AT1A Receptor Research Articles

A different role of angiotensin II type 1a receptor in the development and hypertrophy of plantaris muscle in mice.

The role of angiotensin II type 1 (AT1) receptors in muscle development and hypertrophy remains unclear. This study was designed to reveal the effects that a loss of AT1 receptors has on skeletal muscle development and hypertrophy in mice. Eight-week-old male AT1a receptor knockout (AT1a(-/-)) mice were used for this experiment. The plantaris muscle to body weight ratio, muscle fiber cross-sectional area, and number of muscle fibers of AT1a(-/-) mice was significantly greater than wild type (WT) mice in the non-intervention condition. Next, the functional overload (OL) model was used to induce plantaris muscle hypertrophy by surgically removing the two triceps muscles consisting of the calf, soleus, and gastrocnemius muscles in mice. After 14 days of OL intervention, the plantaris muscle weight, the amount of fiber, and the fiber area increased. However, the magnitude of the increment of plantaris weight was not different between the two strains. Agtr1a mRNA expression did not change after OL in WT muscle. Actually, the Agt mRNA expression level of WT-OL was lower than WT-Control (C) muscle. An atrophy-related gene, atrogin-1 mRNA expression levels of AT1a(-/-)-C, WT-OL, and AT1a(-/-)-OL muscle were lower than that of WT-C muscle. Our findings suggest that AT1 receptor contributes to plantaris muscle development via atrogin-1 in mice.

Inhibition of Sphingosine Kinase 1 Ameliorates Angiotensin II-Induced Hypertension and Inhibits Transmembrane Calcium Entry via Store-Operated Calcium Channel.

Angiotensin II (AngII) plays a critical role in the regulation of vascular tone and blood pressure mainly via regulation of Ca(2+) mobilization. Several reports have implicated sphingosine kinase 1 (SK1)/sphingosine 1-phosphate (S1P) in the mobilization of intracellular Ca(2+) through a yet-undefined mechanism. Here we demonstrate that AngII-induces biphasic calcium entry in vascular smooth muscle cells, consisting of an immediate peak due to inositol tris-phosphate-dependent release of intracellular calcium, followed by a sustained transmembrane Ca(2+) influx through store-operated calcium channels (SOCs). Inhibition of SK1 attenuates the second phase of transmembrane Ca(2+) influx, suggesting a role for SK1 in AngII-dependent activation of SOC. Intracellular S1P triggers SOC-dependent Ca(2+) influx independent of S1P receptors, whereas external application of S1P stimulated S1P receptor-dependent Ca(2+) influx that is insensitive to inhibitors of SOCs, suggesting that the SK1/S1P axis regulates store-operated calcium entry via intracellular rather than extracellular actions. Genetic deletion of SK1 significantly inhibits both the acute hypertensive response to AngII in anaesthetized SK1 knockout mice and the sustained hypertensive response to continuous infusion of AngII in conscious animals. Collectively these data implicate SK1 as the missing link that connects the angiotensin AT1A receptor to transmembrane Ca(2+) influx and identify SOCs as a potential intracellular target for SK1.

Vascular Type 1A Angiotensin II Receptors Control BP by Regulating Renal Blood Flow and Urinary Sodium Excretion

Inappropriate activation of the type 1A angiotensin (AT1A) receptor contributes to the pathogenesis of hypertension and its associated complications. To define the role for actions of vascular AT1A receptors in BP regulation and hypertension pathogenesis, we generated mice with cell-specific deletion of AT1A receptors in smooth muscle cells (SMKO mice) using Loxp technology and Cre transgenes with robust expression in both conductance and resistance arteries. We found that elimination of AT1A receptors from vascular smooth muscle cells (VSMCs) caused a modest (approximately 7 mmHg) yet significant reduction in baseline BP and exaggerated sodium sensitivity in mice. Additionally, the severity of angiotensin II (Ang II)-dependent hypertension was dramatically attenuated in SMKO mice, and this protection against hypertension was associated with enhanced urinary excretion of sodium. Despite the lower BP, acute vasoconstrictor responses to Ang II in the systemic vasculature were largely preserved (approximately 80% of control levels) in SMKO mice because of exaggerated activity of the sympathetic nervous system rather than residual actions of AT1B receptors. In contrast, Ang II-dependent responses in the renal circulation were almost completely eliminated in SMKO mice (approximately 5%-10% of control levels). These findings suggest that direct actions of AT1A receptors in VSMCs are essential for regulation of renal blood flow by Ang II and highlight the capacity of Ang II-dependent vascular responses in the kidney to effect natriuresis and BP control.

Activation of the D4 dopamine receptor attenuates proliferation and migration of vascular smooth muscle cells through downregulation of AT1a receptor expression.

Angiotensin (Ang) II has an important role in the vascular smooth muscle cell (VSMC) proliferation and migration and subsequently in the development of vascular diseases, whereas dopamine has the opposite effect. Previous studies have shown an interaction between dopamine and AT(1) receptors in the kidney. The dopamine D(4) receptor is expressed in arteries and has an inhibitory effect on VSMC proliferation. We hypothesized that the D(4) receptor, through its interaction with the AT(1a) receptor, may have an inhibitory effect on Ang II-mediated VSMC proliferation and migration, which could have a pivotal role in hypertension-induced vascular remodeling. In the current study, we found that Ang II markedly induced the proliferation and migration of A10 cells, which was inhibited by the D(4) receptor agonist PD168077. The activation of the D(4) receptor by PD168077 inhibited AT(1a) receptor expression in a concentration- and time-dependent manner. These effects were attenuated by silencing the D(4) receptor with a D(4) receptor-targeting small interfering RNA. The D(4) receptor-mediated inhibition of AT(1) receptor function involved protein kinase A (PKA). The activation of the D(4) receptor by PD168077 increased PKA activity in A10 cells, and the presence of a PKA inhibitor (PKA inhibitor 14-22, 10(-7) mol l(-1) per 24 h) blocked the inhibitory effect of the D(4) receptor on AT(1) receptor expression and function. The inhibitory effect of the D(4) receptor on AT(1) receptor expression and function was preserved in VSMCs (primary culture) from spontaneously hypertensive rats relative to VSMCs from Wistar-Kyoto rats. In conclusion, our data provide insight into the regulatory role of the D(4) receptor on AT(1a) receptor expression and function in VSMCs and suggest that targeting the action of the D(4) receptor may represent an effective therapeutic approach for the treatment of cardiovascular diseases.

AT1a Receptor Upregulation in the MnPO Following CIH Leads to Sustained Hypertension

Angiotensin type 1 (AT1) receptors play a central role in many forms of neurogenic hypertension. Two distinct subtypes, At1a and AT1b, exist for AT1 receptors. Activation of the AT1a subtype is characterized by increased sympathetic nerve activity and hypertension. The Median Preoptic Nucleus (MnPO) expresses both subtypes of the angiotensin receptor and our lab has previously demonstrated that AT1a message is increased in MnPO brain punches following CIH. This study sought to determine if changes in expression of the angiotensin receptor subtypes play a role in the sustained hypertension that develops from chronic intermittent hypoxia (CIH). We hypothesized that AT1a receptor expression increased and AT1b receptor expression remained unchanged resulting in the sustained elevation in blood pressure from CIH. Rats were microinjected in MnPO with an adeno‐associated virus loaded with a shRNA construct to prevent AT1a expression (shAT1a) or a scramble construct (shSCR). After recovery, rats were exposed to a 7 day CIH or normoxic protocol. Laser Capture Microdissection and qRTPCR was performed to determine the receptor subtype expression in MnPO with ribosomal subunit S18 as the control. shSCR rats exposed to CIH had significantly more AT1a RNA than their normoxic counterparts (P<.05), but their expression of AT1b remained constant. shAT1a CIH rats did not exhibit the sustained component of hypertension found in shSCR CIH animals and their MnPO AT1a message was reduced compared to control. Overall this indicates that the pathophysiological blood pressure increase during CIH is in part mediated by increases in AT1a receptor subtype expression in the MnPO.

Intracellular Renin in the Brain Contributes to Cardiovascular and Metabolic Control

Renin gene expression is regulated by two distinct promoter‐first exon combinations that target renin either for secretion (exon 1a initiates secreted renin) or cytoplasmic retention (exon 1b initiates intracellular renin, icREN). Interestingly, icREN is expressed predominantly in the brain. We selectively deleted exon 1b and developed icREN KO mice to examine the role of icREN in cardiovascular and metabolic control. Systolic blood pressure in the light phase as measured by telemetry was increased in KO mice (126±2 mmHg, n=8 vs 116±2 mmHg, n=7, P<0.01). The low‐ to high‐frequency ratio (LF/HF) derived from power spectral analysis of heart rate variability was increased in KO mice (1.24±0.21, n=7 vs 0.70±0.11, n=7, P<0.05). Body weight was normal in KO mice fed normal chow. However, high fat diet‐induced obesity was attenuated in male KO mice (body weight, 32.4±1.3 g, n=5 vs 36.6±1.4 g, n=9; relative fat mass, 26.1±2.3 %, n=5 vs 34.4±2.3 %, n=9, both P<0.05). The resting metabolic rate measured by respirometry was increased in KO mice (0.156±0.005 kcal/h, n=46, P<0.05) vs controls (0.145±0.003 kcal/h, n=53). We previously reported that the brain renin‐angiotensin system facilitates renal sympathetic nerve activity (RSNA) response to acute intracerebroventricular injection of leptin. Interestingly, the RSNA response to leptin was greater in KO mice compared to controls (214±40 % baseline, n=5 vs 114±18 % baseline, n=10, P<0.01). Further, in KO mice, AT1a receptor mRNA was upregulated in the paraventricular nucleus (P<0.05). These data suggest that this novel icREN isoform contributes to cardiovascular and metabolic control possibly through the modulation of AT1a receptor expression in the brain.

Brain Region‐Specific Expression of Angiotensin AT1A Receptors in Microglia

Through its actions at the AT1A receptor in the hypothalamus, the renin‐angiotensin system contributes to the control of resting metabolic rate and blood pressure. Activation of microglia is required for angiotensin‐dependent hypertension, and exposure to high fat diet increases microglia infiltration in the hypothalamus. We therefore hypothesized that angiotensin AT1A receptors are expressed on microglia within the hypothalamus, and that these cells may play a role in the development of obesity‐hypertension. Brains from mice expressing green fluorescent protein via the angiotensin AT1A promoter (“NZ44” mice from the GenSat Project) were sectioned and immunostained to label microglia via antibodies against allograft inflammatory factor 1 (IBA1) conjugated to red fluorescent protein. When mice were maintained on standard chow diet, we observed dual‐fluorescent cells within the ventromedial hypothalamus, but not in the arcuate nucleus, median eminence, paraventricular nucleus, dorsomedial hypothalamus, or subfornical organ despite substantial numbers of singly‐fluorescent cells in each of these regions. Together, these data support a region‐specific expression of AT1A in microglia. Ongoing studies are focused on investigating the effects of dietary interventions (e.g. – high fat diet) and hypertensive stimuli (e.g. – deoxycorticosterone acetate (DOCA)‐salt treatment) upon region‐specific microglial infiltration, activation, and expression of AT1A.

AVP-induced increase in AQP2 and p-AQP2 is blunted in heart failure during cardiac remodeling and is associated with decreased AT1R abundance in rat kidney.

AimThe objective was to examine the renal effects of long-term increased angiotensin II and vasopressin plasma levels in early-stage heart failure (HF). We investigated the regulations of the V2 vasopressin receptor, the type 1A angiotensin II receptor, the (pro)renin receptor, and the water channels AQP2, AQP1, AQP3, and AQP4 in the inner medulla of rat kidney.MethodsHF was induced by coronary artery ligation. Sixty-eight rats were allocated to six groups: Sham (N = 11), HF (N = 11), sodium restricted sham (N = 11), sodium restricted HF (N = 11), sodium restricted sham + DDAVP (N = 12), and sodium restricted HF + DDAVP (N = 12). 1-desamino-8-D-arginine vasopressin (0.5 ng h-1 for 7 days) or vehicle was administered. Pre- and post-treatment echocardiographic evaluation was performed. The rats were sacrificed at day 17 after surgery, before cardiac remodeling in rat is known to be completed.ResultsHF rats on standard sodium diet and sodium restriction displayed biochemical markers of HF. These rats developed hyponatremia, hypo-osmolality, and decreased fractional excretion of sodium. Increase of AQP2 and p(Ser256)-AQP2 abundance in all HF groups was blunted compared with control groups even when infused with DDAVP and despite increased vasopressin V2 receptor and Gsα abundance. This was associated with decreased protein abundance of the AT1A receptor in HF groups vs. controls.ConclusionEarly-stage HF is associated with blunted increase in AQP2 and p(Ser256)-AQP2 despite of hyponatremia, hypo-osmolality, and increased inner medullary vasopressin V2 receptor expression. Decreased type 1A angiotensin II receptor abundance likely plays a role in the transduction of these effects.

AT1 receptor antagonism to reduce aortic expansion in Marfan syndrome: lost in translation or in need of different interpretation?

Marfan syndrome is an inherited autosomal dominant disorder of the connective tissue caused by mutations in the gene encoding fibrillin-1 ( FBN1 ).1,2 This disease has affected the ATVB community with the loss of 1 of our highly respected members, David Williams.3 The most devastating health issues of patients with Marfan syndrome are dissection and rupture of the proximal aorta. Aortic dimensions are routinely monitored in patients with Marfan syndrome, and surgical graft imposition is performed to sustain life. However, surgical intervention is a formidable process. To abrogate surgical repair, there is a desperate need for a medical approach for attenuating aortic expansion. Presently, patients with Marfan syndrome are routinely provided with β-adrenoceptor blockade. This standard of care is derived from the study of Shores et al.4 However, the benefit derived from administration of β-adrenoceptor blockade is far from established. Although FBN1 is a component of extracellular matrix, mutations in this protein do not directly promote the structural fragility that characterizes the specific aortic regions of patients with Marfan syndrome. Instead, a concept has developed that Fbn1 mutations lead to enhanced transforming growth factor β (TGF-β) activation that is the mechanistic basis of Marfan pathology. This concept originated from the demonstration that Fbn1 deficiency led to excessive activation of TGF-β signaling because of reduced binding, sequestration, and inactivation of TGF-β.5 Activation of TGF-β in Fbn1 -deficient mice was associated with a lung pathology similar to that observed in some patients with Marfan syndrome. This paradigm of FBN1 regulation of TGF-β activation was extended to aortic disease by generating heterozygous mice that express a C1039G mutation.6 These mice develop pronounced dilation of the ascending aorta associated with thickened media containing disrupted elastin fibers. In this model, the major proof of concept for the role of …

Clathrin-dependent internalization of the angiotensin II AT1A receptor links receptor internalization to COX-2 protein expression in rat aortic vascular smooth muscle cells

The major effects of Angiotensin II (AngII) in vascular tissue are mediated by AngII AT1A receptor activation. Certain effects initiated by AT1A receptor activation require receptor internalization. In rat aortic vascular smooth muscle cells (RASMC), AngII stimulates cyclooxygenase 2 protein expression. We have previously shown this is mediated by β-arrestin-dependent receptor internalization and NF-κB activation. In this study, a specific inhibitor of clathrin-mediated endocytosis (CME), pitstop-2, was used to test the hypothesis that clathrin-dependent internalization of activated AT1A receptor mediates NF-κB activation and subsequent cyclooxygenase 2 expression. Radioligand binding assays, real time qt-PCR and immunoblotting were used to document the effects of pitstop-2 on AngII binding and signaling in RASMC. Laser scanning confocal microscopy (LSCM) was used to image pitstop-2׳s effects on AT1 receptor/GFP internalization in HEK-293 cells and p65 NF-κB nuclear localization in RASMC.Pitstop-2 significantly inhibited internalization of AT1A receptor (44.7%±3.1% Control vs. 13.2%±8.3% Pitstop-2; n=3) as determined by radioligand binding studies in RASMC. Studies utilizing AT1A receptor/GFP expressed in HEK 293 cells and LSCM confirmed these findings. Pitstop-2 significantly inhibited AngII-induced p65 NF-κB phosphorylation and nuclear localization, COX-2 message and protein expression in RASMC without altering activation of p42/44 ERK or TNFα signaling.Pitstop-2, a specific inhibitor of clathrin-mediated endocytosis, confirms that internalization of activated AT1A receptor mediates AngII activation of cyclooxygenase 2 expression in RASMC. These data provide support for additional intracellular signaling pathways activated through β-arrestin mediated internalization of G protein-coupled receptors, such as AT1A receptors.

Another chapter in the understanding of angiotensin-catecholamine interactions relevant to blood pressure control.

Another chapter in the understanding of angiotensin-catecholamine interactions relevant to blood pressure control.

Cysteinyl leukotriene 1 receptors as novel mechanosensors mediating myogenic tone together with angiotensin II type 1 receptors-brief report.

Myogenic vasoconstriction is mediated by vascular smooth muscle cells of resistance arteries sensing mechanical stretch. Angiotensin II AT1 receptors and in particular AT1BRs in murine vascular smooth muscle cells have been characterized as mechanosensors that cannot fully account for myogenic vasoconstriction observed. Therefore, we aimed at uncovering novel vascular mechanosensors by expression profiling and functional characterization of candidate proteins. Analyzing myogenic tone of isolated murine mesenteric arteries of AT1A and AT1B receptor double gene-deficient (AT1A/1B (-/-)) mice ex vivo, we observed a decreased myogenic tone at high intraluminal pressures and an unexpected hyper-reactivity at low intraluminal pressures because of upregulation of cysteinyl leukotriene 1 receptors (CysLT1Rs). Pharmacological blockade of CysLT1Rs with pranlukast significantly reduced myogenic tone not only in AT1A/1B (-/-) but also in wild-type arteries. In wild-type arteries, additional blockade of angiotensin II AT1 receptors with candesartan resulted in an additive reduction of myogenic tone. Furthermore, calcium imaging experiments were performed with fura-2-loaded human embryonic kidney 293 cells overexpressing CysLT1Rs and with isolated mesenteric vascular smooth muscle cells. Hypo-osmotically induced membrane stretch provoked calcium transients that were significantly reduced by pranlukast. Incubations of isolated mesenteric vascular smooth muscle cells with the 5-lipoxygenase inhibitor zileuton had no effect. Furthermore, the Gq/11-protein inhibitor YM 254890 profoundly reduced myogenic tone to the same extent as induced by the application of pranlukast plus candesartan. Here, we identify a novel, hitherto unappreciated role of CysLT1Rs in vascular regulation. We identified CysLT1Rs as novel mechanosensors in the vasculature involved in myogenic vasoconstriction. Moreover, our findings suggest that myogenic tone is determined by AT1 and CysLT1 receptors acting together as mechanosensors via Gq/11-protein activation.

Abstract 002: Activation of AT1 receptors on Dendritic Cells Restrains Angiotensin II-Dependent Hypertension Through a CCR7-dependent Mechanism

Dendritic cells (DCs) trigger an adaptive immune response by presenting processed antigens to T lymphocytes. Given the emerging importance of adaptive immunity in hypertension, we posited that DCs may sample neoantigens in the kidney in order to prime pro-hypertensive T cells. Using CD11c-Cre mT/mG mice in which DCs fluoresce green, we detected robust green signals within the kidney after 2 wks of angiotensin (Ang) II-induced hypertension, confirming infiltration of DCs into the hypertensive kidney. As activation of type 1 angiotensin (AT1) receptors on other immune cell lineages has been shown to alter inflammatory responses, we explored the role of the DC AT1 receptor in blood pressure regulation by generating mice with DC-specific deletion of the AT1A receptor (CD11c Cre+ Agtr1aflox/flox = DC KO) and littermate controls (Agtr1aflox/flox = WT). Compared to WTs, the DC KOs had an ≈87% reduction in AT1A receptor mRNA expression in DCs (p<0.0001) but preserved AT1A expression in B and T lymphocytes, kidney, and heart (p=NS). Next, we infused uni-nephrectomized DC KO mice and wild-type (WT) controls with Ang II (500ng/kg/min) for 4 wks. At baseline, the mean arterial blood pressure (MAP) was similar in WT and DC KO mice (123±2 vs. 126±2 mm Hg; p=NS). By contrast, after the 1st week of Ang II infusion, DC KO mice had markedly exaggerated MAP elevations compared to WTs (174±4 vs. 158±5 mm Hg; p=0.026). Expression of the chemokine receptor CCR7 on mature DCs and T cells directs both cell lineages to the lymph node where the DC can display a processed antigen and activate the T cell. We detected higher levels of CCR7 mRNA in the kidneys from the Ang II-infused DC KOs compared to WTs (2.98±0.90 vs. 1.00±0.16 au; p=0.04), and posited that enhanced DC priming of T cells in the DC KOs contributed to their exaggerated hypertension. Consistent with this possibility, uni-nephrectomized CCR7-deficient (KO) mice had a significantly blunted chronic hypertensive response to Ang II (165±4 vs. 178±2 mm Hg; p=0.03) and ameliorated albuminuria (2.1±0.2 vs. 3.7±0.8 mg/24hrs; p=0.02) versus WT controls. We conclude from these data that stimulation of AT1 receptors on DCs constrains Ang II-induced blood pressure elevation, possibly by limiting the CCR7-dependent activation of T lymphocytes by DCs.

Cardiovascular alterations at different stages of hypertension development during ethanol consumption: Time-course of vascular and autonomic changes

The aim of the present work was to establish a time-course correlation between vascular and autonomic changes that contribute to the development of hypertension during ethanol ingestion in rats. For this, male Wistar rats were subjected to the intake of increasing ethanol concentrations in their drinking water during four weeks. Ethanol effects were investigated at the end of each week. Mild hypertension was already observed at the first week of treatment, and a progressive blood pressure increase was observed along the evaluation period. Increased pressor response to phenylephrine was observed from first to fourth week. α1-adrenoceptor protein in the mesenteric bed was enhanced at the first week, whereas β2-adrenoceptor protein in the aorta was reduced after the second week. In the third week, ethanol intake facilitated the depressor response to sodium nitroprusside, whereas in the fourth week it reduced nitrate content in aorta and increased it plasma. The bradycardic component of the baroreflex was impaired, whereas baroreflex tachycardia was enhanced at the third and fourth weeks. AT1A receptor and C-type natriuretic peptide (CNP) mRNAs in the nucleus tractus solitarius were increased at the fourth week. These findings suggest that increased vascular responsiveness to vasoconstrictor agents is possibly a link factor in the development and maintenance of the progressive hypertension induced by ethanol consumption. Additionally, baroreflex changes are possibly mediated by alterations in angiotensinergic mechanisms and CNP content within the brainstem, which contribute to maintaining the hypertensive state in later phases of ethanol ingestion. Facilitated vascular responsiveness to nitric oxide seems to counteract ethanol-induced hypertension.

Telmisartan protects central neurons against nutrient deprivation-induced apoptosis in vitro through activation of PPARγ and the Akt/GSK-3β pathway

To determine whether angiotensin II receptor blockers (ARBs) could protect central neurons against nutrient deprivation-induced apoptosis in vitro and to elucidate the underlying mechanisms. Primary rat cerebellar granule cells (CGCs) underwent B27 (a serum substitute) deprivation for 24 h to induce neurotoxicity, and cell viability was analyzed using LDH assay and WST-1 assay. DNA laddering assay and TUNEL assay were used to detect cell apoptosis. The expression of caspase-3 and Bcl-2, and the phosphorylation of Akt and GSK-3β were detected using Western blot analysis. AT1a mRNA expression was determined using RT-PCR analysis. B27 deprivation significantly increased the apoptosis of CGCs, as demonstrated by LDH release, DNA laddering, caspase-3 activation and positive TUNEL staining. Pretreatment with 10 μmol/L ARBs (telmisartan, candesartan or losartan) partially blocked B27 deprivation-induced apoptosis of CGCs with telmisartan being the most effective one. B27 deprivation markedly increased the expression of AT1a receptor in CGCs, inhibited Akt and GSK-3β activation, decreased Bcl-2 level, and activated caspase-3, which were reversed by pretreatment with 1 μmol/L telmisartan. In addition, pretreatment with 10 μmol/L PPARγ agonist pioglitazone was more effective in protecting CGCs against B27 deprivation-induced apoptosis, whereas pretreatment with 20 μmol/L PPARγ antagonist GW9662 abolished all the effects of telmisartan in CGCs deprived of B27. ARBs, in particular telmisartan, can protect the nutrient deprivation-induced apoptosis of CGCs in vitro through activation of PPARγ and the Akt/GSK-3β pathway.

Inactivation of AT1a Receptor Attenuates Lactate Accumulation and Enhances Cardiac Performance during Exercise Endurance

Inactivation of AT1a Receptor Attenuates Lactate Accumulation and Enhances Cardiac Performance during Exercise Endurance

Endothelin-1 but not angiotensin II contributes to functional aging in murine carotid arteries

AimsAging is a major risk factor for carotid artery disease and stroke. Endothelin-1 (ET-1) and angiotensin II (Ang II) are important modifiers of vascular disease, partly through increased activity of NADPH oxidase and vasoconstrictor prostanoids. Since the renin–angiotensin and endothelin systems become activated with age, we hypothesized that aging affects NADPH oxidase- and prostanoid-dependent contractions to ET-1 and Ang II. Main methodsCarotid artery rings of young (4month-old) and old (24month-old) C57BL6 mice were pretreated with the NO synthase inhibitor L-NAME to exclude differential effects of NO. Contractions to ET-1 and Ang II were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp91ds-tat or the thromboxane-prostanoid receptor antagonist SQ 29,548. Gene expression of endothelin and angiotensin receptors was measured by qPCR. Key findingsAging reduced ET-1-induced contractions and diminished ETA but increased ETB receptor gene expression levels. Gp91ds-tat inhibited contractions to ET-1 in young and to a greater extent in old animals, whereas SQ 29,548 had no effect. Ang II-induced contractions were weak compared to ET-1 and unaffected by aging, gp91ds-tat, and SQ 29,548. Aging had also no effect on AT1A and AT1B receptor gene expression levels. SignificanceAging in carotid arteries decreases ETA receptor gene expression and responsiveness to ET-1, which nevertheless becomes increasingly dependent upon NAPDH oxidase activity with age; responses to Ang II and gene expression of its receptors are however unaffected. These findings suggest that physiological aging differentially regulates functional responses to G protein-coupled receptor agonists and the signaling pathways associated with their activation.

Heterogeneous Downregulation of Angiotensin II AT1-A and AT1-B Receptors in Arterioles in STZ-Induced Diabetic Rat Kidneys

Introduction. The renin granulation of kidney arterioles is enhanced in diabetes despite the fact that the level of angiotensin II in the diabetic kidney is elevated. Therefore, the number of angiotensin II AT1-A and AT1-B receptors in afferent and efferent arteriole's renin-positive and renin-negative smooth muscle cells (SMC) was estimated. Method. Immunohistochemistry at the electron microscopic level was combined with 3D stereological sampling techniques. Results. In diabetes the enhanced downregulation of AT1-B receptors in the renin-positive than in the renin-negative SMCs in both arterioles was resulted: the significant difference in the number of AT1 (AT1-A + AT1-B) receptors between the two types of SMCs in the normal rats was further increased in diabetes and in contrast with the significant difference observed between the afferent and efferent arterioles in the normal animals, there was no such difference in diabetes. Conclusions. The enhanced downregulation of the AT1-B receptors in the renin-negative SMCs in the efferent arterioles demonstrates that the regulation of the glomerular filtration rate by the pre- and postglomerular arterioles is changed in diabetes. The enhanced downregulation of the AT1-B receptors in the renin-positive SMCs in the arterioles may result in an enhanced level of renin granulation in the arterioles.

EFR3s are palmitoylated plasma membrane proteins that control responsiveness to G-protein-coupled receptors.

The yeast Efr3p protein is a main regulator of the Stt4p phosphatidylinositol 4-kinase at contact sites between the endoplasmic reticulum and the plasma membrane. A mutation in its fly homologue Rbo, leads to diminished light responses in the eye attributed to progressively impaired PLC signaling. Here, we find that Efr3s plays a role in maintaining responsiveness to the type-I angiotensin II (AngII) receptors. siRNA-mediated depletion of EFR3A and EFR3B impaired the sustained phase of cytosolic Ca(2+) response to high concentration of AngII in HEK293 cells that express wild type but not truncated AGTR1 (AT1a receptor), missing the phosphorylation sites. Efr3 depletion had minimal effect on the recovery of plasma membrane phosphoinositides during stimulation, and AT1 receptors still underwent ligand-induced internalization. A higher level of basal receptor phosphorylation and a larger response was observed after stimulation. Moreover, Gq activation more rapidly desensitized after AngII stimulation in Efr3 downregulated cells. A similar but less pronounced effect of EFR3 depletion was observed on the desensitization of the cAMP response after stimulation with isoproterenol. These data suggest that mammalian Efr3s contribute to the control of the phosphorylation state and, hence, desensitization of AT1a receptors, and could affect responsiveness of G-protein-coupled receptors in higher eukaryotes.

Telmisartan ameliorates glutamate-induced neurotoxicity: Roles of AT1 receptor blockade and PPARγ activation

Sartans (Angiotensin II AT1 Receptor Blockers, ARBs) are powerful neuroprotective agents in vivo and protect against IL-1β neurotoxicity in vitro. The purpose of this research was to determine the extent of sartan neuroprotection against glutamate excitotoxicity, a common cause of neuronal injury and apoptosis. The results show that sartans are neuroprotective, significantly reducing glutamate-induced neuronal injury and apoptosis in cultured rat primary cerebellar granule cells (CGCs). Telmisartan was the most potent sartan studied, with an order of potency telmisartan > candesartan > losartan > valsartan. Mechanisms involved reduction of pro-apoptotic caspase-3 activation, protection of the survival PI3K/Akt/GSK-3β pathway and prevention of glutamate-induced ERK1/2 activation. NMDA receptor stimulation was essential for glutamate-induced cell injury and apoptosis. Participation of AT1A receptor was supported by glutamate-induced upregulation of AT1A gene expression and AT1 receptor binding. Conversely, AT1B or AT2 receptors played no role. Glutamate-induced neuronal injury and the neuroprotective effect of telmisartan were decreased, but not abolished, in CGCs obtained from AT1A knock-out mice. This indicates that although AT1 receptors are necessary for glutamate to exert its full neurotoxic potential, part of the neuroprotective effect of telmisartan is independent of AT1 receptor blockade. PPARγ activation was also involved in the neuroprotective effects of telmisartan, as telmisartan enhanced PPARγ nuclear translocation and the PPARγ antagonist GW9662 partially reversed the neuroprotective effects of telmisartan. The present results substantiate the therapeutic use of sartans, in particular telmisartan, in neurodegenerative diseases and traumatic brain disorders where glutamate neurotoxicity plays a significant role.