilateral microinjections of the glutamate receptor antagonist kynurenic acid into the rostral ventrolateral medulla had no effect on the responses evoked from the paraventricular nucleus. Conversely, bilateral microinjections of kynurenic acid into the rostral ventrolateral medulla virtually abolished the somatosympathoexcitatory reflex, whereas bilateral microinjections of losartan or L-158,809 had no effect on this reflex. The results indicate that excitatory synaptic inputs to pressor neurons in the rostral ventrolateral medulla arising from activation of the paraventricular nucleus are mediated predominantly by AT(1) receptors.     

Role of angiotensin-(1-7) in rostral ventrolateral medulla in blood pressure regulation via sympathetic nerve activity in Wistar-Kyoto and spontaneous hypertensive rats

Angiotensin (Ang)-(1-7) Ang-(1-7) is formed from angiotensin II by angiotensin-converting enzyme 2 (ACE2) and modulates the renin-angiotensin system. We evaluated whether the Ang-(1-7)-Mas axis in the rostral ventrolateral medulla (RVLM) contributes to neural mechanisms of blood pressure (BP) regulation. We microinjected Ang-(1-7), Ang-(1-7)-Mas receptor antagonist A-779, and ACE2 inhibitor DX600 into the RVLM of anesthetized Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHRs). Unilateral Ang-(1-7) microinjection induced a significantly greater increase in AP (arterial blood pressure) in SHR than in WKY. Bilateral A-779 microinjection induced a significantly greater decrease in AP and renal sympathetic nerve activity in SHR than in WKY. Bilateral DX600 microinjection induced a significantly greater decrease in AP in SHR than in WKY. Our results suggest that endogenous Ang-(1-7) in the RVLM contributes to maintain AP and renal sympathetic nerve activity both in SHR and WKY and that its activity might be enhanced in SHR.     

Involvement of reactive oxygen species in the activation of tyrosine kinase and extracellular signal-regulated kinase by angiotensin II

Reactive oxygen species (ROS) have been proposed to mediate vascular hypertrophy induced by angiotensin II (Ang II). Recently, we and others have shown that growth-promoting signals by Ang II involve protein tyrosine kinase (PTK) and extracellular signal-regulated kinase (ERK). However, whether ROS contribute to the Ang II-induced PTK and/or ERK activation in vascular smooth muscle cells (VSMCs) remains largely unclear. Here, we have investigated the possible involvement of ROS in Ang II-induced PTK and ERK activation. In the presence of a NADH/NADPH oxidase inhibitor, diphenyleneiodonium (DPI) or an antioxidant, alpha-tocopherol, Ang II-induced protein tyrosine phosphorylation of two major proteins (p120, p70) and ERK activation were markedly reduced, whereas ERK activation by epidermal growth factor was unaffected. DPI also inhibited Ang II-induced H2O2 production and PTK activation. In this regard, H2O2 and a membrane permeable thiol-oxidizing agent, diamide, stimulated protein tyrosine phosphorylation of p120 and p70, and ERK activation in VSMCs. H2O2 also enhanced PTK activity. From these data, we conclude that ROS play a critical role in the Ang II-induced PTK and ERK activation in VSMCs, thereby contributing to vascular growth associated with enhanced Ang II activity.     

Tempol attenuates excitatory actions of angiotensin II in the rostral ventrolateral medulla during emotional stress

Superoxide has been shown to be an important intracellular mediator of actions of angiotensin II. Recently, we found that blockade of angiotensin II type-1 receptors in the rostral ventrolateral medulla (RVLM) abrogated the pressor effect of emotional stress in rabbits. In the present study, we examined the influence of superoxide dismutase mimetics, tempol and tiron, in RVLM on cardiovascular stress response in conscious rabbits. Air-jet stress evoked a sustained increase in blood pressure (+14+/-2 mm Hg), tachycardia (+52+/-7 bpm), and renal sympathoactivation (+58+/-8%). Bilateral microinjections of tempol or tiron (20 nmol) into RVLM did not alter resting cardiovascular parameters, but attenuated the pressor, sympathetic, and tachycardiac response to stress by 40% to 55%. By contrast, 3-carbamoylproxyl, which is structurally close to tempol but has a lower superoxide scavenging activity, did not alter the stress response. Neither tempol nor tiron altered the sympathoexcitatory response to glutamate microinjections into RVLM or to baroreceptor unloading. Microinjections of nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 10 nmol) into RVLM did not affect the stress response. Coinjections of tempol and L-NAME decreased the pressor response to stress by 35+/-3%. Tempol attenuated the pressor response to microinjection of angiotensin II into RVLM by 59+/-15%, whereas L-NAME did not alter this response. These results suggest that superoxide dismutase mimetics in RVLM attenuate, partially via a nitric oxide-independent mechanism, the pressor effect of emotional stress in rabbits. Together with our previous studies, these results also indicate that superoxide is a key mediator of excitatory actions of angiotensin II in RVLM during acute stress.     

Expression of constitutively active angiotensin receptors in the rostral ventrolateral medulla increases blood pressure

Angiotensin type 1A (AT(1A)) receptors are expressed within the rostral ventrolateral medulla, and microinjections of angiotensin II into this region increase sympathetic vasomotor tone. To determine the effect of sustained increases in AT(1A) receptor density or activity in rostral ventrolateral medulla, we used radiotelemetry to monitor blood pressure in conscious rats before and after bilateral microinjection into the rostral ventrolateral medulla of adenoviruses encoding the wild-type AT(1A) receptor or a constitutively active version of the receptor (Asn111Gly, [N111G]AT(1A)). The constitutively active receptor signals in the absence of angiotensin II. Adenovirus-directed receptor expression was extensively characterized both in vitro and in vivo. We established that adenoviral infection was limited to the rostral ventrolateral medulla and that receptor expression was sustained for > or =10 days; we also observed that adenoviral transgene expression occurs in glia, with no transgene expression observed in neurons of the rostral ventrolateral medulla. Rats receiving the wild-type AT(1A) receptor showed no change in blood pressure, whereas animals receiving the [N111G]AT(1A) receptor displayed an increase in blood pressure that persisted for 3 to 4 days before returning to basal levels. These data indicate that increased AT(1A) receptor activity (not just overexpression) is a primary determinant of efferent drive from rostral ventrolateral medulla and reveal counterregulatory processes that moderate AT(1A) receptor actions at this crucial relay point. More importantly, they imply that constitutive receptor signaling in glia of the rostral ventrolateral medulla can modulate the activity of adjacent neurons to change blood pressure.     

Effects of angiotensin II type 1 receptor antagonist on pressor responses to pulsatile compression of the rostral ventrolateral medulla in rats.

The rostral ventrolateral medulla (RVLM) is known to be a major center regulating sympathetic and cardiovascular activities. A possible association between neurovascular compression of the RVLM and essential hypertension has been indicated. The present study was performed to determine the role of angiotensin II (AngII) in the pressor and sympathetic responses to pulsatile compression of the RVLM. To determine the role of glutamate and AngII in the RVLM, L-glutamate (Glu) 2 nmol or AngII 100 pmol was injected into the RVLM with or without RVLM pretreatment of kynurenate (Glu receptor antagonist) 3nmol, candesartan (AngII type 1 (AT1) receptor antagonist) 2 nmol, or PD123319 (AngII type 2 (AT2) receptor antagonist) 1 nmol in anesthetized Wistar rats. In addition, to determine the role of glutamate and AngII in the pressor and sympathetic effects to the RVLM compression, kynurenate, candesartan, or PD123319 was locally injected before pulsatile compression of the RVLM. Finally, to determine the effects of peripherally administered AngII antagonists in these pressor and sympathetic excitatory responses, candesartan 0.25 micromol or PD123319 0.05 micromol was intravenously injected before pulsatile compression of the RVLM. Glu injected into the RVLM significantly increased mean arterial pressure (MAP) and splanchnic sympathetic nerve activity (SNA), and these effects were reduced by RVLM pretreatment with kynurenate, but were unaffected by candesartan or PD123319. AngII injected into the RVLM and pulsatile compression of the RVLM also increased MAP and SNA. However, in contrast with Glu injections, these effects were reduced by RVLM pretreatment with candesartan or kynurenate, but were unaffected by PD123319. Pressor and sympathetic excitatory responses to RVLM compression were reduced by intravenous pretreatment with candesartan but not with PD123319. These results indicate that, upon pulsatile compression of the RVLM, AngII may activate RVLM neurons via AT1 receptors and stimulate Glu release to thereby elicit sympathetic activation and pressor effects. Candesartan may exert its hypotensive effect at least in part by affecting the RVLM neurons to reduce sympathetic outflow induced by pulsatile compression of the RVLM.     

Electrophysiological properties of rostral ventrolateral medulla neurons in angiotensin II 1a receptor knockout mice

We compared the electrophysiological properties of neurons in the rostral ventrolateral medulla of neonatal angiotensin II type 1a receptor knockout mice and wild-type mice with responses to angiotensin II, its type-1 receptor blocker candesartan, and its type-2 receptor blocker PD123319. Using the whole-cell patch-clamp technique, we examined the characteristics of rostral ventrolateral medulla neurons in brain stem-spinal cord preparations in which the sympathetic neuronal network is preserved. Baseline membrane potential and firing rate were almost similar between angiotensin II type 1a receptor knockout mice and wild-type mice. Superfusion with angiotensin II depolarized rostral ventrolateral medulla bulbospinal neurons in wild-type mice, whereas it hyperpolarized those in angiotensin II type 1a receptor knockout mice. Because pretreatment with candesartan significantly prevented the angiotensin II-induced depolarization in wild-type mice, the angiotensin II type 1 receptor is crucial for this depolarization. Superfusion with PD123319 depolarized rostral ventrolateral medulla bulbospinal neurons in angiotensin II type 1a receptor knockout mice. PD123319 prevented the angiotensin II-induced hyperpolarization in angiotensin II type 1a receptor knockout mice, and, rather, it induced depolarization. These results suggest that the angiotensin II type 2 receptor in rostral ventrolateral medulla plays an antagonistic role against the angiotensin II type 1a receptor in controlling the neuronal activity of rostral ventrolateral medulla.     

Modulation of angiotensin II-mediated hypertension and cardiac remodeling by lectin-like oxidized low-density lipoprotein receptor-1 deletion

Angiotensin II via type 1 receptor activation upregulates the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), and LOX-1 activation, in turn, upregulates angiotensin II type 1 receptor expression. We postulated that interruption of this positive feedback loop might attenuate the genesis of angiotensin II-induced hypertension and subsequent cardiac remodeling. To examine this postulate, LOX-1 knockout and wild-type mice were infused with angiotensin II or norepinephrine (control for angiotensin II) for 4 weeks. Angiotensin II-, but not norepinephrine-, induced hypertension was attenuated in LOX-1 knockout mice. Angiotensin II-induced cardiac remodeling was also attenuated in LOX-1 knockout mice. Importantly, angiotensin II type 1 receptor expression was reduced, and the expression and activity of endothelial NO synthase were preserved in the tissues of LOX-1 knockout mice given angiotensin II. Reactive oxygen species generation, nicotinamide-adenine dinucleotide phosphate oxidase expression, and phosphorylation of p38 and p44/42 mitogen-activated protein kinases were also much less pronounced in the LOX-1 knockout mice given angiotensin II. These alterations in biochemical and structural abnormalities were associated with preservation of cardiac hemodynamics in the LOX-1 knockout mice. To confirm that fibroblast function is modulated in the absence of LOX-1, cardiac fibroblasts from wild-type and LOX-1 knockout mice were treated with angiotensin II. Indeed, LOX-1 knockout mice cardiac fibroblasts revealed an attenuated profibrotic response on treatment with angiotensin II. These observations provide strong evidence that LOX-1 is a key modulator of the development of angiotensin II-induced hypertension and subsequent cardiac remodeling.     

Redox-sensitive signaling by angiotensin II involves oxidative inactivation and blunted phosphorylation of protein tyrosine phosphatase SHP-2 in vascular smooth muscle cells from SHR

Angiotensin II (Ang II) signaling in vascular smooth muscle cells (VSMCs) involves reactive oxygen species (ROS) through unknown mechanisms. We propose that Ang II induces phosphorylation of growth signaling kinases by redox-sensitive regulation of protein tyrosine phosphatases (PTP) in VSMCs and that augmented Ang II signaling in spontaneously hypertensive rats (SHRs) involves oxidation/inactivation and blunted phosphorylation of the PTP, SHP-2. PTP oxidation was assessed by the in-gel PTP method. SHP-2 expression and activity were evaluated by immunoblotting and by a PTP activity assay, respectively. SHP-2 and Nox1 were downregulated by siRNA. Ang II induced oxidation of multiple PTPs, including SHP-2. Basal SHP-2 content was lower in SHRs versus WKY. Ang II increased SHP-2 phosphorylation and activity with blunted responses in SHRs. Ang II-induced SHP-2 effects were inhibited by valsartan (AT(1)R blocker), apocynin (NAD(P)H oxidase inhibitor), and Nox1 siRNA. Ang II stimulation increased activation of ERK1/2, p38MAPK, and AKT, with enhanced effects in SHR. SHP-2 knockdown resulted in increased AKT phosphorylation, without effect on ERK1/2 or p38MAPK. Nox1 downregulation attenuated Ang II-mediated AKT activation in SHRs. Hence, Ang II regulates PTP/SHP-2 in VSMCs through AT(1)R and Nox1-based NAD(P)H oxidase via two mechanisms, oxidation and phosphorylation. In SHR Ang II-stimulated PTP oxidation/inactivation is enhanced, basal SHP-2 expression is reduced, and Ang II-induced PTP/SHP-2 phosphorylation is blunted. These SHP-2 actions are associated with augmented AKT signaling. We identify a novel redox-sensitive SHP-2-dependent pathway for Ang II in VSMCs. SHP-2 dysregulation by increased Nox1-derived ROS in SHR is associated with altered Ang II-AKT signaling.     

Mitogen-activated protein kinase-activated protein kinase 2 in angiotensin II-induced inflammation and hypertension: regulation of oxidative stress

Vascular oxidative stress and inflammation play an important role in angiotensin II-induced hypertension, and mitogen-activated protein kinases participate in these processes. We questioned whether mitogen-activated protein kinase-activated protein kinase 2 (MK2), a downstream target of p38 mitogen-activated protein kinase, is involved in angiotensin II-induced vascular responses. In vivo experiments were performed in wild-type and Mk2 knockout mice infused intravenously with angiotensin II. Angiotensin II induced a 30 mm Hg increase in mean blood pressure in wild-type that was delayed in Mk2 knockout mice. Angiotensin II increased superoxide production and vascular cell adhesion molecule-1 in blood vessels of wild-type but not in Mk2 knockout mice. Mk2 knockdown by small interfering RNA in mouse mesenteric vascular smooth muscle cells caused a 42% reduction in MK2 protein and blunted the angiotensin II-induced 40% increase of MK2 expression. Mk2 knockdown blunted angiotensin II-induced doubling of intracellular adhesion molecule-1 expression, 2.4-fold increase of nuclear p65, and 1.4-fold increase in Ets-1. Mk2 knockdown abrogated the angiotensin II-induced 4.7-fold and 1.3-fold increase of monocyte chemoattractant protein-1 mRNA and protein. Angiotensin II enhanced reactive oxygen species levels (by 29%) and nicotinamide adenine dinucleotide phosphate oxidase activity (by 48%), both abolished by Mk2 knockdown. Reduction of MK2 blocked angiotensin II-induced p47phox translocation to the membrane, associated with a 53% enhanced catalase expression. Angiotensin II-induced increase of MK2 was prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor Nox2ds-tat. Mk2 small interfering RNA prevented the angiotensin II-induced 30% increase of proliferation. In conclusion, MK2 plays a critical role in angiotensin II signaling, leading to hypertension, oxidative stress via activation of p47phox and inhibition of antioxidants, and vascular inflammation and proliferation.     

Haemorrhage increases the pressor effect of angiotensin-(1-7) but not of angiotensin II at the rat rostral ventrolateral medulla.

OBJECTIVE: To evaluate the effects of angiotensins acting at the rostral ventrolateral medulla (RVLM) on the cardiovascular adjustments following haemorrhage. DESIGN: Changes in mean arterial pressure (MAP) and heart rate (HR) produced by micro-injections of angiotensin II (Ang II) and angiotensin (Ang)-(1-7) and different angiotensin antagonists into the RVLM of anaesthetized rats submitted to haemorrhage, were determined. METHODS: Experiments were performed in 79 urethane-anaesthetized male Wistar rats. Ang-(1-7) (2.5 and 25 pmol), Ang II (25 pmol), [Sar1,Thr8]-Ang II (non-selective angiotensin antagonist, 0.2 nmol), A-779 (Ang-(1-7) antagonist, 0.1 nmol), losartan (AT1 Ang II receptor antagonist, 0.2 nmol) or vehicle (200 nl) were bilaterally micro-injected into the RVLM under basal conditions or 30 min after blood withdrawal (0.6 ml/100 g bodyweight). In additional groups, [Sar1,Thr8]-Ang II, A-779, losartan or vehicle were micro-injected into the RVLM 10 min before bleeding to uncover a possible role of endogenous peptides during haemorrhage. RESULTS: The pressor effect produced by Ang II micro-injection was not altered by haemorrhage. Conversely, haemorrhage significantly increased the magnitude and duration of the pressor effect of Ang-(1-7) at the RVLM. The fall in MAP induced by haemorrhage was similar after micro-injection of vehicle or A-779. However, micro-injection of [Sar1,Thr8]-Ang II significantly reduced the fall in MAP after haemorrhage. A similar finding was obtained with micro-injection of losartan. In addition, while RVLM micro-injection of [Sar1,Thr8]-Ang II or losartan 30 min after blood withdrawn produced MAP changes that were similar to that observed in control animals, micro-injection of A-779 did not significantly alter baseline MAP. CONCLUSIONS: These results suggest that changes in the RVLM reactivity to Ang-(1-7) but not Ang II may contribute to the haemodynamic adjustments triggered by acute reductions in blood volume. The data obtained with [Sar1,Thr8]-Ang II and losartan suggest a primary inhibitory role for endogenous Ang II at the RVLM during haemorrhage.     

Cardiovascular effects of angiotensin II in the rostral ventrolateral medulla: The push-pull hypothesis

Neurons within the rostral ventrolateral medulla (RVLM) play a pivotal role in the tonic and phasic control of blood pressure. This region also contains a high density of angiotensin II type 1 (AT1) receptors. There is evidence that tonic activation of AT1 receptors in the RVLM contributes to an increased sympathetic vasomotor activity in some models of hypertension. At the same time, under certain conditions, activation of AT1 receptors in the RVLM can cause sympathoinhibition. In this review we argue that the effect of endogenous angiotensin II in the RVLM on sympathetic vasomotor activity depends upon the balance between tonic excitatory and inhibitory effects on sympathetic premotor neurons mediated by AT1 receptors within this region, and that this balance may be altered in different physiological or pathophysiological conditions.