AT1 Receptor mRNA Research Articles

Abstract 481: Age-related Hypertension and Salt-sensitivity Are Associated With Specific Kidney Cortico-medullary Distribtion of D1R, AT1R And NADPH-oxidase

We recently reported that oxidative stress has a causal role in increasing kidney angiotensin II AT1 receptor (AT1R) and decreasing dopamine D1 receptor (D1R) functions in the aging Fischer Brown Norway (FBN) rats. These renal receptors by counter-regulating each other’s functions maintain sodium homeostasis and normal blood pressure (BP). Here, we examined systolic BP and kidney distribution (cortex vs medulla) of these receptors as well as of an oxidant producing enzyme (gp 91phox -NADPH-oxidase) in response to 4-week normal-salt (NS, 0.4% NaCl) and high-salt (HS, 8% NaCl) feeding in adult (3-month) and old (21-month) FBNs. BP in conscious animals was measured by radio-telemetry. Distribution of D1R, AT1R and gp 91phox were determined by RT-PCR and western blotting. We found that NS-fed old rats had higher BP (adult vs old: 105±1 vs 118 ±1 mmHg), which further increased with HS feeding in these rats (adult vs old: 111±1 vs 124 ±1 mmHg). The levels of D1R mRNA in cortex or in medulla were not different between NS-fed adult and old rats, but decreased in both cortex [adult vs old: 67 ± 15 vs 54±16 density unit (DU)] and medulla (adult vs old: 22±15 vs 11±3 DU) only in HS-fed old rats. Contrary to this, AT1R mRNA levels were higher only in cortex of old rats fed either NS (adult vs old: 81 ± 23 vs 102± 34 DU) or HS (adult vs old: 80 ± 17vs 98 ± 36 DU). Moreover, there were similar gp 91phox mRNA levels in the cortex and medulla of adult and old rats fed either NS or HS. But, its protein levels were higher in the cortex of old rats fed NS (adult vs old: 60±7 vs 112±8 DU) and HS (adult vs old: 61±3 vs 94±15 DU), and increased only in the medulla with HS feeding in these rats (adult vs old: 12±1 vs 28±2 DU). Our results suggest that there exists a specific cortico-medullary expression pattern of D1R, AT1R and gp 91phox with NS and HS feeding in old rats, which might be contributing to age-related hypertension and salt-sensitivity in FBNs.

Abstract 184: Deterioration of Kidney Function by the (Pro)renin Receptor Blocker Handle Region Peptide in Aliskiren-treated Diabetic Transgenic (mRen2)27 Rats

Objective Renal function in patients with diabetes mellitus (DM) might be influenced by the level of prorenin due to its binding to the (pro)renin receptor ((P)RR) and the subsequently occurring angiotensin production and/or the direct agonistic effects of prorenin mediated via this receptor. Indeed, in DM elevated prorenin levels are correlated with the development of microvascular complications such as nephropathy. Design and Methods In this study we evaluated renal function in diabetic TGR(mREN2)27 rats (a high prorenin hypertensive model), treated with vehicle, the renin inhibitor aliskiren, or aliskiren plus the putative (P)RR antagonist HRP for 3 weeks. 24-hour urine was collected, and blood and kidney were evaluated for renin-angiotensin system components and pathology. Results Increased diuresis and proteinuria due to DM were prevented by aliskiren, but not aliskiren+HRP. Aliskiren+HRP additionally decreased creatinine clearance, and increased the plasma levels of the profibrotic marker plasminogen-activator inhibitor-1. The increased natriuresis and renal collagen-1 expression in this model were unaffected by aliskiren±HRP. Aliskiren increased rat renin expression in the renal cortex. This was associated with a decline in (P)RR and AT 1 receptor mRNA expression, and these changes were unaffected by HRP. Glomerular volume and interlobar arterial lumen diameter modestly increased in the aliskiren+HRP group, and were unaffected by aliskiren alone. Conclusions HRP, when given on top of aliskiren in DM TGR(mREN2)27 rats worsens kidney damage and counteracts the beneficial effects of aliskiren. (P)RR blockade is therefore contraindicated in DM.

Tumor necrosis factor-α inhibits angiotensin II receptor type 1 expression in dorsal root ganglion neurons via β-catenin signaling

Both tumor necrosis factor (TNF)-α and the angiotensin (Ang) II/angiotensin II receptor type 1 (AT1) axis play important roles in neuropathic pain and nociception. In the present study, we explored the interaction between the two systems by examining the mutual effects between TNF-α and the Ang II/AT1 receptor axis in dorsal root ganglion (DRG) neurons. Rat DRG neurons were treated with TNF-α in different concentrations for different lengths of time in the presence or absence of transcription inhibitor actinomycin D, TNF receptor 1 (TNFR1) inhibitor SPD304, β-catenin signaling inhibitor CCT031374, or different kinase inhibitors. TNF-α decreased the AT1 receptor mRNA level as well as the AT1a receptor promoter activity in a dose-dependent manner within 30h, which led to dose-dependent inhibition of Ang II-binding AT1 receptor level on the cell membrane. Actinomycin D (1mg/ml), SPD304 (50μM), p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25μM), and CCT031374 (50μM) completely abolished the inhibitory effect of TNF-α on AT1 receptor expression. TNF-α dose-dependently increased soluble β-catenin and phosphorylated GSK-3β levels, which was blocked by SPD304 and PD169316. In DRG neurons treated with AT2 receptor agonist CGP421140, or Ang II with or without AT1 receptor antagonist losartan or AT2 receptor antagonist PD123319 for 30h, we found that Ang II and Ang II+PD123319 significantly decreased TNF-α expression, whereas CPG421140 and Ang II+losartan increased TNF-α expression. In conclusion, we demonstrate that TNF-α inhibits AT1 receptor expression at the transcription level via TNFR1 in rat DRG neurons by increasing the soluble β-catenin level through the p38 MAPK/GSK-3β pathway. In addition, Ang II appears to inhibit and induce TNF-α expression via the AT1 receptor and the AT2 receptor in DRG neurons, respectively. This is the first evidence of crosstalk between TNF-α and the Ang II/AT receptor axis in DRG neurons.

Expression of angiotensin II and its receptors in activated microglia in experimentally induced cerebral ischemia in the adult rats

Expression of angiotensin II (Ang II) and its receptors (AT1/AT2) is undetected in the mature microglia in normal brain. We report here that the immunoexpression of Ang II and AT1/AT2 was altered in activated microglia notably at 1 week in rats subjected to middle cerebral artery occlusion (MCAO). Immunolabeled activated microglia were widely distributed in the infarcted cerebral tissue after MCAO. By enzyme immunoassay, Ang II protein expression levels of the ischemic tissues were decreased drastically at 12 h after ischemia, then rose rapidly at 3 days and 1 week after MCAO when compared with the control. On the other hand, AT1 and AT2 receptor mRNA and protein levels were up-regulated after MCAO, peaking at 12 h, but declined thereafter. Expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) mRNA and protein levels was concomitantly increased. Edaravone significantly suppressed Ang II and AT1/AT2 receptor expression as well as that of TNF-α and IL-1β suggesting that microglia-derived Ang II can act through an autocrine manner via its receptor that may be linked partly to the production of proinflammatory cytokines. We conclude that neuroinflammation in MCAO may be attenuated by Edaravone which acts through suppression of expression of Ang II and its receptors and proinflammatory cytokines in activated microglia.

Beneficial Effects of the Activation of the Angiotensin-(1–7) Mas Receptor in a Murine Model of Adriamycin-Induced Nephropathy

Angiotensin-(1–7) [Ang-(1–7)] is a biologically active heptapeptide that may counterbalance the physiological actions of angiotensin II (Ang II) within the renin-angiotensin system (RAS). Here, we evaluated whether activation of the Mas receptor with the oral agonist, AVE 0991, would have renoprotective effects in a model of adriamycin (ADR)-induced nephropathy. We also evaluated whether the Mas receptor contributed for the protective effects of treatment with AT1 receptor blockers. ADR (10 mg/kg) induced significant renal injury and dysfunction that was maximal at day 14 after injection. Treatment with the Mas receptor agonist AVE 0991 improved renal function parameters, reduced urinary protein loss and attenuated histological changes. Renoprotection was associated with reduction in urinary levels of TGF-β. Similar renoprotection was observed after treatment with the AT1 receptor antagonist, Losartan. AT1 and Mas receptor mRNA levels dropped after ADR administration and treatment with losartan reestablished the expression of Mas receptor and increased the expression of ACE2. ADR-induced nephropathy was similar in wild type (Mas+/+) and Mas knockout (Mas −/−) mice, suggesting there was no endogenous role for Mas receptor activation. However, treatment with Losartan was able to reduce renal injury only in Mas+/+, but not in Mas −/− mice. Therefore, these findings suggest that exogenous activation of the Mas receptor protects from ADR-induced nephropathy and contributes to the beneficial effects of AT1 receptor blockade. Medications which target specifically the ACE2/Ang-(1–7)/Mas axis may offer new therapeutic opportunities to treat human nephropathies.

Abstract 2628: The multifunctional protein staphylococcal nuclease domain containing-1 (SND1) promotes migration and invasion of hepatocellular carcinoma (HCC) cells by modulating angiotensin II type 1 receptor (AT1R) and transforming growth factor-β (TGF-β) s

Abstract Staphylococcal nuclease domain containing-1 (SND1) is a multifunctional protein that modulates transcription, mRNA splicing and stability, and miRNA function. We previously documented that SND1 is markedly overexpressed in human hepatocellular carcinoma (HCC) patients in a stage-specific manner and inhibition of SND1 in human HCC cells significantly abrogates in vitro proliferation and in vivo tumorigenesis in nude mice. In the present study we document that migration and invasion of human HCC cells are significantly augmented in SND1-overexpressing clones and significantly inhibited in SND1 shRNA-expressing clones. Differential gene expression analysis between control clone and SND1 shRNA expressing clones by Affymetrix microarray revealed downregulation of TGF-β downstream genes upon SND1 inhibition. Additionally, a human angiogenesis array identified plasminogen activator inhibitor-1 (PAI-1), a TGF-β downstream gene, to be upregulated by SND1 and this upregulation was confirmed by ELISA. TGF-β expression is regulated by angiotensin II type 1 receptors (AT1R) and previous studies have documented that SND1 increases AT1R expression by increasing AT1R mRNA stability. We hypothesized that increased AT1R expression, conferred by SND1 overexpression, might facilitate activation of TGF-β signaling and subsequently PAI-1-mediated increase in migration and invasion. Indeed, we observed that AT1R and TGF-β expression was significantly increased in SND1-overexpressing clones and decreased in SND1 knockdown clones. The half-life of AT1R mRNA was significantly longer in SND1-overexpressing clones compared to the control clone confirming that SND1 modulates AT1R post-transcriptionally. The AT1R blocker Losartan Potassium and AT1R siRNA significantly inhibited SND1-induced TGF-β and PAI-1 expression with associated inhibition in migration and invasion. As a corollary, inhibition of PAI-1 by siRNA significantly abrogated migration and invasion of SND1-overexpressing cells. TGF-β induces epithelial-mesenchymal transition (EMT) and we observed increased expression of EMT markers vimentin, snail and slug and decreased expression of E-cadherin in SND1-overexpressing clones and vice versa in SND1-knockdown clones. Our studies thus unravel a novel mechanism in which post-transcriptional regulation of AT1R by SND1, with subsequent activation of TGF-β signaling, promotes invasion of human HCC cells thereby conferring an aggressive phenotype. Small molecule inhibitors of SND1, thus, might be effective therapeutic strategy for late-stage aggressive HCC. Citation Format: Prasanna K. Santhekadur, Rachel Gredler, Maaged Akiel, Paul Dent, Paul B. Fisher, Devanand Sarkar. The multifunctional protein staphylococcal nuclease domain containing-1 (SND1) promotes migration and invasion of hepatocellular carcinoma (HCC) cells by modulating angiotensin II type 1 receptor (AT1R) and transforming growth factor-β (TGF-β) s [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2628. doi:10.1158/1538-7445.AM2013-2628

Improved Glucose-Stimulated Insulin Secretion by Selective Intraislet Inhibition of Angiotensin II Type 1 Receptor Expression in Isolated Islets of db/db Mice

Recent evidence supported the presence of a local renin-angiotensin system (RAS) in the pancreas, which is implicated in many physiological and pathophysiological processes. We utilized small interfering RNA (siRNA) to investigate the effects of angiotensin II type 1 receptor (AT1R) knockdown on glucose-stimulated insulin secretion (GSIS) in isolated islets of db/db mice and to explore the potential mechanisms involved. We found that Ad-siAT1R treatment resulted in a significant decrease both in AT1R mRNA level and in AT1R protein expression level. With downexpression of AT1R, notable increased insulin secretion and decreased glucagon secretion levels were found by perifusion. Simultaneously, significant increased protein levels of IRS-1 (by 85%), IRS-2 (by 95%), PI3K(85) (by 112.5%), and p-Akt2 (by 164%) were found by western blot. And upregulation of both GLUT-2 (by 190%) and GCK (by 121%) was achieved after AT1R inhibition by Ad-siAT1R. Intraislet AT1R expression level is a crucial physiological regulator of insulin sensitivity of β cell itself and thus affects glucose-induced insulin and glucagon release. Therefore, the characteristics of AT1R inhibitors could make it a potential novel therapeutics for prevention and treatment of type 2 diabetes.

Effects of a Novel Bradykinin B1 Receptor Antagonist and Angiotensin II Receptor Blockade on Experimental Myocardial Infarction in Rats

BackgroundThe aim of the present study was to evaluate the cardiovascular effects of the novel bradykinin B1 receptor antagonist BI-113823 following myocardial infarction (MI) and to determine whether B1 receptor blockade alters the cardiovascular effects of an angiotensin II type 1 (AT1) receptor antagonist after MI in rats.Methodology/Principal FindingsSprague Dawley rats were subjected to permanent occlusion of the left descending coronary artery. Cardiovascular function was determined at 7 days post MI. Treatment with either B1 receptor antagonist (BI-113823) or AT1 receptor antagonist (irbesartan) alone or in combination improved post-MI cardiac function as evidenced by attenuation of elevated left ventricular end diastolic pressure (LVEDP); greater first derivative of left ventricular pressure (± dp/dt max), left ventricle ejection fraction, fractional shorting, and better wall motion; as we as reductions in post-MI up-regulation of matrix metalloproteinases 2 (MMP-2) and collagen III. In addition, the cardiac up-regulation of B1 receptor and AT1 receptor mRNA were markedly reduced in animals treated with BI 113823, although bradykinin B2 receptor and angiotensin 1 converting enzyme (ACE1) mRNA expression were not significantly affected by B1 receptor blockade.Conclusions/SignificanceThe present study demonstrates that treatment with the novel B1 receptor antagonist, BI-113823 improves post-MI cardiac function and does not influence the cardiovascular effects of AT1 receptor antagonist following MI.

Effect of lipopolysaccharide on angiotensin II type 1 receptor expression and function in human pulmonary microvascular endothelial cells.

Lipopolysaccharides (LPSs) are an important initiation factor in acute respiratory distress syndrome. The aim of the present study was to investigate the effect of LPSs on the regulation of angiotensinII (AngII) receptors in human pulmonary microvascular endothelial cells (HPMECs). HPMECs were treated with 0, 50, 100 or 200ng/ml LPS and incubated for 4, 8, 12 or 16h. The expression of AngII type1 receptor (AT1R) and AngII type2 receptor (AT2R) was determined using reverse transcription‑polymerase chain reaction and western blot analysis. The affinity to AngII was measured using a radioligand binding assay. No AT2R expression was detected with or without LPS administration in HPMECs, and LPS treatment increased the expression level of AT1R. A time‑dependent increase of AT1R transcription was observed in the 50ng/ml group, while in the 100 and 200ng/ml groups, the AT1R mRNA levels reached peak values at 4h and remained unchanged. The protein level of AT1R increased significantly in a dose‑dependent manner for each incubation time period. A time‑dependent increase in the protein level was observed in the 50 and 100ng/ml groups. As for the 200ng/ml group, the level of AT1R reached a peak value at 8h. Maximal binding (Bmax) significantly increased following LPS treatment and Bmax of the 50ng/ml group exhibited a time‑dependent increase. The Bmax of the100 and 200ng/ml groups reached peak values at 12 and 8h, respectively, and decreased thereafter. The dissociation constant remained unchanged following LPS treatment. Thus, treatment with LPS increased AT1R expression and its Bmax to AngII in HPMECs, however, did not alter the affinity of AT1R to AngII.

Abstract 395: Regulation of G Protein-coupled Receptor Kinase 4 on AT1 Receptor Expression and Function in Artery

G protein-coupled receptor kinases (GRKs) constitute a family of seven mammalian serine/threonine protein kinases that phosphorylate/modify agonist-bound or activated G protein-coupled receptors (GPCRs) as their primary substrates. GRK4 is distinguished from other members of the GPCR family by its constitutive activity and limited tissue expression. Our previous study found that GRK4 variants (A142V, A486V and R65L) increases GRK4 activity; A142V transgenic mice develop hypertension with impaired sodium excretion by impairment of renal D 1 dopamine receptor and augmented renal AT 1 receptor expression and function. Whether there is GRK4 in the artery is not known. Our preliminary study found GRK4 expression in the artery, determined by immunoblotting and histochemistry. We hypothesize that GRK4 might also regulate AT 1 receptor expression and function in vascular smooth muscle cells (VSMCs). A10 cells, an embryonic thoracic aortic smooth muscle cell lines, were transfected with A142V in this study. It resulted A142V increased AT 1 receptor protein and mRNA expressions in A10 cells, meanwhile, decreased AT 1 receptor phosphorylation. The regulation of A142V on AT 1 receptor is physiological significance, because the angiotensin II-mediated VSMC proliferation was greater in the A142V transfected cells than in the control cells. Moreover, the vasoconstrictive effect of angiotensin II was greater in A142 transgenic mice than control mice. The above-mentioned phenomenon is confirmed by siRNA, GRK4 siRNA decreased AT 1 receptor expression, increased AT 1 receptor phosphorylation in primary cultured VSMCs from spontaneously hypertensive rats. Our further study found that there was co-localization between GRK4 and AT 1 receptor in A10 cells, transfected with A142V increased the linkage of GRK4/AT 1 receptor. It indicated that arterial GRK4 regulation of AT 1 receptor expression and function, which might be engaged in the pathogenesis of hypertension.

Regulation of angiotensin II type 2 receptor gene expression in the adrenal medulla by acute and repeated immobilization stress

While the renin-angiotensin system is important for adrenomedullary responses to stress, the involvement of specific angiotensin II (Ang II) receptor subtypes is unclear. We examined gene expression changes of angiotensin II type 1A (AT(1A)) and type 2 (AT(2)) receptors in rat adrenal medulla in response to immobilization stress (IMO). AT(2) receptor mRNA levels decreased immediately after a single 2-h IMO. Repeated IMO also decreased AT(2) receptor mRNA levels, but the decline was more transient. AT(1A) receptor mRNA levels were unaltered with either single or repeated IMO, although binding was increased following repeated IMO. These effects of stress on Ang II receptor expression may alter catecholamine biosynthesis, as tyrosine hydroxylase and dopamine β-hydroxylase mRNA levels in PC12 cells are decreased with Ang II treatment in the presence of ZD7155 (AT(1) receptor antagonist) or with CGP42112 (AT(2) receptor agonist) treatment. Involvement of stress-triggered activation of the hypothalamic-pituitary-adrenocortical or sympathoadrenal axis in AT(2) receptor downregulation was examined. Cultured cells treated with the synthetic glucocorticoid dexamethasone displayed a transcriptionally mediated decrease in AT(2) receptor mRNA levels. However, glucocorticoids are not required for the immediate stress-triggered decrease in AT(2) receptor gene expression, as demonstrated in corticotropin-releasing hormone knockout (Crh KO) mice and hypophysectomized rats, although they can regulate basal gene expression. cAMP and pituitary adenylate cyclase-activating polypeptide also reduced AT(2) receptor gene expression and may mediate this response. Overall, the effects of stress on adrenomedullary AT(1A) and AT(2) receptor expression may contribute to allostatic changes, such as regulation of catecholamine biosynthesis.

Angiotensin II Type 2 Receptor–Mediated Inhibition of NaCl Absorption Is Blunted in Thick Ascending Limbs From Dahl Salt-Sensitive Rats

NO reduces NaCl absorption by thick ascending limbs (TALs) by inhibiting the Na/K/2Cl cotransporter (NKCC2). We have shown that NO-induced inhibition of Na transport is reduced in Dahl salt-sensitive rat (SS) TALs. Angiotensin II increases NO production in TALs via angiotensin II type 2 receptor (AT(2)R). It is unknown whether AT(2)Rs regulate TAL NaCl absorption and whether this effect is reduced in SS rats. We hypothesized that AT(2)R activation decreases TAL Na transport via NO, and this effect is blunted in SS rats. In the presence of angiotensin II type 1 receptor antagonist losartan, AT(2)R activation with angiotensin II inhibited NKCC2 activity by 32±7% (P<0.03). AT(2)R antagonist PD-123319 abolished the effect of angiotensin II. Activation with the AT(2)R-selective agonist CGP42112A (10 nmol/L) decreased NKCC2 activity by 29±6% (P<0.03). The effect of CGP42112A on NKCC2 activity was blocked by PD-123319 and by NO synthase inhibitor N(G)-nitro-l-arginine methyl ester. In Dahl salt-resistant rat TALs, 1 nmol/L of CGP42112A decreased NKCC2 activity by 23±4% (P<0.01). In SS TALs, it had no effect. TAL AT(2)R mRNA did not differ in SS versus salt-resistant rats. We conclude the following: (1) TAL AT(2)R activation decreases Na absorption; (2) this effect is mediated by AT(2)R-induced stimulation of NO; (3) AT(2)R-induced reduction of NKCC2 activity is blunted in SS rats; and (4) defects in AT(2)R/NO signaling rather than decreased AT(2)R expression likely account for the blunted effect in SS TALs. Impaired AT(2)R-mediated signaling in TALs could contribute to the Na retention associated with salt-sensitive hypertension.

Telmisartan directly ameliorates the neuronal inflammatory response to IL-1β partly through the JNK/c-Jun and NADPH oxidase pathways

BackgroundBlockade of angiotensin II type 1 (AT1) receptors ameliorates brain inflammation, and reduces excessive brain interleukin-1 beta (IL-1β) production and release from cortical microglia. The aim of this study was to determine whether, in addition, AT1 receptor blockade directly attenuates IL-1β-induced inflammatory responses in neuronal cultures.MethodsSK-N-SH human neuroblasts and primary rat cortical neurons were pretreated with telmisartan followed by exposure to IL-1β. Gene expression was determined by reverse transcriptase (RT)-PCR, protein expression and kinase activation by western blotting, NADPH oxidase activity by the lucigenin method, prostaglandin E2 (PGE2) release by enzyme immunoassay, reactive oxygen species (ROS) generation by the dichlorodihydrofluorescein diacetate fluorescent probe assay, and peroxisome proliferator-activated receptor gamma (PPARγ) involvement was assessed with the antagonists GW9662 and T0070907, the agonist pioglitazone and the expression of PPARγ target genes ABCG1 and CD36.ResultsWe found that SK-N-SH neuroblasts expressed AT1 but not AT2 receptor mRNA. Telmisartan reduced IL-1β-induced cyclooxygenase-2 (COX-2) expression and PGE2 release more potently than did candesartan and losartan. Telmisartan reduced the IL-1β-induced increase in IL-1R1 receptor and NADPH oxidase-4 (NOX-4) mRNA expression, NADPH oxidase activity, and ROS generation, and reduced hydrogen peroxide-induced COX-2 gene expression. Telmisartan did not modify IL-1β-induced ERK1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation or nuclear factor-κB activation but significantly decreased IL-1β-induced c-Jun N-terminal kinase (JNK) and c-Jun activation. The JNK inhibitor SP600125 decreased IL-1β-induced PGE2 release with a potency similar to that of telmisartan. The PPARγ agonist pioglitazone reduced IL-1β-induced inflammatory reaction, whereas telmisartan did not activate PPARγ, as shown by its failure to enhance the expression of the PPARγ target genes ABCG1 and CD36, and the inability of the PPARγ antagonists GW9662 and T0070907 to modify the effect of telmisartan on COX-2 induction. The effect of telmisartan on IL-1β-stimulated COX-2 and IL-1R1 mRNA expression and ROS production was replicated in primary rat cortical neurons.ConclusionsTelmisartan directly ameliorates IL-1β-induced neuronal inflammatory response by inhibition of oxidative stress and the JNK/c-Jun pathway. Our results support the hypothesis that AT1 receptor blockers are directly neuroprotective, and should be considered for the treatment of inflammatory conditions of the brain.

New insight into the mechanisms associated with the rapid effect of T3 on AT1R expression

The angiotensin II type 1 receptor (AT1R) is involved in the development of cardiac hypertrophy promoted by thyroid hormone. Recently, we demonstrated that triiodothyronine (T₃) rapidly increases AT1R mRNA and protein levels in cardiomyocyte cultures. However, the molecular mechanisms responsible for these rapid events are not yet known. In this study, we investigated the T₃ effect on AT1R mRNA polyadenylation in cultured cardiomyocytes as well as on the expression of microRNA-350 (miR-350), which targets AT1R mRNA. The transcriptional and translational actions mediated by T₃ on AT1R levels were also assessed. The total content of ubiquitinated proteins in cardiomyocytes treated with T₃ was investigated. Our data confirmed that T₃ rapidly raised AT1R mRNA and protein levels, as assessed by real-time PCR and western blotting respectively. The use of inhibitors of mRNA and protein synthesis prevented the rapid increase in AT1R protein levels mediated by T₃. In addition, T₃ rapidly increased the poly-A tail length of the AT1R mRNA, as determined by rapid amplification of cDNA ends poly-A test, and decreased the content of ubiquitinated proteins in cardiomyocytes. On the other hand, T₃ treatment increased miR-350 expression. In parallel with its transcriptional and translational effects on the AT1R, T₃ exerted a rapid posttranscriptional action on AT1R mRNA polyadenylation, which might be contributing to increase transcript stability, as well as on translational efficiency, resulting to the rapid increase in AT1R mRNA expression and protein levels. Finally, these results show, for the first time, that T₃ rapidly triggers distinct mechanisms, which might contribute to the regulation of AT1R levels in cardiomyocytes.

Role of mitochondria in paricalcitol-mediated cytoprotection during obstructive nephropathy

Vitamin D slows the progression of chronic kidney disease. Furthermore, activators of vitamin D receptors (VDR) have suppressant effects on the renin-angiotensin system, as well as anti-inflammatory and antifibrotic actions. This study aimed to evaluate the cytoprotective effects of paricalcitol, a VDR activator, at the mitochondrial level using an obstructive nephropathy model [unilateral ureteral obstruction (UUO)]. Rats subjected to UUO and controls were treated daily with vehicle or paricalcitol. The control group underwent a sham surgery. The treatment was done for 15 days (30 ng/kg). The following were determined: biochemical parameters; fibrosis; apoptosis; mitochondrial morphology; VDR, AT(1) receptor, and NADPH oxidase 4 expression; and NADPH oxidase activity (in total and in mitochondrial fractions from the renal cortex). VDR activation prevented fibrosis (20 ± 5 vs. 60 ± 10%) and the number of TUNEL-positive apoptotic cells (10 ± 3 vs. 25 ± 4) in UUO. Biochemical, histological, and molecular studies suggest mitochondrial injury. Electron microscopy revealed in UUO electronically luminous material in the nucleus. Some mitochondria were increased in size and contained dilated crests and larger than normal spaces in their interiors. These changes were not present with paricalcitol treatment. Additionally, high AT(1)-receptor mRNA and NADPH activity was reverted in mitochondrial fractions from obstructed paricalcitol-treated animals (0.58 ± 0.06 vs. 0.95 ± 0.05 relative densitometry units and 9,000 ± 800 vs. 15,000 ± 1,000 relative fluorescence units·μg protein(-1)·min(-1), respectively). These changes were consistent with an improvement in VDR expression (0.75 ± 0.05 vs. 0.35 ± 0.04 relative densitometry units). These results suggest that paricalcitol confers a protective effect and reveal, as well, a possible AT(1) receptor-dependent protective effect that occurs at the mitochondrial level.

Regulation of AT1R expression through HuR by insulin

Angiotensin II type 1 receptor (AT1R) has a pathophysiological role in hypertension, atherosclerosis and heart failure. Type 2 diabetes is hyperinsulinemic state and a major risk factor for atherosclerosis and hypertension. It is known that hyperinsulinemia upregulates AT1R expression post-transcriptionally by increasing the half-life of AT1R mRNA, but little is known about the mechanism of this effect. In the present study, we first identified AT1R 3′-UTR as a mediator of insulin effect. Using 3′-UTR as a bait, we identified through analysis of insulin-stimulated cell lysates by affinity purification and mass spectrometry HuR as an insulin-regulated AT1R mRNA binding protein. By ribonucleoprotein immunoprecipitation, we found HuR binding to AT1R to be increased by insulin. Overexpression of HuR leads to increased AT1R expression in a 3′-UTR-dependent manner. Both insulin and HuR overexpression stabilize AT1R 3′-UTR and their responsive element within 3′-UTR are located within the same region. Cell fractionation demonstrated that insulin induced HuR translocation from nucleus to cytoplasm increased HuR binding to cytoplasmic AT1R 3′-UTR. Consistent with HuR translocation playing a mechanistic role in HuR effect, a reduction in the cytoplasmic levels of HuR either by silencing of HuR expression or by inhibition of HuR translocation into cytoplasm attenuated insulin response. These results show that HuR translocation to cytoplasm is enhanced by insulin leading to AT1R upregulation through HuR-mediated stabilization of AT1R mRNA.

Testosterone improves cardiac function and alters angiotensin II receptors in isoproterenol-induced heart failure

The renin-angiotensin-aldosterone system is known to play an important role in the pathophysiology and development of heart failure. Several studies have reported the benefits of testosterone in heart failure. However, the mechanisms of testosterone-induced effects on heart failure require further study. To determine the effects of castration and testosterone administration on cardiac function and angiotensin II receptor function in rats with isoproterenol-induced heart failure. Wistar rats were divided randomly into control and heart failure groups. The heart failure groups were further divided into the following groups: castration; castration+testosterone replacement; and sham castration. Echocardiography and haemodynamic measurements were used to evaluate cardiac function. Cardiocyte apoptosis and fibrosis were determined using terminal deoxyribonucleotide transferase-mediated dUTP nick-end labelling (TUNEL) staining and Masson's Trichrome staining, respectively. Angiotensin II receptor (AT1 and AT2) messenger ribonucleic acid (mRNA) expression levels were assayed using real-time reverse transcriptase-polymerase chain reactions, while Western immunoblotting was used to estimate Bcl-2 protein expression levels. Castration significantly increased cardiomyocyte apoptosis and fibrosis that was normally induced by isoproterenol (P<0.05). AT2 receptor mRNA expression in the castration group was increased and Bcl-2 protein expression was decreased compared with the castration+testosterone replacement group (P<0.05). These data suggest that androgen therapy could play an important role in pathophysiological changes in heart failure and have beneficial effects for its treatment.

Inhibition of cerebrovascular raf activation attenuates cerebral blood flow and prevents upregulation of contractile receptors after subarachnoid hemorrhage

BackgroundLate cerebral ischemia carries high morbidity and mortality after subarachnoid hemorrhage (SAH) due to reduced cerebral blood flow (CBF) and the subsequent cerebral ischemia which is associated with upregulation of contractile receptors in the vascular smooth muscle cells (SMC) via activation of mitogen-activated protein kinase (MAPK) of the extracellular signal-regulated kinase (ERK)1/2 signal pathway. We hypothesize that SAH initiates cerebrovascular ERK1/2 activation, resulting in receptor upregulation. The raf inhibitor will inhibit the molecular events upstream ERK1/2 and may provide a therapeutic window for treatment of cerebral ischemia after SAH.ResultsHere we demonstrate that SAH increases the phosphorylation level of ERK1/2 in cerebral vessels and reduces the neurology score in rats in additional with the CBF measured by an autoradiographic method. The intracisternal administration of SB-386023-b, a specific inhibitor of raf, given 6 h after SAH, aborts the receptor changes and protects the brain from the development of late cerebral ischemia at 48 h. This is accompanied by reduced phosphorylation of ERK1/2 in cerebrovascular SMC. SAH per se enhances contractile responses to endothelin-1 (ET-1), 5-carboxamidotryptamine (5-CT) and angiotensin II (Ang II), upregulates ETB, 5-HT1B and AT1 receptor mRNA and protein levels. Treatment with SB-386023-b given as late as at 6 h but not at 12 h after the SAH significantly decreased the receptor upregulation, the reduction in CBF and the neurology score.ConclusionThese results provide evidence for a role of the ERK1/2 pathway in regulation of expression of cerebrovascular SMC receptors. It is suggested that raf inhibition may reduce late cerebral ischemia after SAH and provides a realistic time window for therapy.

Inhibition of Prolyl Hydroxylase Domain-Containing Protein Downregulates Vascular Angiotensin II Type 1 Receptor

Inhibition of prolyl hydroxylase domain-containing protein (PHD) by hypoxia stabilizes hypoxia-inducible factor 1 and increases the expression of target genes, such as vascular endothelial growth factor. Although the systemic renin-angiotensin system is activated by hypoxia, the role of PHD in the regulation of the renin-angiotensin system remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin II type 1 receptor (AT(1)R). Hypoxia, cobalt chloride, and dimethyloxalylglycine, all known to inhibit PHD, reduced AT(1)R expression in vascular smooth muscle cells. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT(1)R expression. Cobalt chloride diminished angiotensin II-induced extracellular signal-regulated kinase phosphorylation. Cobalt chloride decreased AT(1)R mRNA through transcriptional and posttranscriptional mechanisms. Oral administration of cobalt chloride (14 mg/kg per day) to C57BL/6J mice receiving angiotensin II infusion (490 ng/kg per minute) for 4 weeks significantly attenuated perivascular fibrosis of the coronary arteries without affecting blood pressure level. These data suggest that PHD inhibition may be beneficial for the treatment of cardiovascular diseases by inhibiting renin-angiotensin system via AT(1)R downregulation.

Involvement of angiotensin II type 1 receptors in interleukin-1β-induced interleukin-6 production in human gingival fibroblasts

The renin-angiotensin system is thought to be involved in inflammatory processes such as periodontitis. However, its precise role is still unclear. Therefore, in the present study the expression of the angiotensin II type 1 receptor (AT1R) was investigated in inflamed human gingival tissue, and the possible involvement of the AT1R in interleukin-1β (IL-1β)-induced interleukin-6 (IL-6) production by cultured human gingival fibroblasts (HGFs) was also studied. Immunohistochemical staining revealed that inflammatory cells and fibroblast-like cells were positive for the AT1R. However, in healthy gingival tissue, AT1R staining was very weak. The levels of AT1R mRNA and AT1R protein increased in HGFs after stimulation with IL-1β. The levels of IL-1β-induced IL6 mRNA and IL-6 protein were significantly reduced in AT1R gene-silenced HGFs compared with control HGFs. The data suggest that the AT1R may be involved in the regulation of gingival inflammation by modulating IL-1β-induced IL-6 production in HGFs.