Characterization of Binding Sites for the Angiotensin II Antagonist 125I-[Sarc1,Ile8]-Angiotensin II on Murine Neuroblastoma N1E-115 Cells

The murine neuroblastoma N1E-115 cell line contains binding sites for the angiotensin II (Ang II) receptor antagonist 125I-[Sarc1,Ile8]-Ang II (125I-SARILE). Binding of 125I-SARILE to N1E-115 membranes was rapid, reversible, and specific for Ang II-related peptides. The rank order potency of 125I-SARILE binding was the following: [Sarc1]-Ang II = [Sarc1,Ile8]-Ang II greater than Ang II greater than Ang III = [Sarc1,Thr8]-Ang II much greater than Ang I. Scatchard analysis of membranes prepared from confluent monolayers revealed a homogenous population of high affinity (KD = 383 +/- 60 pM) binding sites with a Bmax of 25.4 +/- 1.6 fmol/mg of protein. Moreover, the density, but not the affinity, of the binding sites increased as the cells progressed from logarithmic to stationary growth in culture. Finally, agonist, but not antagonist, binding to N1E-115 cells was regulated by guanine nucleotides. Collectively, these results suggest that the murine neuroblastoma N1E-115 cell line may provide a useful model in which to investigate the signal transduction mechanisms utilized by neuronal Ang II receptors.     

Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.     

Effects of [Sar1] Angiotensin II on Proenkephalin Gene Expression and Secretion of [Met5]Enkephalin in Bovine Adrenal Medullary Chromaffin Cells

We have studied the effect of [Sar1]angiotensin II [S1-AII; a degradation-resistant analogue of angiotensin II (AII) on the release of [Met5]enkephalin (ME) and proenkephalin A (proENK) gene expression. Short-term (15-min to 1-h) stimulation of bovine adrenal medullary chromaffin (BAMC) cells with S1-AII at concentrations from 0.1 to 100 nM had no significant effect on secretion of ME, whereas high concentrations of S1-AII (3 to 100 microM) produced a concentration-dependent increase in the concentration of ME in the incubation media. In contrast, long-term (3- to 24-h) stimulation with low concentrations (0.1 nM-1 microM) of S1-AII increased the secretion of ME in a concentration-dependent manner (EC50 = 1 nM). The intracellular level of ME was not changed by long-term treatment with S1-AII (100 nM). In addition to increased ME secretion, long-term (24-h) stimulation with S1-AII increased the expression of proENK mRNA in a concentration-dependent manner (EC50 = 4 nM). Losartan (2-n-butyl-4 chloro-5-hydroxymethyl-1-[(2'-(1 H-tetrazol-5-yl)biphenyl-4-yl)- methyl]imidazole potassium salt, a type 1 AII receptor antagonist) inhibited these effects, whereas PD123319 (50 microM, a type 2 AII receptor antagonist) was inactive. Our results suggest that AII in BAMC cells exerts a major effect on the long-term regulation of expression of proENK mRNA and secretion of ME. These effects appear to be mediated by type 1-like AII receptors.     

Neuronal Localization of Specific Angiotensin II Binding Sites in the Rat Inferior Olivary Nucleus

Adult male Sprague-Dawley rats were treated with 3-acetylpyridine, a neurotoxin selective for the inferior olivary nucleus. Following treatment, the rats exhibited deficits in locomotor behavior indicative of destruction in the inferior olivary nucleus. The rats were sacrificed 3 weeks later, and the binding of 125I-sarcosine, isoleucine angiotensin II to brain homogenates and slide-mounted sections of brainstem was determined. Treatment with 3-acetylpyridine significantly decreased specific 125I-sarcosine, isoleucine angiotensin II binding in homogenates of posteroventral brainstem (containing the inferior olivary nucleus) by approximately 50%. Homogenates of dorsal brainstem and hypothalamus-thalamus-midbrain showed no significant changes in specific binding. Treatment with 3-acetylpyridine did not significantly alter the radioligand binding affinity which was determined in the hypothalamus-thalamus-midbrain. Autoradiographic analysis of 125I-sarcosine, isoleucine angiotensin II binding in the brainstem sections indicated that specific angiotensin II binding sites in the inferior olivary nucleus were virtually eliminated by the 3-acetylpyridine treatment. In addition, a comparatively small, but significant, decrease in specific 125I-sarcosine, isoleucine angiotensin II binding occurred in the solitary tract nucleus/dorsal vagal motor nucleus complex. These results indicate that specific angiotensin II binding sites in the inferior olivary nucleus occur exclusively on neuronal perikarya and/or dendrites.     

Angiotensin II Induces Vascular Endocannabinoid Release, Which Attenuates Its Vasoconstrictor Effect via CB1 Cannabinoid Receptors

In the vascular system angiotensin II (Ang II) causes vasoconstriction via the activation of type 1 angiotensin receptors. Earlier reports have shown that in cellular expression systems diacylglycerol produced during type 1 angiotensin receptor signaling can be converted to 2-arachidonoylglycerol, an important endocannabinoid. Because activation of CB(1) cannabinoid receptors (CB(1)R) induces vasodilation and reduces blood pressure, we have tested the hypothesis that Ang II-induced 2-arachidonoylglycerol release can modulate its vasoconstrictor action in vascular tissue. Rat and mouse skeletal muscle arterioles and mouse saphenous arteries were isolated, pressurized, and subjected to microangiometry. Vascular expression of CB(1)R was demonstrated using Western blot and RT-PCR. In accordance with the functional relevance of these receptors WIN55212, a CB(1)R agonist, caused vasodilation, which was absent in CB(1)R knock-out mice. Inhibition of CB(1)Rs using O2050, a neutral antagonist, enhanced the vasoconstrictor effect of Ang II in wild type but not in CB(1)R knock-out mice. Inverse agonists of CB(1)R (SR141716 and AM251) and inhibition of diacylglycerol lipase using tetrahydrolipstatin also augmented the Ang II-induced vasoconstriction, suggesting that endocannabinoid release modulates this process via CB(1)R activation. This effect was independent of nitric-oxide synthase activity and endothelial function. These data demonstrate that Ang II stimulates vascular endocannabinoid formation, which attenuates its vasoconstrictor effect, suggesting that endocannabinoid release from the vascular wall and CB(1)R activation reduces the vasoconstrictor and hypertensive effects of Ang II.     

Inhibition of intracellular Angiotensin II formation blocks high glucose effect on mesangial matrix

High glucose causes increased matrix synthesis by glomerular mesangial cells and angiotensin II (Ang II) has been shown to mediate this effect of glucose. These studies investigate whether inhibition of Ang II formation can block high glucose-induced increase in mesangial matrix. Human mesangial cells were incubated with 25 mM glucose (HG) along with captopril, an ACE inhibitor, to block Ang II formation. In other experiments, cells were nucleofected with siRNA to knockdown angiotensinogen (Agt), the precursor of Ang II, and then exposed to high glucose. Captopril blocked high glucose-induced increase in Ang II levels in the cell media (extracellular) but failed to inhibit it in the cell lysate (intracellular). Moreover, captopril treatment did not block the stimulatory effect of high glucose on TGF-β1 and fibronectin. In contrast, knockdown of the Agt gene prevented high glucose-induced increase in both extracellular and intracellular Ang II levels, and was accompanied by normalization of TGF-β1 and fibronectin. These data suggest that intracellular Ang II may play an important role in the mediation of the high glucose effect on matrix and that ACE inhibitors may not be effective in blocking intracellular Ang II formation in mesangial cells.     

Inability of [125I]Sar1, Ile8-Angiotensin II to Move Between the Blood and Cerebrospinal Fluid Compartments

The angiotensin II competitive antagonist [125I]-Sar1, Ile8-angiotensin II was not transported from the vascular space to the cerebroventricular space in either intact or nephrectomized rats. In addition [125I]Sar1, Ile8-angiotensin II lacked the capacity to move in the opposite direction over a 20-min collection period following cerebroventricular infusion. These data suggest that angiotensins lack the capacity to move freely between the blood and cerebrospinal fluid compartments and are consistent with the notion that blood-borne and cerebroventricular angiotensins access different receptor populations.     

Angiotensin II Receptors Are Coupled to ω-Conotoxin-Sensitive Calcium Influx in Bovine Adrenal Medullary Chromaffin Cells

The contribution of an omega-conotoxin GVIA (omega Cgtx)-sensitive Ca2+ influx pathway to the effects of angiotensin II (AII) receptor activation was examined in bovine adrenal medullary (BAM) cells. Pretreatment of BAM cells with 10(-6) M omega Cgtx blocked stimulation of exocytosis by the degradation-resistant analogue, sarcosine1-angiotensin II (S1-AII). In contrast, omega Cgtx had no effect on basal secretion, nor did it inhibit [3H]norepinephrine and [32P]ATP release in response to bradykinin, another phospholipase C-linked receptor agonist. Similarly, omega Cgtx pretreatment inhibited the stimulation of 45Ca2+ uptake by S1-AII, but did not affect the response to bradykinin. This selective inhibition did not appear to be due to blockade of AII receptors by omega Cgtx, as the accumulation of 3H-labeled inositol phosphates in response to S1-AII was not inhibited. The peak S1-AII-stimulated increase in the intracellular free Ca2+ concentration (Cai) in fura 2-loaded BAM cells also was not significantly reduced by omega Cgtx (or by stimulating in nominally Ca(2+)-free buffer), indicating that this response is dependent on intracellular Ca2+ pools. However, a small omega Cgtx-sensitive Cai response was detected after depletion of intracellular Ca2+ pools with ionomycin. This study shows that AII receptors, but not bradykinin receptors, are linked to an omega Cgtx-sensitive Ca2+ influx pathway in BAM cells.     

Angiotensin II induces nephrin dephosphorylation and podocyte injury: role of caveolin-1

Nephrin, an important structural and signal molecule of podocyte slit-diaphragm (SD), has been suggested to contribute to the angiotensin II (Ang II)-induced podocyte injury. Caveolin-1 has been demonstrated to play a crucial role in signaling transduction. In the present study, we evaluated the role of caveolin-1 in Ang II-induced nephrin phosphorylation in podocytes. Wistar rats-receiving either Ang II (400 ng/kg/min) or normal saline (via subcutaneous osmotic mini-pumps, control) were administered either vehicle or telmisartan (3 mg/kg/min) for 14 or 28 days. Blood pressure, 24-hour urinary albumin and serum biochemical profile were measured at the end of the experimental period. Renal histomorphology was evaluated through light and electron microscopy. In vitro, cultured murine podocytes were exposed to Ang II (10⁻⁶ M) pretreated with or without losartan (10⁻⁵ M) for variable time periods. Nephrin and caveolin-1 expression and their phosphorylation were analyzed by Western-blotting and immunofluorescence. Caveolar membrane fractions were isolated by sucrose density gradient centrifugation, and then the distribution and interactions between Ang II type 1 receptor (AT1), nephrin, C-terminal Src kinase (Csk) and caveolin-1 were evaluated using Western-blotting and co-immunoprecipitation. Podocyte apoptosis was evaluated by cell nucleus staining with Hoechst-33342.Ang II-receiving rats displayed diminished phosphorylation of nephrin but enhanced glomerular/podocyte injury and proteinuria when compared to control rats. Under control conditions, podocyte displayed expression of caveolin-1 in abundance but only a low level of phospho moiety. Nonetheless, Ang II stimulated caveolin-1 phosphorylation without any change in total protein expression. Nephrin and caveolin-1 were co-localized in caveolae fractions. AT1 receptors and Csk were moved to caveolae fractions and had an interaction with caveolin-1 after the stimulation with Ang II. Transfection of caveolin-1 plasmid (pEGFPC3-cav-1) significantly increased Ang II-induced nephrin dephosphorylation and podocyte apoptosis. Furthermore, knockdown of caveolin-1 expression (using siRNA) inhibited nephrin dephosphorylation and prevented Ang II-induced podocyte apoptosis. These findings indicate that Ang II induces nephrin dephosphorylation and podocyte injury through a caveolin-1-dependent mechanism.     

Down-Regulation of Angiotensin II Receptor Subtypes and Desensitization of Cyclic GMP Production in Neuroblastoma N1E-115 Cells

Abstract: Murine neuroblastoma N1E-115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E-115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either ¹²⁵I-AngII or the high-affinity antagonist ¹²⁵I-[Sarc¹,Ile⁸]-AngII. This apparent down-regulation was dose related with an ED₅₀ of 1 nM, and maximal decreases of ～90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist-exposed cells with non-hydrolyzable analogues of GTP for 60 min at 37°C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT₁ receptor subtype during the first 5 min, followed by a decline in both AT₁ and AT₂ receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long-term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down-regulation of receptors, and the high-affinity antagonist [Sarc¹, Thr⁸]-AngII blocked agonist-induced down-regulation. Finally, the apparent loss of cell surface Angll receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E-115 cells. These results suggest that differentiated N1E-115 cells are an excellent cell line in which to examine the factors regulating the expression of AngII receptor subtypes in the nervous system.     

Effects of Angiotensin II on [3H]Noradrenaline Release and Phosphatidylinositol Hydrolysis in the Parietal Cortex and Locus Coeruleus of the Rat

Angiotensin II (ANGII) (3-100 nM) facilitated the potassium-evoked (22.5 mM) release of [3H]-noradrenaline ([3H]NA) from slices of parietal cortex in a concentration-dependent manner, but did not significantly alter the release of [3H]NA evoked in a similar manner from locus coeruleus slices. The facilitatory action of ANGII was blocked by saralasin (0.1-3 microM). Neither nimodipine (10-30 microM) nor phenylmethylsulphonyl fluoride (1 mM) altered either [3H]NA release or the facilitatory action of ANGII in the parietal cortex. Carbachol (0.01-3 mM) and raised potassium (22.5 mM), but not ANGII (3-100 nM), stimulated the production of inositol phosphates in parietal cortex slices. The potassium-evoked increase in inositol phosphate production was unaffected by ANGII (3-100 nM). In the locus coeruleus, ANGII (3-100 nM) did not stimulate inositol phosphate production. The mechanism underlying the ANGII facilitation of [3H]NA release from the parietal cortex does not appear to involve either nimodipine-sensitive calcium channels, or, as far as we have been able to determine, the release of calcium from intracellular stores following the breakdown of phosphoinositides.     

The impact of ANG II and IV on INS-1 cells and on blood glucose and plasma insulin

The impact of angiotensin (ANG) for peripheral, global effects is well known. Local ANG systems including that of the insulin-releasing β cell are not well investigated. In insulin-secreting cell line (INS-1), AT₁ and AT₄ receptors for ANG II and IV were demonstrated by Western blots. Only small amounts of ANG II-binding sites of low affinity were observed. ANG II and SARILE displaced binding of ¹²⁵I-ANG II. ANG II and IV as well as their non-degradable analogs SARILE and Nle-ANG IV increased the glucose-induced insulin release in a bell-shaped way; the maximum effect was at ～1?nM. The increase was antagonized by 1 µM losartan or 10 µM divalinal (AT₁ and AT₄ receptor antagonists, respectively). The insulin release was accompanied by a ⁴⁵Ca²⁺ uptake in the case of ANG II and ANG IV. Divalinal abolished the effect of ANG IV and Nle-ANG IV on this parameter. ANG IV reduced the increase in blood glucose during a glucose tolerance test with corresponding, albeit smaller effects on plasma insulin. Using confocal laser scanning microscopy, transfected insulin-regulated aminopeptidase (IRAP) with AT₄ receptors was shown to be accumulated close to the nucleus and the cytosolic membrane, whereas GLUT4 was not detectable. IRAP was inhibited by ANG IV. In conclusion, AT₁ and AT₄ receptors may be involved in diabetic homeostasis. Effects are mediated by insulin release, which is accompanied by an influx of extracellular Ca²⁺. The impact of ANG IV/IRAP agonists may be worth being used as antidiabetics.     

Effects of Bisphenol A and its Derivatives on the Response of GABAA Receptors Expressed in Xenopus Oocytes

To study the effects of bisphenol-A (BPA) known to have estrogenic actions, and its derivatives, 3,5-dimethylphenol (DMP) and p-t-butylphenol (TBP), on ionotropic γ-aminobutyric acid (GABA) receptors, GABA_A receptors were expressed in Xenopus oocytes by injecting both poly(A)⁺RNA prepared from rat whole brain and cRNAs synthesized from cloned cDNAs of α₁ and β₁ subunit of the bovine receptors, and their electrical responses were measured by the voltage clamping method. BPA caused the potentiation and inhibition of the former receptor-responses, while it caused only inhibition of the latter ones. In the presence of low concentrations of GABA, DMP and TBP potentiated the responses of both receptors. DMP and TBP also increased the rate of decay of the response, possibly by desensitization of the receptors when GABA solution was continuously bath-applied. Diethyl terephthalate (DTP), which is also known to have estrogenic actions, had little effect on both the responses and the decay of both receptors.     

Angiotensin II type 1 receptor-associated protein regulates carotid intimal hyperplasia through controlling apoptosis of vascular smooth muscle cells

Intimal hyperplasia is the main cause of restenosis after carotid artery injury, and the underlying mechanism involves the proliferation and migration of vascular smooth muscle cells (VSMCs). Angiotensin II Type 1 Receptor-Associated Protein (ATRAP) has been reported to withstand intimal hyperplasia by inhibiting VSMCs proliferation and migration; however, whether the beneficial effect of ATRAP associates with VSMCs apoptosis remains unclarified. We demonstrated that the adenoviral-mediated overexpression of ATRAP induced VSMC apoptosis, alleviating the balloon injury-induced neointima formation in rats. Under the condition of Angiotensin-II stimulation, ATRAP overexpression induced the apoptosis of rat VSMCs by depressing the PI3K-Akt signaling; whereas up-regulation of Akt by PTEN inhibitor abolished the apoptotic death. Thus, ATRAP regulates carotid intimal hyperplasia through controlling the PI3K-Akt signal-mediated VSMCs apoptosis.     

The Epithelial Phenotype of Human Neuroblastoma Cells Expresses Bradykinin, Endothelin, and Angiotensin II Receptors That Stimulate Phosphoinositide Hydrolysis

The neuroblastoma line SK-N-SH consists of distinct and interconverting cell types, which include a neuroblast phenotype (SH-SY5Y), an epithelial phenotype (SH-EP), and an intermediate cell type (SH-IN). In SH-SY5Y cells, only muscarinic receptor activation produced stimulation of phosphoinositide turnover, whereas in SH-EP cells, where muscarinic receptors are not present, the peptides bradykinin, endothelin, and angiotensin II stimulated phosphoinositide hydrolysis with EC50 values of 16, 6, and 0.7 nM, respectively, and a rank order of maximal effects of bradykinin greater than endothelin greater than angiotensin II. Fetal calf serum at concentrations between 1 and 10% was also a potent stimulator of phosphoinositide hydrolysis in SH-EP cells but not in SH-SY5Y cells. In the intermediate cell clone, SH-IN, phosphoinositide hydrolysis was stimulated not only by muscarinic receptors, but also by endothelin, bradykinin, and serum, an indication that this cell type harbors all the kinds of receptors that are differentially expressed in the other two cell types. The effects of the three peptides--bradykinin, endothelin, and angiotensin II--on phosphoinositide hydrolysis in SH-EP cells were additive, a result suggesting that the three kinds of receptors may activate distinct transducer proteins and/or phospholipase C subtypes. Pretreatment of intact SH-EP cells with pertussis toxin under conditions sufficient to ADP-ribosylate 90-95% of the endogenous guanine nucleotide regulatory protein substrates did not impair the ability of any of the receptors to stimulate phosphoinositide hydrolysis in any of the cell types. In contrast, short-term exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (1 microM) abolished the stimulation of phosphoinositide hydrolysis mediated by peptide receptors in SH-EP cells and partially inhibited that by muscarinic receptors in SH-SY5Y cells. Prolonged incubation of SH-EP cells with phorbol ester resulted in a recovery of receptor responsiveness, the extent and rate of which were different for each receptor type. In contrast, there was no recovery of responsiveness for muscarinic receptors in SH-SY5Y cells. The pattern of phorbol ester-mediated effects depended on the cell rather than on the receptor type. In fact, muscarinic receptor responsiveness in SH-IN, the intermediate cell type, was desensitized by and recovered from treatment with phorbol esters in a manner more similar to peptide receptors in SH-EP than to muscarinic receptors in SH-SY5Y. These data suggest that the transduction mechanisms by which distinct receptor types are coupled to phosphoinositide hydrolysis in the three cell phenotypes differ in sensitivity to feedback regulation by protein kinase C.     

Guanine Nucleotide Effects on 8-Cyclopentyl-1,3-[3H]Dipropylxanthine Binding to Membrane-Bound and Solubilized A1 Adenosine Receptors of Rat Brain

The effects of guanine nucleotides on binding of 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX), a highly selective A1 adenosine receptor antagonist, have been investigated in rat brain membranes and solubilized A1 receptors. GTP, which induces uncoupling of receptors from guanine nucleotide binding proteins, increased binding of [3H]DPCPX in a concentration-dependent manner. The rank order of potency for different guanine nucleotides for increasing [3H]DPCPX binding was the same as for guanine nucleotide-induced inhibition of agonist binding. Therefore, a role for a guanine nucleotide binding protein, e.g., Gi, in the regulation of antagonist binding is suggested. This was confirmed by inactivation of Gi by N-ethylmaleimide (NEM) treatment of membranes, which resulted in an increase in [3H]DPCPX binding similar to that seen with addition of GTP. Kinetic and equilibrium binding studies showed that the GTP- or NEM-induced increase in antagonist binding was not caused by an affinity change of A1 receptors for [3H]DPCPX but by an increased Bmax value. Guanine nucleotides had similar effects on membrane-bound and solubilized receptors, with the effects in the solubilized system being more pronounced. In the absence of GTP, when most receptors are in a high-affinity state for agonists, only a few receptors are labeled by [3H]DPCPX. It is suggested that [3H]DPCPX binding is inhibited when receptors are coupled to Gi. Therefore, uncoupling of A1 receptors from Gi by guanine nucleotides or by inactivation of Gi with NEM results in an increased antagonist binding.     

Effects of Angiotensin II type I receptor shRNA on blood pressure and left ventricular remodeling in spontaneously hypertensive rats

This study was designed to investigate the effects of the Angiotensin II type I receptor (AT1R) shRNA on blood pressure and left ventricular remodeling in spontaneously hypertensive rats. Ten Wistar Kyoto (WKY) rats were used as a normal blood pressure control group, and 20 spontaneously hypertensive rats (SHR) were randomly divided into the experimental and hypertension control groups. The rats in the experimental group were injected with AT1R shRNA recombinant adenovirus (Ad5-AT1R-shRNA) via a tail vein, and the rats in the other two groups were injected with recombinant adenovirus (Ad5-EGFP). The systolic blood pressure (SBP) at rat arteria caudalis was measured before and after the injection, and the heart, kidney, aorta, and adrenal tissues were obtained two days after repeated injection to observe the distribution of Ad5-AT1R-shRNA under a fluorescence microscope. Before the injection of Ad5-AT1R-shRNA, the blood pressure of the experimental group and the hypertension control group was significantly higher than that of the normal blood pressure control group (P<0.01). After two injections, the blood pressure in the experimental group decreased significantly, and the duration of blood pressure reduction reached 19 days. In the experimental group, the kidney, heart, aorta, and adrenal gland tissues showed vigorous fluorescence expression under the fluorescence microscope. Repeated administration of Ad5-AT1R-shRNA has a long-lasting hypotensive effect on SHR and can significantly improve ventricular remodeling.     

Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.