Journal: Journal of the American Heart Association

Article Title: Connexin37‐Dependent Mechanisms Selectively Contribute to Modulate Angiotensin II‐Mediated Hypertension

doi: 10.1161/jaha.118.010823

Figure Lengend Snippet: Figure 6. Loss of Cx37 selectively alters the expression of the AT2 receptors in the aortas of Cx37/ mice. A, Immunofluorescence showed a similar staining for AT1R (upper panels) in the aortas of all WT and Cx37/ mice. In contrast, the staining for AT2R was more intense in the aortas of Cx37/ than in WT mice. Bar=20 lm. L indicates lumen; M, media. B, Western blot showed that the aortas of WT and Cx37/ mice expressed similar levels of the AT1R protein. C, In contrast, the basal levels of the AT2R protein were higher in Cx37/ than in WT mice, and were not increased after the 2K1C surgery. WT sham N=21; Cx37/ sham N=17; WT 2K1C N=19; Cx37/ 2K1C N=17. D and E, Comparable observations were made for both AT1R (D) and AT2R (E) in mice infused daily with 1 mg Ang II/kg body weight WT NaCl N=4; Cx37/ NaCl, Cx37/ Ang II N=3, WT Ang II N=6. Results are means+SEM. °P≤0.05 vs WT mice, as given by 2-way ANOVA. 2K1C indicates 2-kidney, 1-clip; AngII, angiotensin II; AT1R, Ang II type 1 receptors; AT2R, Ang II type 2 receptors; Cx37, Connexin37; DAPI, 40,6-diamidino-2-phenylindole; Sham, sham- operated controls; WT, wild type.

Article Snippet: The membranes were then incubated overnight at 4°C with 1 of the following primary antibodies: rabbit polyclonal antibodies against AT1R (Alomone labs, AAR-011, 1:1000), AT2R (Alomone labs, AAR-012, 1:1000), MLC2 (Cell Signaling, 3672, 1:1000), P-MLC2 (Cell Signaling, 3674, 1:1000), ERK (Cell Signaling, 4695, 1:1000), P-ERK (Cell Signaling, 9101, 1:1000), AKT (Cell Signaling, 9272S, 1:1000), Cx40 (Chemicon, AB1726; 1:250), Cx37 (Biotrend Chemikalien, Cx37A11-A; 1:500), Cx43 (Cell Signaling, 3512S, 1:500) or Cx45 (Millipore, AB1745, 1:500); mouse monoclonal antibodies against eNOS (BD Biosciences, 610297, 1:500), PeNOS (BD Biosciences, 612392, 1:500), P-AKT (Cell Signaling, 4051, 1:500), and atubulin (Sigma-Aldrich, T5168; 1:2500).

Techniques: Expressing, Staining, Western Blot

Journal: Journal of the American Heart Association

Article Title: Connexin37‐Dependent Mechanisms Selectively Contribute to Modulate Angiotensin II‐Mediated Hypertension

doi: 10.1161/jaha.118.010823

Figure Lengend Snippet: Figure 7. Loss of Cx37 does not alter the expression of several signals involved in the Ang II effects. The levels of transcripts coding for the chymase (Cma1), which converts Ang I to Ang II, the Regulator of G-protein signaling 2 (Rgs2), which negatively regulates AT1R function by modulating its Gaq subunit activity, the Dual specificity protein phosphatase 1 (Dusp1), an AT2R- associated phosphatase that contributes to AT2R-mediated inactivation of the ERK cascade, and Mas1, the receptor for Ang-(1–7) that promotes vasodilatation, were similar in the aortas of WT and Cx37/ mice. WT N=9; Cx37/ N=8. Results are means+SEM. Statistical analysis was performed using t test. Ang indicates angiotensin; AT1R, Ang II type 1 receptors; AT2R, Ang II type 2 receptors; Cx37, Connexin37; WT, wild type.

Article Snippet: The membranes were then incubated overnight at 4°C with 1 of the following primary antibodies: rabbit polyclonal antibodies against AT1R (Alomone labs, AAR-011, 1:1000), AT2R (Alomone labs, AAR-012, 1:1000), MLC2 (Cell Signaling, 3672, 1:1000), P-MLC2 (Cell Signaling, 3674, 1:1000), ERK (Cell Signaling, 4695, 1:1000), P-ERK (Cell Signaling, 9101, 1:1000), AKT (Cell Signaling, 9272S, 1:1000), Cx40 (Chemicon, AB1726; 1:250), Cx37 (Biotrend Chemikalien, Cx37A11-A; 1:500), Cx43 (Cell Signaling, 3512S, 1:500) or Cx45 (Millipore, AB1745, 1:500); mouse monoclonal antibodies against eNOS (BD Biosciences, 610297, 1:500), PeNOS (BD Biosciences, 612392, 1:500), P-AKT (Cell Signaling, 4051, 1:500), and atubulin (Sigma-Aldrich, T5168; 1:2500).

Techniques: Expressing, Activity Assay

Journal: Drug Design, Development and Therapy

Article Title: Liraglutide attenuates cardiac remodeling and improves heart function after abdominal aortic constriction through blocking angiotensin II type 1 receptor in rats

doi: 10.2147/DDDT.S213910

Figure Lengend Snippet: Effects of liraglutide and telmisartan on the expression of AT1R, AT2R, AT1R/AT2R ratio, and ACE2 after AAC. ( A and B ) Protein levels of AT1R and AT2R were detected by Western blot assay. ( C ) AT1R/AT2R ratio was calculated from the intensity of each individual bands. ( D ) ACE2 expression in the intermyocardium and intracardiac vessels was determined using immunohistochemical staining and calculated as mean optical density (MOD) in the tissue section (magnification: 200; scale bars: 100 μm). Values are mean±SEM (n=6/group). * p <0.05 AAC vs Sham; # p <0.05 Lira or Telmi vs AAC. Abbreviation: AAC, abdominal aortic constriction.

Article Snippet: The protein contents of the AT1R and AT2R, TGF-β1, Smads, and collagens were determined by Western blot assay., In brief, the heart tissue samples from different groups were homogenized in a lysis buffer and the protein concentration was measured by the BCA Protein Assay reagent kit (Boster Biotech, Wuhan, People’s Republic of China).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: The Journal of Biological Chemistry

Article Title: The Angiotensin II Type 1 Receptor (AT1R) Closely Interacts with Large Conductance Voltage- and Ca 2+ -activated K + (BK) Channels and Inhibits Their Activity Independent of G-protein Activation *

doi: 10.1074/jbc.M114.595603

Figure Lengend Snippet: BK and AT1R co-localization in HEK293T cells and in arterial myocytes. A–C, live labeling of BK (green) and AT1R (red) in transiently expressing HEK293T cells displaying co-localization at the plasma membrane. HEK293T cells were co-transfected with N-terminally tagged FLAG-AT1R and c-Myc-BK. Anti-FLAG mAb and anti-c-Myc pAb were used for labeling. D–F, labeling of BK (red) and AT1R (green) after permeabilization showing total expression and subplasmalemma co-localization in rat renal arterial smooth muscle cells. Anti-BK mAb and anti-AT1R pAb were used for labeling. G, a representative three-dimensional cross-correlation plot of BK → AT1R as a function of pixel shift in the x and y axes (arrows in A and B indicate the cell used for the plot). The cross-correlation surface has a peak at zero pixel shift that decays abruptly by shifting the image few pixels indicative of specific co-localization. H, quantification of co-localization in HEK293T cells and smooth muscle cells (SMCs) by PPI analysis (see “Experimental Procedures”). In HEK293T cells, PPI is 0.68 ± 0.08 for BK → AT1R and 0.87 ± 0.08 for AT1R → BK (n = 16 cells, three independent transfections). In rat renal arterial SMCs, PPI is 0.62 ± 0.08 for AT1R → BK and 0.71 ± 0.08 for BK → AT1R (n = 18 cells, two independent cell isolations). Error bars indicate S.D. values.

Article Snippet: Anti-AT1R pAb was custom-made by AnaSpec and raised against N-terminal residues 10 GIKRIQDDCPKAGRH 24 of AT1R.

Techniques: Labeling, Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: The Angiotensin II Type 1 Receptor (AT1R) Closely Interacts with Large Conductance Voltage- and Ca 2+ -activated K + (BK) Channels and Inhibits Their Activity Independent of G-protein Activation *

doi: 10.1074/jbc.M114.595603

Figure Lengend Snippet: Molecular analysis of BK-AT1R association. A, BK topology (left) and truncated BK constructs that were used for immunocytochemistry (right). RCK, regulator of conductance for K+. B, overlaid confocal images of cells co-expressing AT1R (red) and full-length or truncated BK (green) molecules: BK, BK1–711, BK1–441, BK1–343, and BK323–1113. Note that BK323–1113 appears intracellular. Larger yellow squares show 5× magnification of the regions in the smaller yellow squares. C, specific co-localization analysis by PPI (25) demonstrating that the first 1–343 amino acids of BK are sufficient for its association with AT1R. PPI values were 0.68 ± 0.1 for BK to AT1R and 0.87 ± 0.08 for AT1R to BK (n = 16 cells, four independent experiments); 0.63 ± 0.06 for BK1–711 to AT1R and 0.83 ± 0.12 for AT1R to BK1–711 (n = 9 cells, two independent experiments); 0.75 ± 0.1 for BK1–441 to AT1R and 0.88 ± 0.12 for AT1R to BK1–441 (n = 13 cells, two independent experiments); 0.83 ± 0.11 for BK1–343 to AT1R and 0.91 ± 0.07 for AT1R to BK1–343 (n = 15 cells, three independent experiments); and 0.19 ± 0.08 for BK322–1113 to AT1R and 0.26 ± 0.09 for AT1R to BK322–1113 (n = 17 cells, three independent experiments). Error bars indicate S.D. values. *, p < 0.05.

Article Snippet: Anti-AT1R pAb was custom-made by AnaSpec and raised against N-terminal residues 10 GIKRIQDDCPKAGRH 24 of AT1R.

Techniques: Construct, Immunocytochemistry, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Angiotensin II Type 1 Receptor (AT1R) Closely Interacts with Large Conductance Voltage- and Ca 2+ -activated K + (BK) Channels and Inhibits Their Activity Independent of G-protein Activation *

doi: 10.1074/jbc.M114.595603

Figure Lengend Snippet: ANG-II inhibits whole cell BK currents in freshly dissociated rat renal arterial smooth muscle cells via losartan-sensitive AT1R and independent of G-protein activation. A and B, whole cell BK currents at baseline (control) and after 1 μm ANG-II treatment in the same arterial myocyte. Whole cell currents were elicited by 20-ms pulses from −90 to 160 mV from a holding potential of 0 mV. C, mean percentage values normalized to control. The currents were reduced to 62 ± 7% (n = 5 cells, four independent experiments) of the original current after ANG-II treatment. D and E, ANG-II failed to inhibit BK whole cell currents when the same myocyte was pretreated with 10 μm losartan (20 min), an AT1R inhibitor. F, mean percentage BK current values were the same in cells pretreated with losartan and after stimulation with ANG-II (n = 9 cells, three independent experiments). G and H, superimposed traces with test pulses of 100 mV before and after ANG-II (1 μm) treatment in a control cell and in a cell intracellularly perfused with 500 μm GDPβS, an inhibitor of G-protein activation. I, mean values of paired experiments as in H showing that in the presence of GDPβS, ANG-II reduced currents to 68 ± 5% (n = 5 cells, two independent cell isolations) of its original value. Measurements were at the end of test pulses to 100 mV. In this and the following figures, the current traces are just prior to drug application and after the effect of drug treatment reached steady state. Error bars indicate S.D. values. *, p < 0.05.

Article Snippet: Anti-AT1R pAb was custom-made by AnaSpec and raised against N-terminal residues 10 GIKRIQDDCPKAGRH 24 of AT1R.

Techniques: Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: The Angiotensin II Type 1 Receptor (AT1R) Closely Interacts with Large Conductance Voltage- and Ca 2+ -activated K + (BK) Channels and Inhibits Their Activity Independent of G-protein Activation *

doi: 10.1074/jbc.M114.595603

Figure Lengend Snippet: AT1R expression is essential for ANG-II inhibitory effect on BK channels. A and C, representative traces before and after application of 1 μm ANG-II in BK and AT1R-IRES-BK transfected cells, respectively. B and D, corresponding mean % values ± S.D. (n = 6 cells, four independent transfections for BK alone, and n = 3 cells, one transfection for AT1R + BK). Only when AT1R is expressed, ANG-II reduced BK currents to 66 ± 10% of its original value. E, example of time course of ANG-II action on BK currents. A similar time course was collected for other drugs. F, current traces before and after 1 μm ANG-II in AT1R-IRES-BK transfected cells pretreated with 10 μm losartan. G, corresponding mean % values (n = 5 cells, one transfection). H, example of immunoblot analysis of pERK1/2 in lysates from cells expressing AT1R and BK that were untreated (lane 1), treated with ANG-II alone (lane 2), treated with losartan alone (lane 3), and pretreated with losartan and stimulated with ANG-II (lane 4). pERK1/2 levels were higher in ANG-II treated cells (lane 2) and returned to near baseline in cells preincubated with losartan (lane 4) (n = 3 experiments). Loading and expression controls were ERK1/2, AT1R, and BK blots. All lanes were loaded with 30 μg of protein. Antibody concentrations were 186 ng/ml pERK1/2 pAb, 2.85 ng/ml ERK1/2 pAb, 250 ng/ml c-Myc pAb, and 525 ng/ml BK mAb. Error bars indicate S.D. values. *, p < 0.05. Ctrl, control; Los, losartan.

Article Snippet: Anti-AT1R pAb was custom-made by AnaSpec and raised against N-terminal residues 10 GIKRIQDDCPKAGRH 24 of AT1R.

Techniques: Expressing, Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The Angiotensin II Type 1 Receptor (AT1R) Closely Interacts with Large Conductance Voltage- and Ca 2+ -activated K + (BK) Channels and Inhibits Their Activity Independent of G-protein Activation *

doi: 10.1074/jbc.M114.595603

Figure Lengend Snippet: Constitutive inhibition of BK channel activity by AT1R expression. A, voltage stimulation protocol. B and C, representative inside-out patch recordings of type 1 and 3 currents in HEK293T cells co-transfected with BK and AT1R. Fitted V½ values were 0.4 and 36.8 mV, respectively. V½ values were calculated using instantaneous tail currents, I, to obtain FPo (G/Gmax = I/Imax) as a function of the preceding test pulse voltage and fitting the data to a Boltzmann distribution as in F (see “Experimental Procedures”). D, examples of normalized BK tail currents (I/Imax) in cells expressing BK alone or BK + AT1R. The scheme (top trace) shows the pulse protocol. Current traces demonstrate that peak I/Imax (FPo) magnitude followed the trend BK ≈ type 1 AT1R + BK > type 2 AT1R + BK ≈ type 3 AT1R + BK. E, BK activation kinetics was slowed down by AT1R co-expression in the order type 3 > type 2 > type 1 (test pulse = 60 mV) consistent with a larger decrease in Po in type 3 AT1R + BK channels. F, average voltage activation curves (FPo versus voltage) of BK expressed alone or in combination with AT1R. The error bars are within symbols and indicate S.E. Continuous lines are the means of the fitted curves of each experiment. Average V½ values were −0.4 ± 3.2 for BK (n = 24 cells), −0.3 ± 2 for AT1R + BK type 1 (n = 22 cells), 13 ± 2 for AT1R + BK type 2 (n = 17 cells), and AT1R + BK type 3 = 35 ± 2 (n = 12 cells). Type 3 channels displayed the highest inhibition by AT1R co-expression (FPo is lower at a given potential when compared with BK expressed alone). G, V½ distribution in all patches (n = 79) from cells co-expressing BK and AT1R. Ca2+ concentration facing the intracellular side of the channels was 6.7 μm.

Article Snippet: Anti-AT1R pAb was custom-made by AnaSpec and raised against N-terminal residues 10 GIKRIQDDCPKAGRH 24 of AT1R.

Techniques: Inhibition, Activity Assay, Expressing, Transfection, Activation Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: The Angiotensin II Type 1 Receptor (AT1R) Closely Interacts with Large Conductance Voltage- and Ca 2+ -activated K + (BK) Channels and Inhibits Their Activity Independent of G-protein Activation *

doi: 10.1074/jbc.M114.595603

Figure Lengend Snippet: AT1R forms a complex with BK in HEK293T cells. A, cartoons of seven-transmembrane domain BK α-subunit (left) and AT1R (right). B, AT1R pulls down BK (n = 3 independent experiments). Lanes 1–4, IP using anti-c-Myc pAb recognizing AT1R-c-Myc in lysates from transfected (+) or untransfected (−) cells with AT1R-c-Myc and/or BK; lanes 5 and 6, IP with rabbit IgG or protein G beads using lysates from cells co-transfected with AT1R-c-Myc and BK; lane 7, IP using a mixture of lysates from cells independently expressing AT1R-c-Myc or BK. Loading was equal in all lanes (25 μl). Lower panel, control immunoblot of BK expression in input cell lysates (30 μg of protein/lane). Immunoblots were with 525 ng/ml anti-BK mAb. C, control showing effective IP of AT1R in same cell lysates or mixture of lysates. Loading was equal in all lanes (5 μl). Lower panel, AT1R expression in input cell lysates (30 μg of protein/lane) probed with 250 ng/ml anti-c-Myc pAb. D, BK also pulls down AT1R (n = 2 independent experiments). Lanes 1–3, IP of lysates from cells expressing only AT1R, expressing only BK or co-expressing both proteins. IP was with anti-c-Myc pAb recognizing c-Myc-BK, and immunoblot was with 495 ng/ml anti-FLAG mAb recognizing AT1R. Lane 4, negative control using IgG to IP lysates from cells expressing both AT1R and BK. Lower panel, immunoblot of input lysates. E, control of effective IP of BK. Lower panel, expression of BK in corresponding input cell lysates. Immunoblot was with 525 ng/ml anti-BK mAb. IB, immunoblot; IE, independently expressing.

Article Snippet: Anti-AT1R pAb was custom-made by AnaSpec and raised against N-terminal residues 10 GIKRIQDDCPKAGRH 24 of AT1R.

Techniques: Transfection, Expressing, Western Blot, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The Angiotensin II Type 1 Receptor (AT1R) Closely Interacts with Large Conductance Voltage- and Ca 2+ -activated K + (BK) Channels and Inhibits Their Activity Independent of G-protein Activation *

doi: 10.1074/jbc.M114.595603

Figure Lengend Snippet: AT1R and BK channels co-localized FRET. A, co-localized FRET in cells co-expressing AT1R-YFP and BK-CFP fusion proteins, expressing CFP-linker-YFP (positive control), and co-expressing CFP and YFP independently (negative control). Bleed-through was calculated using cells individually expressing each molecule (data not shown). B, correlation of CFP-YFP co-localization and FRET signal. Red dots indicate high correlation, and blue dots indicate no correlation. C, mean values of normalized co-localized FRET were as follows for cells expressing: (i) CFP-linker-YFP, 46 ± 10.3 (n = 14); (ii) CFP + YFP 0.5 ± 0.8 (n = 16, two independent experiments); (iii) BK-CFP + AT1R-YFP, 28 ± 7.5 (n = 20); (iv) CFP + AT1R-YFP, 2.4 ± 3 (n = 10); and (v) BK-CFP + YFP, 1.1 ± 1.6 (n = 11). Co-localized FRET was analyzed with a pixel by pixel method using a FRET analyzer from ImageJ. *, p < 0.05 with respect to controls. coFRET, co-localized FRET.

Article Snippet: Anti-AT1R pAb was custom-made by AnaSpec and raised against N-terminal residues 10 GIKRIQDDCPKAGRH 24 of AT1R.

Techniques: Expressing, Positive Control, Negative Control

Journal: Arthritis

Article Title: Expression of Angiotensin II Receptor-1 in Human Articular Chondrocytes

doi: 10.1155/2012/648537

Figure Lengend Snippet: Expression of angiotensin receptors in human chondrocytes. Representative results of RT-PCR using 5 OA, 5 RA, and 4 fracture (control) samples are shown. AT1R: 255 bp, AT2R: 191 bp, GAPDH: 598 bp.

Article Snippet: Rabbit polyclonal anti-human AT1R antibodies (Abs) were purchased from Assay Designs (Ann Arbor, MI, USA; for immunohistochemistry) and from Alomone Labs, Ltd. (Jerusalem, Israel; for western blotting and flow cytometry).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Arthritis

Article Title: Expression of Angiotensin II Receptor-1 in Human Articular Chondrocytes

doi: 10.1155/2012/648537

Figure Lengend Snippet: Immunohistochemical analysis of AT1R expression in human cartilage. Representative results are shown. (a) Original magnification: ×4. (b) An OA sample (×40). (c) An RA sample (×40).

Article Snippet: Rabbit polyclonal anti-human AT1R antibodies (Abs) were purchased from Assay Designs (Ann Arbor, MI, USA; for immunohistochemistry) and from Alomone Labs, Ltd. (Jerusalem, Israel; for western blotting and flow cytometry).

Techniques: Immunohistochemical staining, Expressing

Journal: Arthritis

Article Title: Expression of Angiotensin II Receptor-1 in Human Articular Chondrocytes

doi: 10.1155/2012/648537

Figure Lengend Snippet: IL-1-induced upregulation of AT1R/AT2R mRNA expression in chondrocytes. Results of the quantitative PCR analyses are shown. (a) Normal ( n = 3) (b) OA ( n = 4) (c) RA ( n = 4).

Article Snippet: Rabbit polyclonal anti-human AT1R antibodies (Abs) were purchased from Assay Designs (Ann Arbor, MI, USA; for immunohistochemistry) and from Alomone Labs, Ltd. (Jerusalem, Israel; for western blotting and flow cytometry).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Arthritis

Article Title: Expression of Angiotensin II Receptor-1 in Human Articular Chondrocytes

doi: 10.1155/2012/648537

Figure Lengend Snippet: AT1R protein expression in chondrocytes. (a) Western Blotting . Chondrocytes with or without IL-1 stimulation were analyzed: AT1R: approximately 60 kD (see text), GAPDH: 36 kD. (b) Flow Cytometry . A representative result is shown, shaded area: control staining, solid line: nonstimulated chondrocytes, dotted line: IL-1-stimulated chondrocytes. Representative results of 2 OA samples are shown.

Article Snippet: Rabbit polyclonal anti-human AT1R antibodies (Abs) were purchased from Assay Designs (Ann Arbor, MI, USA; for immunohistochemistry) and from Alomone Labs, Ltd. (Jerusalem, Israel; for western blotting and flow cytometry).

Techniques: Expressing, Western Blot, Flow Cytometry, Control, Staining