Ramchandran et al. 10.1073/pnas.0602715103. |
Fig. 6. Comparison of basal, agonist-activated, and antagonist-inhibited function of the WT and N111G mutant AT1R in COS-1 cells. The Kd of [Sar-1]Ang II for WT is 0.6 ± 0.01 nM, N111G is 0.2 ± 0.01 nM. The Kd of L-158809 for WT is 2.6 ± 0.1 nM and N111G is 46 ± 5 nM.
Fig. 7. Relative levels of endogenous WT and constitutively active transgenic N111G receptors expressed in isolated endothelial cells from newborn mice (n = 3). Total specific radioligand binding in the presence of the AT2 receptor antagonist PD123319 (1 mM) did not significantly differ between different strains. The bars represent averages (n = 3) ± SD. The decrease in binding in the presence of 1 mM [Sar-1,Ile-8]Ang II represents the total AT1 receptor, and the decrease in binding in the presence of 1 mM SII-Ang II represents the proportion of constitutively active N111G receptor. The fraction of displacement by SII-Ang II is specific for endothelial cells derived from TG lines, with »25-40% in the two TG lines and <5% in the WT. The difference between the TG lines is not significant and that between NTL and TG is significant; **, P < 0.01. The Kd of 125I-[Sar-1,Ile-8]Ang II for WT is 0.1 ± 0.01 nM and for N111G is 0.09 ± 0.01 nM. The Kd of [Sar-1,Ile-8]Ang II for WT is 0.4 ± 0.01 nM and for N111G is 0.1 ± 0.01 nM. The Kd of SII-Ang II for WT is 300 ± 10 nM and for N111G is 3.1 ± 0.5 nM.
Fig. 8. Age-dependent changes in heart rate. *, P < 0.05; **, P < 0.01.
Fig. 9. Age-related changes in cardiac function assessed by echocardiography. **, P < 0.01.
Supporting Methods
Generation of Tie1-AT1R Transgenic (TG) Mice.
The mouse tyrosine kinase receptor (Tie1) promoter (ref. 1; a gift from Kari Alitalo, University of Helsinki, Finland) was amplified by PCR and cloned into the expression vector pcDNA1.1 (Invitrogen, Carlsbad, CA). The synthetic rat AT1R wild-type (WT) and N111G mutant genes (2) with 1D4 epitope were cloned into EcoRI-NotI sites located downstream from the Tie1 promoter. Correct orientation of the transgene and the presence of the N111G mutation were confirmed by DNA sequencing. The SfiI restriction-digested linear DNA fragment consisted of the 735-bp region of the mouse Tie1 promoter, the synthetic rat N111G mutant AT1R gene, the 1D4 epitope tag, and the SV40 intronic/polyadenylation signals in sequence (Fig. 1A). This gene construct is referred to as the transgene hereafter. The transgene fragment was purified using a Geneclean Kit (BIO 101, Morgan Irvine, CA). The Transgenic Core Facility at Cleveland Clinic Foundation (CCF) injected »200 gene copies of transgene into the pronuclei of one-cell B6/CBA mouse embryos (Jackson Laboratory, Bar Harbor, ME), which were surgically implanted into "pseudopregnant" female mice. Founder Tie1-AT1R mice were identified by transgene-specific PCR (559-bp product; see Fig. 1) of genomic DNA extracted from tail biopsies using the following primers: sense, 5'-ATGCATAGCGTATTTTAACAACTGTT-3'; and antisense, 5'-GAGAGTCAGCAGTAGCCTCATCATC-3'. PCR conditions with Taq polymerase were 94°C/1 min, 58°C/1.5 min, and 72°C/2 min for 30 cycles (Fig. 1A). Southern blotting was done using a BamHI-XbaI fragment as a probe (Fig. 1A) labeled with [a-32P]dCTP (PerkinElmer Life Sciences, Wellesley, MA) using a Random-Primed DNA labeling kit (Roche Applied Science, Indianapolis, IN).Quantitative RT-PCR.
Total RNA was extracted by using RNAzol method (TelTest Inc., Friendswood, TX). Twenty-five micrograms of RNA was treated with 25 units of RNase-free DNase for 30 min at 37°C with reaction buffer (50 mM Tris·Cl, pH 8.3/75 mM KCl/3 mM MgCl2/60 units of RNasin) and purified on an oligo(dT)-cellulose column. One microgram of poly(A)+ RNA was reverse transcribed using murine Moloney virus reverse transcriptase and random hexamer priming. The TaqMan probe for quantitative real-time PCR of mouse AT1R was from Applied Biosystems (Foster City, CA). The transgene-specific TaqMan probe complement to the 3' UTR of the transgene (Fig. 1A) was synthesized by the Gene Expression Core Facility at CCF.Membrane Preparation and Radioligand-Binding Experiments.
Tissues were placed in ice-cold buffer A (10 mM Hepes, pH 7.4/250 mM sucrose/5 mM EGTA/12.5 mM MgCl2/protease inhibitor mixture). The tissue was disrupted for 30 s with a Polytron, diluted 1:7 in buffer A, and homogenized 10 times using a Dounce homogenizer. Homogenates were centrifuged for 5 min at 300 ´ g to remove debris, for 5 min at 1,250 ´ g to remove nuclei, and for 15 min at 35,000 ´ g to pellet the membranes. Pellets were resuspended in ice-cold buffer B (20 mM Hepes, pH 7.4/100 mM NaCl/5 mM EGTA/12.5 mM MgCl2/protease inhibitors). This centrifugation resuspension procedure was repeated twice, and the pellet was resuspended in buffer B containing 10% (vol/vol) glycerol before protein estimation using the Bradford method. Saturation binding using the Ang II peptide antagonist, 125I-[Sar-1, Ile-8]Ang II as the radioligand has been described in ref. 1.Cell Culture and Transfection.
Bovine aortic endothelial cells, passage 10 (a gift from Paul DiCorleto, CCF), were cultured in DMEM/F12 medium supplemented with 10% FCS, and they were transfected with expression vectors containing the Tie1-AT1R WT or the Tie1-AT1R N111G mutant genes using FuGENE 6 reagent (Roche Diagnostics, Indianapolis, IN). For evaluation of acute effects, cells were treated with vehicle or 10-6 M Ang II or SII-Ang II for 45 min. For evaluation of chronic effects, cells were treated for 24 h (see Fig. 5). The receptor expression was assessed in each case by Western blot analysis and by 125I-[Sar-1,Ile-8]Ang II-binding analysis.Rabbit 1D4 Antibody.
A polyclonal antibody for the 1D4 epitope peptide (KTETSQVAPA) conjugated to keyhole limpet hemocyanin was produced in rabbits (HTI; FMC BioProducts, Rockland, ME) and purified by an ImmunoPure IgG protein A affinity column (Pierce Biotechnology, Rockford, IL) to obtain 10 mg/ml IgG preparation. In Western blot analysis with 50 mg of membranes prepared from COS-1 cells transfected with pMT3-AT1R-N111G, a 1:500 dilution of this antibody showed specific staining of the tagged receptor but not untagged WT receptor. A 1:150 dilution was used for immunohistochemical staining of tissue sections.Western Blot Analysis.
Cell extracts were made in lysis buffer (buffer A containing 1% Nonidet P-40/0.1% SDS/0.1% deoxycholic acid) and separated on 7.5% SDS/polyacrylamide gels, and proteins were electroblotted onto polyvinylidene difluoride membrane. Membranes were blocked and probed with rabbit 1D4 polyclonal antibody (1:500 dilution) or with anti-endothelial nitric oxide synthase (eNOS) polyclonal antibody (1:1,000, Transduction Laboratories, San Jose, CA). Membranes were exposed to horseradish peroxidase-conjugated secondary antibody followed by development in ECL Plus reagent (Amersham Pharmacia Biosciences, Piscataway, NJ).Immunohistochemistry.
General histological and immunohistochemical methods were performed as described in refs. 3 and 4. Tissues were fixed in 10% neutral buffered formalin or HistoCHOICE tissue fixative (AMRESCO, Solon, OH). Fixed tissue was blocked and embedded in paraffin, and serial 5- to 7-mm sections were deparaffinized and stained with hematoxylin/eosin. Sections were incubated for 1 h with rabbit polyclonal 1D4 antibody at 4°C. After endogenous peroxidases were quenched with 3% H2O2 in methanol, immunostaining was performed with standard streptavidin/biotin/horseradish peroxidase detection (Vector Laboratories, Burlingame, CA) and with 3,3'-diaminobenzidine and counterstained.Blood Pressure Measurement, Ang II Infusion, and Echocardiography.
Systolic blood pressure was determined in conscious mice using a RTBP007 computerized tail-cuff blood pressure analysis system (Harvard Apparatus, Holliston, MA) in a blinded fashion, as described previously in ref. 5. Mice were trained before measurement as described in ref. 5. For drug treatment experiments to determine the effect of NOS inhibition or AT1R inhibition, mice were fed drinking water containing NOS inhibitor Nw-nitro-L-arginine methyl ester (L-NAME) (1 mg/ml) or AT1R antagonist, and L-158809 (0.025 mg/ml) ad libitum for 4 weeks, and blood pressure was monitored.Male mice (6 months old) were anesthetized with ketamine (50 mg/g of body weight) and inactin (100 mg/g of body weight) for the Ang II infusion experiments as described by Lorenz and Robbins (6). Throughout the experiment, mice were assessed for simultaneous increases in heart rate, respiratory efforts, and arterial pressure in response to foot pinch, and if these were present, supplemental doses of inactin (25 mg/g) were given. A tracheotomy was performed, and the mouse was intubated using PE90 tubing over which a steady stream of 100% oxygen was blown. A high-fidelity 1.4F transducer-tipped catheter (Millar Instruments, Houston, TX) was calibrated in room-temperature saline, and it was introduced into the carotid artery for pressure recordings. A second cannula using a PE10 catheter was introduced into the femoral vein for infusing drugs using a constant-flow pump.
Echocardiographic assessments were performed as described in ref. 7. Mice were lightly anesthetized with pentobarbital sodium (40-50 mg/kg of body weight), and they were examined by transthoracic echocardiography using the Acuson Sequoia 512 system (Siemens, Malvern, PA) with 13 MHz symmetrical annular transducer.
Measurement of NOS Activity.
Tissues or bovine aortic endothelial cells were homogenized in a cold lysis buffer (50 mM Tris·HCl, pH 7.4/0.1 mM EGTA/0.1 mM EDTA/1 mM phenylmethylsulfonyl fluoride/0.1% protease inhibitor mixture), and they were centrifuged at 1,000 ´ g for 5 min. The protein concentration of samples was determined by using the Bradford method. NOS activity was assayed by the conversion of L-[3H]arginine to L-citrulline (8). The homogenates (30 ml of 1 mg/ml) were incubated at 23°C for 30 min with reaction buffer (250 mM Tris·HCl, pH 7.4/4 mM tetrahydrobiopterin, 4 mM FAD/1 mM NADPH/1 mM CaCl2/0.1 mM calmodulin) and 20 mCi/ml L-[3H]arginine. The reaction was terminated by adding 500 ml of stop buffer containing 50 mM Hepes, pH 5.5/5 mM EDTA. The L-citrulline formed was separated from arginine substrate by cation-exchange chromatography on a Dowex AG 50WX-8 resin column (Bio-Rad Laboratories, Hercules, CA), and it was measured by liquid scintillation counting. The activities are expressed in pmol of citrulline per mg of protein per min. To determine calcium-independent activity, 5 mM EGTA was added, and CaCl2 and calmodulin were omitted. For blank value, 1 mM L-NAME was added, and CaCl2 and calmodulin were omitted. The calcium-independent fraction of NOS activity, constitutive NOS was calculated.Measurement of Blood Biochemistry.
Plasma electrolytes, creatinine, and blood urea nitrogen were measured by a multiparameter autoanalyzer (9). Plasma endothelin-1 was measured by a commercial ELISA kit (R&D Systems, Minneapolis, MN). Plasma atrial natriuretic peptide was assayed by ELISA by using a kit obtained from SPI Bio (Montigny le Bretonneux, France). The plasma renin activity measurement was done by using a kit supplied by DiaSorin (Stillwater, MN). The blood NO level was estimated from the measurement of nitrosyl hemoglobin by electron spin resonance (10).Isolation and Culture of Endothelial Cells.
Endothelial cells were isolated from 1- to 3-day-old mouse fetus using collagenase disruption similar to the protocol based on Kobayashi et al. (11). The cells were FACS-sorted to enrich for endothelial cells, and the sorted cells were plated in gelatin-coated dishes. The contamination by vascular smooth muscle cells was <5-8%. Endothelial cells were seeded into 12-well plates and cultured until cells reached confluence. The cells were washed with 2 ml of DMEM containing 0.006% BSA and then DMEM and 1 mM PD123319 were added to block AT2R, and 0.05-5 ´10-5 M [Sar-1,Ile-4,Ile-8]Ang II or [Sar-1, Ile-8]Ang II and 300 pM 125I-[Sar-1,Ile-8]Ang II were added, and the cells were incubated for 1 h. After incubation, cells were washed twice with ice-cold PBS. Then 0.5 M NaOH/0.05% SDS was added, and the solubilized cells were collected and transferred into a 5-ml tube. Radioactivity was measured by a gamma counter.Statistical Analysis.
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