Nuclear accumulation of the AT1 receptor in a rat vascular smooth muscle cell line: effects upon signal transduction and cellular proliferation

The objective of the study was to identify the functional outcome of intracellular versus extracellular angiotensin II-AT(1) receptor interactions in vascular cells. Rat vascular smooth muscle cell line A10 was transfected, independently and concurrently, with plasmids encoding fluorescent fusion proteins of rat angiotensin II (pECFP/AII, encodes AII fused downstream of enhanced cyan fluorescent protein) and the rat AT(1a) receptor (pAT(1)R/EYFP, encodes the rat AT(1a) receptor fused upstream of enhanced yellow fluorescent protein). The AII fluorescent fusion protein possesses no secretory signal peptide and deconvolution microscopy established that is maintained within these cells predominantly in the nucleus. AT(1)R/EYFP was absent from the nucleus when expressed exclusively or in untreated cells but accumulated in the nucleus following exogenous AII treatment or when co-expressed with ECFP/AII. Furthermore, expression of ECFP/AII stimulated proliferation of A10 vascular smooth muscle cells (VSMCs) 1.6-fold (P < 0.05). Transfection of a control, pECFP/AII(C) (which encodes a scrambled AII peptide fused to ECFP) had no growth effect. In light of the intracellular growth effects of ECFP/AII, we sought to elucidate the underlying signaling pathways. We found that extracellular AII treatment of A10 cells activated cAMP response element-binding protein (CREB) as determined by one-hybrid assays and immunoblots. Expression of intracellular ECFP/AII similarly activated CREB. However, intracellular and extracellular AII activated CREB through different phosphorylation pathways. Exogenous AII treatment of A10 cells activated p38MAPK and ERK1/2 phosphorylation as determined by Western blot analyses and one-hybrid assays. The p38MAPK inhibitor, SB203580, and the ERK kinase inhibitor, PD98059 each partially inhibited exogenous AII-conferred CREB activation confirming that p38MAPK and ERK1/2 mediate CREB phosphorylation in this system. In contrast, expression of ECFP/AII (intracellular AII) in A10 VSMCs activated p38MAPK but not ERK1/2; inhibition of p38MAPK by SB203580 inhibited intracellular AII-induced CREB phosphorylation. In summary, extracellular AII stimulates at least one pathway common to intracellular AII. This common pathway, in the case of exogenous AII, likely reflects intracellular signaling following internalization of receptor-ligand complex. Extracellular AII also stimulates a unique pathway, apparently reflecting interaction with plasma membrane-associated AT(1)R.     

AT1 receptors mediate the release of prostaglandins in porcine smooth muscle cells and rat astrocytes.

Angiotensin II (AII) can release arachidonic acid metabolites such as prostacyclin (PGI2) and PGE2 from cells in cultures. It has recently been reported that the AT1 selective nonpeptide AII receptor antagonist losartan had similar effects. The present study was undertaken to further evaluate the effects of AII and losartan on cells which synthesize prostaglandins, including vascular smooth muscle, endothelial, and glial cells. Inhibition of specific [125I]AII binding was demonstrated in porcine smooth muscle cell (PSMC) suspensions with unlabeled AII and losartan. The IC50 values were 1.3 x 10(-9) mol/L and 7.7 x 10(-9) mol/L, respectively. PD123177 (an AT2 selective antagonist) had no effect on binding. AII produced a concentration-related increase in calcium mobilization (fura-2 fluorescence) which was blocked by losartan (IC50 = 8.4 x 10(-8) mol/L) but not by PD123177 (10(-6) mol/L). AII (10(-7) to 10(-5) mol/L) stimulated the basal release of PGI2 by 100%. This response was blocked by losartan (10(-6) to 10(-5) mol/L) but not by PD123177 (10(-6) to 10(-5) mol/L) and neither agent stimulated basal release in PSMC. Similar effects of AII and antagonists were observed upon receptor binding and PGE2 release in primary rat astrocyte (RA) cultures. AII did not release PGI2 from porcine endothelial cells, bovine pulmonary arterial endothelial cells, or rat C6 glioma cells. Losartan had no significant effect at 10(-5) mol/L. By contrast, bradykinin or the calcium ionophore A23187 dramatically increased PGI2 release in each of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of AT1 receptors in functional adaptation to ischemia-reperfusion in solated rat hearts in relationship to oxidative stress

Implication of AT1 receptors (AT1R) in functional and metabolic modifications associated with ischemia-reperfusion is not clearly defined. The aim of this study was:--to evaluate the role of AT1R in isolated rat hearts subjected to a reversible ischemia:--to establish possible relationships between functional parameters and oxidative stress during reperfusion period. Isolated hearts perfused by the Langendorff method underwent 30 min of a global total ischemia followed by 30 min of reperfusion. Functional parameters and LDH release were recorded under AT1R stimulation by angiotensin II (AII) (10(-7) M) and/or AT1R blockade by losartan (10(-6) M). Quantification of oxidative stress was performed in coronary effluents 1) directly, using ESR spectroscopy associated with PBN spin trapping and 2) indirectly, using HPLC method to detect glutathione (GSH + GSSG) release. Our results showed that All induced vasoconstrictive and negative inotropic effects during control period. During reperfusion. All reduced incidence of reperfusion arrhythmia and LDH release. From the onset of reperfusion, a large and long lasting release of alkyl/alkoxyl radicals and glutathione was detected and the intensity of the oxidative stress was not significantly changed in the groups treated will All and/or losartan. In conclusion, no relationship has been clearly demonstrated between the oxidative stress intensity and AT1R activation, but these results couldn't exclude the contribution of free radical in some myocardial effects of AT1R stimulation such as vasoconstriction and negative inotropic effect.     

Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer

Excitatory synaptic activity can evoke transient and substantial elevations of postsynaptic calcium. Downstream effects of elevated calcium include the activation of the calcium-dependent protease calpain. We have developed a reagent that identifies dendritic spines in which calpain has been activated. A fusion protein was expressed that contained enhanced yellow and enhanced cyan fluorescent protein (EYFP and ECFP, respectively) linked by a peptide that included the micro-calpain cleavage site from alpha-spectrin. A PDZ-binding site fused to ECFP anchored this protein to postsynaptic densities. The fusion protein exhibited fluorescence resonance energy transfer (FRET), and diminution of FRET by proteolysis was used to localize calpain activity in situ by fluorescence microscopy. Incubation of the fusion protein with calpain in the presence of calcium resulted in the separation of EYFP and ECFP into monomeric fluorophores. In transiently transfected cell lines and dissociated hippocampal neurons, FRET was diminished by raising intracellular calcium levels with an ionophore or with glutamatergic agonists. Calpain inhibitors blocked these changes. Under control conditions, FRET levels in different dendritic spines of cultured neurons and in hippocampal slices were heterogeneous but showed robust decreases upon treatment with glutamatergic agonists. Immunostaining of cultured neurons with antibodies to a spectrin epitope produced by calpain-mediated digestion revealed an inverse correlation between the amount of FRET present at postsynaptic elements and the concentration of spectrin breakdown products. These results suggest that the FRET methodology identifies sites of synaptically induced calpain activity and that it may be useful in analyzing synapses undergoing changes in efficacy.     

Angiotensin II dilates bovine adrenal cortical arterioles: role of endothelial nitric oxide

Adrenal steroidogenesis is modulated by humoral and neuronal factors and blood flow. Angiotensin II (AII) stimulates adrenal cortical aldosterone and cortisol production and medullary catecholamine release. However, AII regulation of adrenal vascular tone has not been characterized. We examined the effect of AII on diameters of cannulated bovine adrenal cortical arteries. Cortical arteries (average internal diameter = 230 microm) were constricted with U46619 and concentration-diameter responses to AII (10(-13) to 10(-8) mol/liter) were measured. In endothelium-intact arteries, AII induced dilations at low concentrations (maximum dilation = 25 +/- 6% at 10(-10) mol/liter) and constrictions at high concentrations (maximum constriction = 25 +/- 18% at 10(-8) mol/liter). AII constrictions were blocked by the angiotensin type 1 (AT1) receptor antagonist, losartan (10(-6) mol/liter). AII dilations were enhanced by losartan (maximal dilation = 48 +/- 8%), abolished by endothelial cell removal or N-nitro-L-arginine (L-NA, 3 x 10(-5) mol/liter) and inhibited by the angiotensin type 2 (AT2) receptor antagonist, PD123319 (10(-6) mol/liter, maximal dilation = 18 +/- 4%). In a 4,5-diaminofluorescein diacetate nitric oxide (NO) assay of isolated cortical arteries, AII stimulated NO production, which was abolished by PD123319, L-NA, or endothelial cell removal. Western immunoblot of arterial homogenates and endothelial and zona glomerulosa cell lysates revealed 48-kD and 50-kD bands corresponding to AT1 and AT2 receptors, respectively, in all three and a 140-kD band corresponding to endothelial NO synthase in endothelial cells and arteries. Our results demonstrate that AII stimulates adrenal cortical arterial dilation through endothelial cell AT2 receptor activation and NO release and AT1 receptor-dependent constriction.     

Angiotensin II enhances insulin sensitivity in vitro and in vivo

The renin-angiotensin system plays a critical role in the pathogenesis of obesity, obesity-associated hypertension, and insulin resistance. However, the biological actions of angiotensin II (AII) on insulin sensitivity remain controversial. Because angiotensinogen and AII receptors are expressed on adipose tissue, we investigated the effect of AII on the insulin sensitivity of isolated rat adipocytes. The results of a receptor binding assay showed the maximal AII binding capacity of adipocytes to be 8.3 +/- 0.9 fmol/7 x 10(6) cells and the dissociation constant to be 2.72 +/- 0.11 nM. Substantial expression of both type 1 and 2 AII (AT1 and AT2) receptors was detected by RT-PCR. AII had no effect on basal glucose uptake, but significantly potentiated insulin-stimulated glucose uptake; this effect was abolished by the AT1 antagonist, losartan. In addition, AII did not alter the insulin binding capacity of adipocytes, but increased insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit, Akt phosphorylation, and translocation of glucose transporter 4 to the plasma membrane. AII potentiated insulin-stimulated glucose uptake through the AT1 receptor and by alteration of the intracellular signaling of insulin. Intraperitoneal injection of Sprague Dawley rats with AII increased insulin sensitivity in vivo. In conclusion, we have shown that AII enhances insulin sensitivity both in vitro and in vivo, suggesting that dysregulation of the insulin-sensitizing effect of AII may be involved in the development of insulin resistance.     

Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion

Herpes simplex virus entry into cells requires four glycoproteins, gB, gD, gH, and gL. Binding of gD to one of its receptors triggers steps requiring the core fusion proteins, gB and the gH/gL heterodimer. There is evidence that gH/gL initiates hemifusion of cells, but whether this complex interacts physically with gB to cause complete fusion is unknown. We used bimolecular complementation (BiMC) of enhanced yellow fluorescent protein (EYFP) to detect glycoprotein interactions during cell-cell fusion. The N- or C-terminal half of EYFP was fused to the C terminus of gD, gB, and gH to form six chimeric proteins (Dn, Dc, Bn, Bc, Hn, and Hc). BiMC was detected by confocal microscopy. Receptor-bearing (C10) cells cotransfected with Dn and Bc or Dn, Hc, and untagged gL exhibited EYFP fluorescence, indicative of interactions between gD and gB and between gD and gH/gL. EYFP complementation did not occur in cells transfected with gL, Bc, and Hn. However, when gD was coexpressed with these other three proteins, cell-cell fusion occurred and the syncytia exhibited bright EYFP fluorescence. To separate glycoprotein expression from fusion, we transfected C10 cells with gL, Bc, and Hn for 20 h and then added soluble gD to trigger fusion. We detected fluorescent syncytia within 10 min, and both their number and size increased with exposure time to gD. Thus, when gD binds its receptor, the core fusion machinery is triggered to form a multiprotein complex as a step in fusion and possibly virus entry.     

Delivery of angiotensin II type 1 receptor antisense inhibits angiotensin action in neurons from hypertensive rat brain.

Increased brain angiotensin II (AII) type 1 receptor (AT1R) expression has been implicated in the hyperactive brain angiotensin system and the development and maintenance of hypertension in the genetically spontaneously hypertensive (SH) rat. Neuronal cells in primary culture from the cardioregulatory-relevant brain areas (hypothalamus/brainstem) mimic increased brain AT1R gene expression and AT1R function of the adult SH rat. They have been utilized in the present study to determine whether cellular actions of AII could be regulated by the transfer of AT1R antisense (AT1R-AS) with the use of a retroviral-mediated gene delivery system developed for the central nervous system cultures. AII stimulates norepinephrine (NE) uptake in neuronal cultures of both normotensive (Wistar Kyoto) and SH rat brains. This neuromodulatory action is mediated by the AT1R subtype, is significantly higher in SH neurons, and is associated with a parallel stimulation of mRNAs for c-fos and NE transporter. Infection of neuronal cultures with a retrovirus vector that contains AT1R-AS (LNSV-AT1R-AS) results in an inhibition of AT1R-mediated stimulation of both c-fos and NE transporter mRNA, as well as NE uptake in both strains of rats; however, the inhibition is more pronounced in SH neurons compared with Wistar Kyoto rat brain neurons. The higher sensitivity of the SH rat brain neurons is further supported by our observation that a certain dose of LNSV-AT1R-AS that fails to induce inhibition of cellular actions of AII in WKY neurons causes a significant inhibition of AII actions in SH neurons. These observations show that retrovirally mediated delivery of AT1R-AS could be used to selectively control the actions of AII in primary neuronal cultures from SH rat brain.     

Opposing actions of angiotensin II type 1 and 2 receptors on plasma cholesterol levels in rats

OBJECTIVE: There have been very few studies to examine how angiotensin II (AII) affects lipid metabolism. We examined the roles of AII type 1 and type 2 receptors (AT1R and AT2R) in cholesterol metabolism in rats fed either normal chow or high-fructose diets. METHODS AND RESULTS: AII (100 ng/kg per min) or vehicle (saline) was continuously infused through an osmotic mini-pump to normal chow-fed or 60% fructose-fed rats for 2 weeks. AII infusion markedly elevated both the systolic and diastolic blood pressure in the two animal groups. AII did not affect the plasma total cholesterol (TC) in the chow-fed rats. In the AII-infused rats treated with olmesartan medoxomil, an AT1R blocker, we were interested to observe significant decreases in plasma TC and non-high-density lipoprotein (HDL)-cholesterol (C) (TC minus HDL-C), and liver cholesterol content were also decreased. Simultaneous infusion of AII and PD123319, an AT2R blocker, markedly increased non-HDL-C and hepatic cholesterol. The infusion of CGP42112A, an AT2R agonist, decreased non-HDL-C by 30% in normal rats. The AII infusion led to significant elevations in TC and non-HDL-C in the fructose-fed rats, and olmesartan treatment completely rectified this AII-induced hypercholesterolemia. CONCLUSIONS: These results suggest that the AII receptors exert opposing effects on the plasma cholesterol level; that is, AT1R increases plasma cholesterol while AT2R decreases it. Fructose feeding may selectively augment the action of AT1R and thereby enhance the increase in plasma cholesterol levels in response to AII infusion.     

Estrogen activates mitogen-activated protein kinase in native, nontransfected CHO-K1, COS-7, and RAT2 fibroblast cell lines

CHO-K1, COS-7, and Rat2 fibroblast cell lines are generally believed to be devoid of estrogen receptors (ERs) and have been widely used to study the functions of ER-alpha and ER-beta after transfection of their cDNAs. Numerous studies have demonstrated that transfected ER-alpha or ER-beta mediates estradiol-induced activation of multiple signaling pathways, including the MAPK/ERK pathways. We report here for the first time that both 17alpha-estradiol and 17beta-estradiol elicit activation of MAPK/ERK in native, nontransfected CHO-K1, COS-7, and Rat2 fibroblast cell lines. We further report that, contrary to the generally held belief, these cell lines are not unresponsive to estradiol in their native, nontransfected state, and that this estrogen responsiveness is associated with estrogen binding. Using multiple ER antibodies, we failed to find ER-alpha or ER-beta isoforms or even ER-X. In view of these findings, researchers, using such cells as models to investigate mechanisms of estrogen action, must always take into account the existence of endogenous estrogen binding proteins other than ER-alpha, ER-beta, or ER-X.     

Anti-cancer actions of a recombinant antibody (R6313/G2) against the angiotensin II AT1 receptor

Although several tumour types express both AT1 and AT2 angiotensin II receptors, and angiotensin II stimulates cell proliferation, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are not effective anti-cancer agents. Development of a biologically active monoclonal antibody (6313/G2) against the AT1 receptor prompted the testing of a recombinant short-chain variable fragment form (R6313/G2) against breast cancer cells in vitro and in vivo. Cell lines MCF-7, MDA-MB-231 and T47D all expressed both receptor subtypes. In vitro, R6313/G2 suppressed cell proliferation in the presence of 100 nM angiotensin II, with IC50s of 30 nM, 153 nM and 2.8 microM for the three cell types respectively; in contrast, the AT1 receptor blocker losartan was effective only in T47D cells, at 25 microM. Studies on MCF-7 and T47D cells showed R6313/G2 also opposed the angiotensin II-induced inhibition of caspase-3/7 activity. In vivo, hollow fibres containing the cell lines were implanted in nu/nu balb-c mice at two sites, s.c. and i.p. Treatments of R6313/G2 at 2.5 nmol/kg and 25 nmol/kg twice per day for 7 days dose dependently reduced cell numbers for all three cell lines, but here MCF-7 cells responded most sensitively and MDA-MB-231 cells least. Although T47D cells were refractory at the s.c. site, growth was inhibited at the i.p. location, and otherwise results were similar at the two sites. In xenografts, MCF-7 cell tumours were dose dependently reduced by R6313/G2, and 13 and 27 nmol/kg R6313/G2 twice/day gave means of 74 and 76% tumour regression after 7 days. The data suggest that the anti-cancer action of R6313/G2 is considerably more effective than AT1 antagonists.     

Functional cross-talk between angiotensin II and epidermal growth factor receptors in NIH3T3 fibroblasts

BACKGROUND: The main angiotensin (Ang) II subtype receptors (AT1R and AT2R) are involved in cellular growth processes and exert functionally antagonistic effects. OBJECTIVE: To characterize the mechanisms by which Ang II receptors influence growth, by investigating the interactions between Ang II subtype receptors and epidermal growth factor (EGF) receptors on mitogen-activated protein kinase (MAPK) pathway activation. DESIGN AND METHODS: The experiments were performed using a mouse fibroblast cell line, NIH3T3, by transient co-transfection with rat AT1R or AT2R expression vectors, or both. Extracellular-signal-regulated kinase (ERK)1/2 phosphorylation was analysed by western blot and the ERK activity was evaluated using PathDetect, an in-vivo signal transduction pathway trans-reporting system. Selective Ang II receptor antagonists (losartan for AT1R and PD123319 for AT2R) were used to investigate the contributions of each receptor to the response observed. RESULTS: Our data show that, in this cellular model, both Ang II receptors phosphorylate ERK1/2. However, in the cells expressing AT1R, the EGF-induced MAPK pathway was enhanced in the presence of Ang II in a synergistic fashion. In contrast, a reduction of EGF-induced MAPK activation was observed in the cells expressing AT2R. In cells expressing both Ang II subtype receptors, Ang II promoted an enhancement of EGF-induced MAPK activation. However, in the presence of the AT1R antagonist, losartan, the effect of EGF was reduced. CONCLUSION: These data indicate the existence of an opposite cross-talk of AT1R and AT2R with EGF receptors, and suggest a complex functional interaction between these pathways in the regulation of cellular growth processes.     

Contrasting effects of angiotensin type 1 and 2 receptors on nitric oxide release under pressure.

This study was designed to test the hypothesis that increased pressure itself could cause endothelial dysfunction and lead to decreased nitric oxide (NO) release, partly through effects on the tissue renin angiotensin system in hypertension. Cultured endothelial cells (ECs) isolated from the aortas of WKY rats were continuously exposed to a pressure of 150 mmHg in a CO2 incubator for 72 h using a pressure system, and the NOx (NO2 and NO3) and angiotensin II (Ang II) concentrations in the supernatant were measured. An Ang II type 1 receptor (AT1R) antagonist (losartan) and an Ang II type 2 receptor (AT2R) antagonist (PD123319) were added to the medium. The expression of AT1R and AT2R mRNAs was also examined. Pressure loading significantly decreased the NO release from ECs. Concomitant administration of losartan restored NO release to the level before the application of pressure (p<0.001). This effect of losartan was blocked by simultaneous administration of PD123319, bradykinin type 2 receptor antagonist, and NO synthase inhibitor (p<0.05). The Ang II concentration was increased by pressure and was further increased by losartan. The gene expression of AT1R was not changed by pressure, but AT2R mRNA was increased almost 2-fold. These results indicate that high pressure itself attenuates NO release from ECs, and that losartan improves NO release by activating the bradykinin system via AT2R stimulation. In addition, the increase of AT2R gene expression in ECs during exposure to pressure may compensate for the reduction of NO.     

The trafficking protein GABARAP binds to and enhances plasma membrane expression and function of the angiotensin II type 1 receptor

Proteins that bind to the intracellular expanses, particularly cytoplasmic tail regions, of heptahelical integral membrane receptors are of particular interest in that they can mediate or modulate trafficking or intracellular signaling. In an effort to distinguish new proteins that might promote angiotensin II type 1 (AT(1)) receptor intracellular events, we screened a yeast 2-hybrid mouse brain library with the rat AT(1A) receptor (AT(1)R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABA(A) receptor, as a binding partner for the AT(1)R. Interaction of GABARAP with the AT(1)R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT(1)R to GABARAP was verified using coimmunoprecipitation. Bioluminescence resonance energy transfer assays further confirmed specific interaction of GABARAP with AT(1)R. Moreover, GABARAP clearly increased the steady-state level of plasma membrane-associated AT(1)R in PC-12 cells. Cotransfection of GABARAP with an AT(1)R fluorescent fusion protein increased PC-12 cell surface expression of the AT(1)R more than 6-fold when standardized to the level of intracellular expression. Furthermore, GABARAP overexpression in CHO-K1 cells engineered to express AT(1)R increased angiotensin II binding sites 3.7-fold and angiotensin II-induced phospho-extracellular signal-regulated kinase 1/2 and cellular proliferation significantly over levels obtained with AT(1)R overexpression alone. In addition, small interfering RNA-mediated knockdown of GABARAP reduced the steady-state levels of the AT(1)R fluorescent fusion protein by 43% and its cell surface expression by 84%. Immunoblot analyses confirmed the quantitative image data. We conclude that GABARAP binds to and promotes trafficking of the AT(1)R to the plasma membrane.     

Overexpression of angiotensin II type I receptor in cardiomyocytes induces cardiac hypertrophy and remodeling

Angiotensin II (AII) is a major determinant of arterial pressure and volume homeostasis, mainly because of its vascular action via the AII type 1 receptor (AT1R). AII has also been implicated in the development of cardiac hypertrophy because angiotensin I-converting enzyme inhibitors and AT1R antagonists prevent or regress ventricular hypertrophy in animal models and in human. However, because these treatments impede the action of AII at cardiac as well as vascular levels, and reduce blood pressure, it has been difficult to determine whether AII action on the heart is direct or a consequence of pressure-overload. To determine whether AII can induce cardiac hypertrophy directly via myocardial AT1R in the absence of vascular changes, transgenic mice overexpressing the human AT1R under the control of the mouse alpha-myosin heavy chain promoter were generated. Cardiomyocyte-specific overexpression of AT1R induced, in basal conditions, morphologic changes of myocytes and nonmyocytes that mimic those observed during the development of cardiac hypertrophy in human and in other mammals. These mice displayed significant cardiac hypertrophy and remodeling with increased expression of ventricular atrial natriuretic factor and interstitial collagen deposition and died prematurely of heart failure. Neither the systolic blood pressure nor the heart rate were changed. The data demonstrate a direct myocardial role for AII in the development of cardiac hypertrophy and failure and provide a useful model to elucidate the mechanisms of action of AII in the pathogenesis of cardiac diseases.