Angiotensin II induces hyperresponsiveness of bronchial smooth muscle via an activation of p42/44 ERK in rats

Angiotensin II (Ang II) might be an important mediator in the pathogenesis of bronchial asthma, although the mechanisms of airway hyperresponsiveness caused by Ang II are not yet clear. Whether p42/44 ERK contributes to the Ang II-elicited bronchial smooth muscle (BSM) hyperresponsiveness in rats was presently examined. The RT-PCR analyses revealed that Ang II AT_1A, AT_1B, and AT₂ receptors, angiotensinogen, angiotensin-converting enzyme, but not renin, were expressed in the lungs, trachea, and main bronchi of rats. Only a small and transient contraction was induced by the application of Ang II in the main bronchial smooth muscle; the contraction was inhibited by losartan, an AT₁ receptor antagonist. The contractions induced by carbachol (CCh), high K⁺ depolarization, and sodium fluoride (NaF; a G protein activator) were augmented by pretreatment with Ang II. The BSM hyperresponsiveness induced by Ang II was abolished by losartan. Furthermore, the Ang II-induced BSM hyperresponsiveness to CCh was attenuated by pretreatment with U-0126, a p42/44 ERK kinase (MEK-1/2) inhibitor. In conclusion, Ang II-induced BSM hyperresponsiveness through the activation of p42/44 ERK may play an important role in the pathophysiology of bronchial asthma, although Ang II itself caused a small force development in the bronchial smooth muscle.     

Angiotensin III modulates the nociceptive control mediated by the periaqueductal gray matter

Endogenous angiotensin (Ang) II and/or an Ang II-derived peptide, acting on Ang type 1 (AT₁) and Ang type 2 (AT₂) receptors, can carry out part of the nociceptive control modulated by periaqueductal gray matter (PAG). However, neither the identity of this putative Ang-peptide, nor its relationship to Ang II antinociceptive activity was clarified. Therefore, we have used tail-flick and incision allodynia models combined with an HPLC time course of Ang metabolism, to study the Ang III antinociceptive effect in the rat ventrolateral (vl) PAG using peptidase inhibitors and receptor antagonists. Ang III injection into the vlPAG increased tail-flick latency, which was fully blocked by Losartan and CGP 42,112A, but not by divalinal-Ang IV, indicating that Ang III effect was mediated by AT₁ and AT₂ receptors, but not by the AT₄ receptor. Ang III injected into the vlPAG reduced incision allodynia. Incubation of Ang II with punches of vlPAG homogenate formed Ang III, Ang (1–7) and Ang IV. Amastatin (AM) inhibited the formation of Ang III from Ang II by homogenate, and blocked the antinociceptive activity of Ang II injection into vlPAG, suggesting that aminopeptidase A (APA) formed Ang III from Ang II. Ang III can also be formed from Ang I by a vlPAG alternative pathway. Therefore, the present work shows, for the first time, that: (i) Ang III, acting on AT₁ and AT₂ receptors, can elicit vlPAG-mediated antinociception, (ii) the conversion of Ang II to Ang III in the vlPAG is required to elicit antinociception, and (iii) the antinociceptive activity of endogenous Ang II in vlPAG can be ascribed preponderantly to Ang III.     

Angiotensin II stimulates proliferation of adventitial fibroblasts cultured from rat aortic explants

It has been proposed that the local renin-angiotensin system is activated in the adventitia after vascular injury. However, the physiological role of Angiotensin II (Ang II) in the adventitia has not been studied at a cellular level. This study was designed to assess the role of Ang II in the growth response of cultured adventitial fibroblasts (AFs). Adventitial explants of the rat thoracic aorta showed outgrowth of AFs within 5-7 days. Ang II caused hyperplastic response of AF cultures. The Ang II-induced mitogenic response of AFs was mediated primarily by the AT1 receptor. Ang II caused a rapid induction of immediate early genes (c-fos, c-myc and jun B). Induction of c-fos expression was fully blocked by an AT1 receptor antagonist but not by an AT2 receptor antagonist. Epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) induced DNA synthesis in AFs. Co-stimulation of AFs with the growth factors and Ang II potentiated the incorporation of 3H-thymidine into DNA. Results from this study indicate that Ang II causes mitogenesis of AFs via AT1 receptor stimulation and potentiates the responses to other mitogens. These data suggest that the Ang II may play an important role in regulating AF function during vascular remodeling following arterial injury.     

AT1 receptor-mediated angiotensin II activation and chemotaxis of T lymphocytes

Angiotensin II (Ang II), a central renin–angiotensin system (RAS) effector molecule, and its receptors, AT₁ and AT₂, have been shown to be involved in the inflammatory aspects of different diseases, however the cellular mechanisms underlying the regulation of immunity are not fully understood. In this work, using spleen-derived CD4⁺ and CD8⁺ T lymphocytes activated in vitro, we tested the influence of Ang II on different aspects of the T cell function, such as activation and adhesion/transmigration through endothelial basal membrane proteins. The addition of 10⁻⁸ M Ang II did not change any of the parameters evaluated. However, 10⁻⁶ M losartan, an AT₁ receptor antagonist: (i) reduced the percentage of CD25⁺ and CD69⁺ cells of both subsets; (ii) inhibited adhesion of these cells to fibronectin or laminin by 53% or 76%, respectively and (iii) significantly reduced transmigration through fibronectin or laminin by 57% or 43%, respectively. In addition, 10⁻⁶ M captopril, an angiotensin-converting enzyme inhibitor had similar effects to Ang II, however its effects were reverted by exogenous Ang II (10⁻⁸ M). None of these responses was modified by 10⁻⁷ M PD123319, an AT₂ antagonist. These data reinforce the notion of endogenous production of Ang II by T cells, which is important for T cell activation, and adhesion/transmigration induced on interaction with basal membrane proteins, possibly involving AT₁ receptor activation. Moreover, AT₁ receptor expression is 10-fold higher in activated T lymphocytes compared with naive cells, but AT₂ receptor expression did not change after T cell receptor triggering.     

Angiotensin receptors as determinants of life span

Angiotensin II (Ang II), the central product of renin-angiotensin system, has a role in the etiology of hypertension and in pathophysiology of cardiac and renal diseases in humans. Other functions of Ang II include effects on immune response, inflammation, cell growth and proliferation, which are largely mediated by Ang II type 1 receptor (AT₁). Several experimental studies have demonstrated that Ang II acts through AT₁ as a mediator of normal aging processes by increasing oxidant damage to mitochondria and in consequences by affecting mitochondrial function. Recently, our group has demonstrated that the inhibition of Ang II activity by targeted disruption of the Agtr1a gene encoding Ang II type 1A receptor (AT_1A) in mice translates into marked prolongation of life span. The absence of AT_1A protected multiple organs from oxidative damage and the alleviation of aging-like phenotype was associated with increased number of mitochondria and upregulation of the prosurvival gene sirtuin 3. AT₁ receptor antagonists have been proven safe and well-tolerated for chronic use and are used as a key component of the modern therapy for hypertension and cardiac failure, therefore Ang II/AT₁ pathway represents a feasible therapeutic strategy to prolong life span in humans.     

Angiotensin-(1–7) and angiotension II in the rostral ventrolateral medulla modulate the cardiac sympathetic afferent reflex and sympathetic activity in rats

The rostral ventrolateral medulla (RVLM) plays a pivotal role in regulating sympathetic vasomotor activity. The cardiac sympathetic afferent reflex (CSAR) contributes to the enhanced sympathetic outflow in chronic heart failure and hypertension. The aim of the present study was to determine whether angiotensin (Ang) II and Ang-(1–7) in the RVLM modulate the CSAR and sympathetic activity. Bilateral sinoaortic denervation and vagotomy were carried out in anesthetized rats. The CSAR was evaluated as the renal sympathetic nerve activity (RSNA) response to epicardial application of capsaicin. The effects of bilateral microinjection of Ang II, Ang-(1–7), the AT₁ receptor antagonist losartan or the Mas receptor antagonist d-alanine-Ang-(1–7) (A-779) into the RVLM were determined. Either Ang II or Ang-(1–7) enhanced the CSAR as well as increased RSNA and mean arterial pressure (MAP) in a dose-dependent manner. Pretreatment with losartan but not the A-779 abolished the effects of Ang II, while A-779 but not the losartan eliminated the effects of Ang-(1–7). The RVLM microinjection of losartan alone had no direct effect on the CSAR, RSNA, and MAP, but A-779 alone attenuated the CSAR and decreased RSNA and MAP. These results indicate that Ang-(1–7) is as effective as Ang II in sensitizing the CSAR and increasing sympathetic outflow. In contrast to Ang II, the effects of Ang-(1–7) are not mediated by AT₁ receptors but by Mas receptors. Mas receptors, but not the AT₁ receptors, in the RVLM are involved in the tonic control of the CSAR.     

血管紧张素Ⅱ和血管紧张素-(1-7)对大鼠血管平滑肌细胞肾素(原)受体表达的影响

目的: 研究血管紧张素Ⅱ(Ang II)和血管紧张素-(1-7) 对大鼠血管平滑肌细胞(VSMCs)肾素(原)受体 表达的影响。方法: 将VSMCs按以下分组:(1)对照组:不加干预因素;(2)不同浓度AngⅡ组:分别加入AngⅡ10、100、1 000 nmol/L;(3)不同浓度Ang-(1-7)组:分别加入Ang-(1-7) 10、100、1 000 nmol/L; (4)AngⅡ+ losartan(AT₁受体拮抗剂)组:losartan 10^-6 mol/L预处理30 min后,再加入AngⅡ100 nmol/L; (5)AngⅡ+ PD123319(AT₂受体拮抗剂)组: 先用10^-5 mol/LPD123319预处理30 min后,再用终浓度为100 nmol/L AngⅡ;(6)CGP42112A(AT₂受体激动剂)组:加入10^-7 mol/L CGP42112A。各组用real-time PCR法和Western blotting法检测(P)RR的表达情况。结果: 与对照组比,不同浓度AngⅡ可以促进(P)RR mRNA和蛋白的表达,并且呈浓度依赖性(均P<0.01);不同浓度Ang-(1-7)可抑制 (P)RR mRNA和蛋白表达(均P<0.01),各浓度之间比较无显著差异(均P>0.05);加入CGP42112A可以促进(P)RR mRNA和蛋白的表达(均P<0.01),与AngⅡ处理组比较,加入PD123319可抑制(P)RR mRNA和蛋白表达(均P<0.01),加入losartan不能抑制(P)RR mRNA和蛋白表达(P>0.05)。结论: AngⅡ可通过AT₂受体促进(P)RR mRNA和蛋白表达,而Ang-(1-7) 可抑制(P)RR mRNA和蛋白的表达。     

Endothelium-derived contracting factors mediate the Ang II-induced endothelial dysfunction in the rat aorta: preventive effect of red wine polyphenols

Angiotensin II (Ang II)-induced hypertension is associated with vascular oxidative stress and an endothelial dysfunction. This study examined the role of reactive oxygen species (ROS) and endothelium-derived contracting factors in Ang II-induced endothelial dysfunction and whether these effects are prevented by red wine polyphenols (RWPs), a rich source of natural antioxidants. Rats were infused with Ang II for 14 days. RWPs were administered in the drinking water 1 week before and during the Ang II infusion. Arterial pressure was measured in conscious rats. Vascular reactivity was assessed in organ chambers and cyclooxygenase-1 (COX-1) and COX-2 expression by Western blot and immunofluorescence analyses. Ang II-induced hypertension was associated with blunted endothelium-dependent relaxations and induction of endothelium-dependent contractions in the presence of nitro-L-arginine in response to acetylcholine (Ach). These effects were not affected by the combination of membrane permeant analogs of superoxide dismutase and catalase but were abolished by the thromboxane A₂ (TP) receptor antagonist GR32191B and the COX-2 inhibitor NS-398. The COX-1 inhibitor SC-560 also prevented contractile responses to Ach. Ang II increased the expression of COX-1 and COX-2 in the aortic wall. RWPs prevented Ang II-induced hypertension, endothelial dysfunction, and upregulation of COX-1 and COX-2. Thus, Ang II-induced endothelial dysfunction cannot be explained by an acute formation of ROS reducing the bioavailability of nitric oxide but rather by COX-dependent formation of contracting factors acting on TP receptors. RWPs are able to prevent the Ang II-induced endothelial dysfunction mostly due to their antioxidant properties.     

Pigment epithelium-derived factor (PEDF) blocks angiotensin II-induced T cell proliferation by suppressing autocrine production of interleukin-2

Angiotensin II (Ang II) elicits numerous inflammatory-proliferative responses in vascular cells, thereby being involved in atherosclerosis. We have previously shown that pigment epithelium-derived factor (PEDF) blocks the Ang II-induced endothelial cell activation, thus suggesting that PEDF may play a role in atherosclerosis. However, effects of PEDF on T cell activation, another key steps of atherosclerosis, remain to be elucidated. In this study, we examined whether PEDF could inhibit the Ang II-induced MOLT-3 T cell proliferation in vitro and the way that it might achieve this effect. Ang II significantly stimulated DNA synthesis in MOLT-3 T cells, which was inhibited by PEDF, olmesartan, an Ang II type I receptor blocker, an anti-oxidant N-acetylcysteine (NAC), or antibodies directed against IL-2. PEDF or NAC suppressed gene expression of interleukin-2 (IL-2) in Ang II-exposed MOLT-3 T cells. Furthermore, PEDF blocked the Ang II-induced reactive oxygen species (ROS) generation and NADPH oxidase activity in MOLT-3 T cells. These results demonstrate that PEDF inhibits the Ang II-induced T cell proliferation by blocking autocrine production of IL-2 via suppression of NADPH oxidase-mediated ROS generation. Blockade by PEDF of T cell activation may become a novel therapeutic target for atherosclerosis.     

Evolving concepts on regulation and function of renin in distal nephron

Sustained stimulation of the intrarenal/intratubular renin–angiotensin system in a setting of elevated arterial pressure elicits renal vasoconstriction, increased sodium reabsorption, proliferation, fibrosis, and eventual renal injury. Activation of luminal AT₁ receptors in proximal and distal nephron segments by local Ang II formation stimulates various transport systems. Augmented angiotensinogen (AGT) production by proximal tubule cells increases AGT secretion contributing to increased proximal Ang II levels and leading to spillover of AGT into the distal nephron segments, as reflected by increased urinary AGT excretion. The increased distal delivery of AGT provides substrate for renin, which is expressed in principal cells of the collecting tubule and collecting ducts, and is also stimulated by AT₁ receptor activation. Renin and prorenin are secreted into the tubular lumen and act on the AGT delivered from the proximal tubule to form more Ang I. The catalytic actions of renin and or prorenin may be enhanced by binding to prorenin receptors on the intercalated cells or soluble prorenin receptor secreted into the tubular fluid. There is also increased luminal angiotensin converting enzyme in collecting ducts facilitating Ang II formation leading to stimulation of sodium reabsorption via sodium channel and sodium/chloride co-transporter. Thus, increased collecting duct renin contributes to Ang II-dependent hypertension by augmenting distal nephron intratubular Ang II formation leading to sustained stimulation of sodium reabsorption and progression of hypertension.     

Extracellular signal regulated kinase and SMAD signaling both mediate the angiotensin II driven progression towards overt heart failure in homozygous TGR(mRen2)27

Angiotensin (Ang) II is a key player in left ventricular (LV) remodeling and cardiac fibrosis. Its effects are thought to be transferred at least in part by mitogen-activated protein kinases (MAPK), transforming growth factor (TGF) ₁, and the Smad pathway. In this study we sought to elucidate whether Ang II related effects on LV dysfunction and fibrosis in vivo are mediated via MAPK or rather via Smad stimulation. We treated homozygous REN2 rats (7–11 weeks) with placebo, Ang II type 1 (AT₁) receptor blocker or tyrphostin A46 (TYR), an inhibitor of epidermal growth factor receptor tyrosine kinase that blocks extracellular signal-regulated kinase (ERK) activity. REN2 rats had LV hypertrophy (LVH) and LV dysfunction that progressed to heart failure between 10 and 13 weeks. Blood pressure normalized over time. Renin, N-terminal atrial natriuretic peptide (N-ANP), and ERK were activated while p38 MAPK was not. Treatment with AT₁ receptor blockade prevented LVH and right ventricular hypertrophy, normalized systolic and diastolic dP/dt, N-ANP levels, and reduced collagen apposition. Similarly, TYR reduced LVH, N-ANP levels, and collagen apposition. Myocardial ERK activation did not depend on AT₁ receptor signaling as it was not affected by AT₁ receptor blockade. TYR abolished myocardial ERK activity. Smad2 activation was inhibited by AT₁ receptor blockade but was unaltered by TYR. Ang II induced LV remodeling and fibrosis are dependent on both ERK and Smad2 activation. This process is prevented by both AT₁ receptor blockade and TYR, and therefore inhibition of either pathway is equally efficacious in restoring LV function and architecture.R.A.D.B. and S.P. contributed equally to this work     

Microinjection of angiotensin II in the caudal ventrolateral medulla induces hyperalgesia

Nociceptive transmission from the spinal cord is controlled by supraspinal pain modulating systems that include the caudal ventrolateral medulla (CVLM). The neuropeptide angiotensin II (Ang II) has multiple effects in the CNS and at the medulla oblongata. Here we evaluated the expression of angiotensin type 1 (AT₁) receptors in spinally-projecting CVLM neurons, and tested the effect of direct application of exogenous Ang II in the CVLM on nociceptive behaviors. Although AT₁-immunoreactive neurons occurred in the CVLM, only 3% of AT₁-positive neurons were found to project to the dorsal horn, using double-immunodetection of the retrograde tracer cholera toxin subunit B. In behavioral studies, administration of Ang II (100 pmol) in the CVLM gave rise to hyperalgesia in both the tail-flick and formalin tests. This hyperalgesia was significantly attenuated by local administration of the AT₁ antagonist losartan. The present study demonstrates that Ang II can act on AT₁ receptors in the CVLM to modulate nociception. The effect on spinal nociceptive processing is likely indirect, since few AT₁-expressing CVLM neurons were found to project to the spinal cord. The renin-angiotensin system may also play a role in other supraspinal areas implicated in pain modulation.     

Angiotensin II increases L-type Ca2+ current in gramicidin D-perforated adult rabbit ventricular myocytes: comparison with conventional patch-clamp method

The effects of angiotensin II (Ang II) on L-type Ca2+ current (I(Ca,L)) remains controversial. We studied the effects of Ang II on I(Ca,L) in single adult rabbit ventricular myocytes using a perforated patch-clamp technique with gramicidin D. Ang II increased I(Ca,L) in a concentration-dependent manner (EC(50)=0.75 nM). In contrast, in conventional whole-cell patch-calmp, I(Ca,L)ran down gradually and the I(Ca,L) response to Ang II was variable, suggesting the potential loss of diffusible components crucial for the Ang II-induced signaling process. An AT(1) antagonist, CV11974 (0.1 microM), completely inhibited the increase in I(Ca,L) induced by Ang II (0.1 microM), whereas an AT(2) antagonist, PD123319 (10 microM), did not influence the I(Ca,L) increase. Neither pre- nor after-treatment with a Na+/H+ exchange (NHE) inhibitor HOE642 (1 microM) affected the Ang II-induced increase in I(Ca,L). The protein kinase C (PKC) inhibitor chelerythrine (1 microM) did not affect the Ang II-induced I(Ca,L) increase. The present findings indicate that Ang II increases I(Ca,L) via AT(1) receptors in adult rabbit ventricular myocytes. Neither the activation of NHE nor PKC may contribute to the Ang II-induced activation of I(Ca,L).     

Angiotensin II Modulates p130Cas of Podocytes by the Suppression of AMP-Activated Protein Kinase

Angiotensin II (Ang II) induces the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. In kidneys, Ang II plays an important role in the development of proteinuria by the modification of podocyte molecules. We have previously found that Ang II suppressed podocyte AMP-activated protein kinase (AMPK) via Ang II type 1 receptor and MAPK signaling pathway. In the present study, we investigated the roles of AMPK on the changes of p130Cas of podocyte by Ang II. We cultured mouse podocytes and treated them with various concentrations of Ang II and AMPK-modulating agents and analyzed the changes of p130Cas by confocal imaging and western blotting. In immunofluorescence study, Ang II decreased the intensity of p130Cas and changed its localization from peripheral cytoplasm into peri-nuclear areas in a concentrated pattern in podocytes. Ang II also reduced the amount of p130Cas in time and dose-sensitive manners. AMPK activators, metformin and AICAR, restored the suppressed and mal-localized p130Cas significantly, whereas, compound C, an AMPK inhibitor, further aggravated the changes of p130Cas. Losartan, an Ang II type 1 receptor antagonist, recovered the abnormal changes of p130Cas suppressed by Ang II. These results suggest that Ang II induces the relocalization and suppression of podocyte p130Cas by the suppression of AMPK via Ang II type 1 receptor, which would contribute to Ang II-induced podocyte injury.     

Effects of Angiotensin II on sustained outward currents in rat ventricular myocytes

We investigated the effects of angiotensin II (Ang II) on the sustained outward current (I _sus) and action potential of rat ventricular myocytes using the whole-cell patch-clamp technique. Ang II at 30 nM~3 µM inhibited I _sus with an IC₅₀ of 240 nM, a Hill coefficient of 1.0 and maximum inhibition of 19.4%. Ang II-mediated inhibition of I _sus was voltage independent, was due to a decrease in the K⁺ current and was abolished by the Ang II type-I (AT₁) receptor blocker, valsartan. The protein kinase C (PKC) inhibitors PKC19–36 or calphostin C, abolished Ang II-mediated inhibition of I _sus. In contrast, pretreatment with the protein kinase A (PKA) inhibitor PKA6–22 (100 µM) significantly enhanced the suppression of I _sus by 1 µM Ang II: (33.7±5.1% vs. control 17.1±2.3%). These results indicate that Ang II inhibits I _sus via the AT₁ receptor and activation of PKC. Ang II significantly prolonged action potential duration (APD) when the control APD was lengthened by a Ca²⁺ channel activator, BAY K8644. In myocytes with a relatively long APD, Ang II may prolong APD by inhibiting I _sus.     

Angiotensin II inhibits GABAergic synaptic transmission in dorsolateral periaqueductal gray neurons

The purpose of this study was to determine the role of angiotensin II (Ang II) in modulating inhibitory and excitatory synaptic inputs to the dorsolateral periaqueductal gray (dl-PAG). The whole cell voltage-clamp recording was performed to examine inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs) of the dl-PAG neurons. Ang II, at the concentration of 2 μM, decreased the frequency of miniature IPSCs from 0.83 ± 0.02 to 0.45 ± 0.03 Hz (P < 0.05) in 10 tested neurons. This did not significantly affect the amplitude and decay time constant. The effect of Ang II on miniature IPSCs was blocked by the prior application of Ang II AT1 receptor antagonist losartan, but not by AT2 receptor antagonist PD123319. Additionally, Ang II decreased the amplitude of evoked IPSCs from 148 ± 15 to 89 ± 7 pA (P < 0.05), and increased the paired-pulse ratio from 96 ± 5% to 125 ± 7% (P < 0.05) in eight tested neurons. In contrast, Ang II had no distinct effects on the EPSCs. Our data suggest that Ang II inhibits GABAergic synaptic inputs to the dl-PAG through activation of presynaptic AT1 receptors.