rabbit α α1b  (Alomone Labs)

Journal: JCI Insight

Article Title: L-DOPA sensitizes vasomotor tone by modulating the vascular alpha1-adrenergic receptor

doi: 10.1172/jci.insight.90903

Figure Lengend Snippet: (A) Schematic representation of the experimental design. Measurement of blood pressure (BP) and heart rate (HR) was performed on anesthetized mice, and drugs were administered i.v. (B) Typical traces of the effects of phenylephrine on BP and HR in anesthetized WT (blue) and Gpr143–/y (red) mice. (C) Summarized effects of phenylephrine on BP and HR in WT and Gpr143–/y mice (F1,52 = 42.39, P < 0.001, n = 7 and 8). (D) Effects of phenylephrine on BP under pretreatment with saline or L-DOPA cyclohexyl ester (CHE) (10 mg/kg) (F1,40 = 46.75, P < 0.001, n = 6). (E) Effects of vasopressin on BP in WT and Gpr143–/y mice (F1,32 = 0.209, P > 0.05, n = 5). (F) Effects of phenylephrine on BP in SM22-cre; Gpr143WT and SM22-cre; Gpr143fl/y mice (F1,44 = 20.99, P < 0.005, n = 6 and 7). (G) Effects of L-DOPA CHE alone on BP in WT mice with or without prazosin (1 mg/kg, i.p.) and in Gpr143–/y mice (F1,28 = 10.18, P < 0.005, n = 4–7). All values are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: The sections were incubated with anti-TH (#22941, ImmunoStar) antibody, anti-GPR143 antibody ( 12 ), or anti-ADRA1B (#AAR-018, Alomone Labs) antibody at 4°C for 24 hours.

Techniques:

Journal: JCI Insight

Article Title: L-DOPA sensitizes vasomotor tone by modulating the vascular alpha1-adrenergic receptor

doi: 10.1172/jci.insight.90903

Figure Lengend Snippet: Contractile response in dissected aorta. (A) Contractile responses to phenylephrine (Phe) in WT and Gpr143–/y aortas (F1,68 = 20.29, P < 0.001, n = 9 and 10). (B) Contractile response to phenylephrine, combined with control buffer or L-DOPA cyclohexyl ester (CHE) (1 mM) in WT aortas (F1,56 = 4.363, P < 0.01, n = 8). (C) Contractile response to phenylephrine (100 nM) in the presence of increasing concentrations of L-DOPA in WT and Gpr143–/y aortas (F1,45 = 10.16, P < 0.01, n = 6–11). Contractile response in dissected mesenteric arteries. (D) Contractile response to phenylephrine in WT and Gpr143–/y arteries (F1,44 = 9.684, P < 0.001, n = 6). (E) Contractile response to phenylephrine (1 μM) in the presence of increasing concentration of L-DOPA in WT and Gpr143–/y arteries (F1,38 = 4.565, P < 0.05, n = 6–11). (F) Contractile response to vasopressin (Vaso) in WT and Gpr143–/y. L-DOPA (10 nM) did not augment the effect of vasopressin (n = 6). Contractility is expressed as the percentage of the contraction induced by KCl (10 mM). **P < 0.01, ***P < 0.001, compared with WT (A and D). *P < 0.05, compared with 0 nM L-DOPA in WT (C and E), 1-way ANOVA with Dunnett’s multiple comparisons test. #P < 0.05, compared with 10 nM L-DOPA in WT (C and E), 2-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: The sections were incubated with anti-TH (#22941, ImmunoStar) antibody, anti-GPR143 antibody ( 12 ), or anti-ADRA1B (#AAR-018, Alomone Labs) antibody at 4°C for 24 hours.

Techniques: Concentration Assay

Journal: JCI Insight

Article Title: L-DOPA sensitizes vasomotor tone by modulating the vascular alpha1-adrenergic receptor

doi: 10.1172/jci.insight.90903

Figure Lengend Snippet: Intracellular Ca2+ response in VSMCs. (A) Concentration-response curve of phenylephrine-induced (Phe-induced) intracellular Ca2+ increase in WT and Gpr143–/y VSMCs (n = 9–17 cells/3–6 dishes). (B) Concentration-response curve of vasopressin-induced (Vaso-induced) intracellular Ca2+ ([Ca2+]i) increase in WT and Gpr143–/y VSMCs (n = 6–9 cells/3–4 dishes). L-DOPA–induced augmentation of Ca2+ response in VSMCs. (C) Representative images of cytosolic Ca2+ increase detected with Fura-2 in WT and Gpr143–/y VSMCs before and after phenylephrine in the presence of L-DOPA or control buffer. Scale bar: 25 μm. The magnitude of Fura-2 fluoresce (340/380 nm) intensity is expressed by pseudocolor in a linear scale. (D) Intracellular Ca2+ increase to phenylephrine (1 μM) in the presence of L-DOPA in WT and Gpr143–/y VSMCs (n = 7–12 cells/4–6 dishes). (E) Intracellular Ca2+ increase to vasopressin (1.5 nM) in the presence of L-DOPA in WT and Gpr143–/y VSMCs (n = 6–16 cells/3–6 dishes). All values are mean ± SEM. **P < 0.01, compared with 10 nM L-DOPA in WT, 2-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: The sections were incubated with anti-TH (#22941, ImmunoStar) antibody, anti-GPR143 antibody ( 12 ), or anti-ADRA1B (#AAR-018, Alomone Labs) antibody at 4°C for 24 hours.

Techniques: Concentration Assay

Journal: JCI Insight

Article Title: L-DOPA sensitizes vasomotor tone by modulating the vascular alpha1-adrenergic receptor

doi: 10.1172/jci.insight.90903

Figure Lengend Snippet: Phenylephrine-induced (Phe-induced) myosin light chain 2 (MLC2) and ERK phosphorylation in dissected blood vessels. Protein levels of pMLC2 and MLC2 (A) and pERK and ERK (B and C) were detected after 3-minute treatment with phenylephrine in the dissected vessels from WT and Gpr143–/y, or SM22-cre; Gpr143WT and SM22-cre; Gpr143fl/y mice. Summarized data of pMLC2/MLC2 (A) and pERK/ERK (B and C) (% of WT control) in WT and Gpr143–/y, or SM22-cre; Gpr143WT and SM22-cre; Gpr143fl/y mice (n = 5–6) are shown. *P < 0.05, **P < 0.01, 1-way ANOVA with Tukey’s multiple comparisons test. Phenylephrine-induced ERK phosphorylation in VSMCs. (D) Protein levels of pERK and ERK were determined after 10-minute treatment with phenylephrine in WT and Gpr143–/y mice. (E) Summarized data of pERK/ERK (% of WT control) (F1,42 = 7.803, n = 8). *P < 0.05, compared with 30 μM phenylephrine in WT mice, 2-way ANOVA with Bonferroni’s multiple comparisons test. n.s., not significant.

Article Snippet: The sections were incubated with anti-TH (#22941, ImmunoStar) antibody, anti-GPR143 antibody ( 12 ), or anti-ADRA1B (#AAR-018, Alomone Labs) antibody at 4°C for 24 hours.

Techniques:

Journal: JCI Insight

Article Title: L-DOPA sensitizes vasomotor tone by modulating the vascular alpha1-adrenergic receptor

doi: 10.1172/jci.insight.90903

Figure Lengend Snippet: (A) Co-immunoprecipitation (Co-IP) of ADRA1B-Myc and GPR143-HA. Bands were detected by anti-Myc (left) or by anti-HA (right) antibodies. (B) Protein levels of pERK and whole ERK 10 minutes after the treatment with phenylephrine. (C) Summarized data of pERK/ERK (% of control) in HEK293 cells coexpressing ADRA1B-Myc and free-EGFP or ADRA1B-Myc and GPR143-EGFP (F1,32 = 7.899, P < 0.001, n = 5). (D) Interaction between ADRA1B-Myc and GPR143-HA in the presence or absence of L-DOPA. Note that the interaction was enhanced in the presence of L-DOPA for 1 minute. The arrowhead indicates predicted ADRA1B. (E) TIRF microscopic images of GPR143-EGFP (left), ADRA1B-mCherry (center), and merged images (right) as indicated by white arrowheads (n = 3, independent experiments). Scale bar: 3.5 μm. (F) FRET efficiency between Venus and CFP at the plasma membrane. *P < 0.05, **P < 0.01, 1-way ANOVA with Bonferroni’s multiple comparisons test (n = 36–59). n.s., not significant. (G) Displacement binding curve of [H3]-prazosin (0.4 nM) by phenylephrine in HEK293 cells expressing ADRA1B-Myc and GPR143-EGFP or ADRA1B-Myc and free-EGFP (F1,40 = 19.31, ***P < 0.001, n = 3). Two-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: The sections were incubated with anti-TH (#22941, ImmunoStar) antibody, anti-GPR143 antibody ( 12 ), or anti-ADRA1B (#AAR-018, Alomone Labs) antibody at 4°C for 24 hours.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Expressing

Journal: JCI Insight

Article Title: L-DOPA sensitizes vasomotor tone by modulating the vascular alpha1-adrenergic receptor

doi: 10.1172/jci.insight.90903

Figure Lengend Snippet: Expression of GPR143 (A) and ADRA1B (C) in the liver (arrowheads). No GPR143 signals were observed in the liver of Gpr143–/y mice (B). No ADRA1B signals were observed in sections incubated with the antigenic peptide–preadsorbed antibody (D). Scale bar: 100 μm. Endogenous interaction between GPR143 and ADRA1B. (E) The GPR143-ADRA1B interacting signals (green) were detected by in situ proximity ligation assay technology in WT liver (arrowheads). (F) No interacting signals were observed in Gpr143–/y liver (n = 2, independent experiments). The nucleus was counterstained by DAPI (blue). Scale bars: 5 μm.

Article Snippet: The sections were incubated with anti-TH (#22941, ImmunoStar) antibody, anti-GPR143 antibody ( 12 ), or anti-ADRA1B (#AAR-018, Alomone Labs) antibody at 4°C for 24 hours.

Techniques: Expressing, Incubation, In Situ, Proximity Ligation Assay

Journal: JCI Insight

Article Title: L-DOPA sensitizes vasomotor tone by modulating the vascular alpha1-adrenergic receptor

doi: 10.1172/jci.insight.90903

Figure Lengend Snippet: Tail-pinch-induced pressor response in WT, Gpr143–/y, and SM22-cre; Gpr143fl/y anesthetized and conscious mice. (A) Typical trace of the effects of tail-pinch (10 seconds) on BP and heart rate (HR) in anesthetized WT (left) and Gpr143–/y (right) mice using invasive BP and HR monitoring. (B) Summarized effects of prazosin (1 mg/kg, i.p.) on tail-pinch-induced pressor response in anesthetized WT and Gpr143–/y mice (n = 9 and 6). (C) Tail-pinch-induced pressor response in anesthetized SM22-cre; Gpr143WT and SM22-cre; Gpr143fl/y mice (n = 8 and 4). (D) Mean BP (MBP) before and after tail-pinch in SM22-cre; Gpr143WT and SM22-cre; Gpr143fl/y mice using the radiotelemetry method (n = 2). Circadian BP during the light and dark phases in conscious WT, Gpr143–/y, SM22-cre; Gpr143WT, and SM22-cre; Gpr143fl/y mice. (E) An L-DOPA/GPR143 signaling mechanism that regulates blood pressure by sensitizing vascular ADRA1. NA, noradrenaline. (F) Systolic BP (BPs) in WT and Gpr143–/y mice during the light and dark phase using the tail-cuff method (n = 9–10). Circadian pattern of MBP (G and H); summarized average of MBP during the light and dark phases (I and J); and the percent of change in MBP from the light to dark phases (K and L) in mice using the radiotelemetry method (n = 3–4). *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA with Tukey’s multiple comparisons test (B, F, I, and J), 2-way ANOVA with Bonferroni’s multiple comparisons test (G and H), or 2-tailed unpaired Student’s t test (C, K, and L). n.s., not significant.

Article Snippet: The sections were incubated with anti-TH (#22941, ImmunoStar) antibody, anti-GPR143 antibody ( 12 ), or anti-ADRA1B (#AAR-018, Alomone Labs) antibody at 4°C for 24 hours.

Techniques:

Journal: JCI Insight

Article Title: L-DOPA sensitizes vasomotor tone by modulating the vascular alpha1-adrenergic receptor

doi: 10.1172/jci.insight.90903

Figure Lengend Snippet: (A) Co-immunoprecipitation (Co-IP) of ADRA1B-Myc and GPR143-HA. Bands were detected by anti-Myc (left) or by anti-HA (right) antibodies. (B) Protein levels of pERK and whole ERK 10 minutes after the treatment with phenylephrine. (C) Summarized data of pERK/ERK (% of control) in HEK293 cells coexpressing ADRA1B-Myc and free-EGFP or ADRA1B-Myc and GPR143-EGFP (F1,32 = 7.899, P < 0.001, n = 5). (D) Interaction between ADRA1B-Myc and GPR143-HA in the presence or absence of L-DOPA. Note that the interaction was enhanced in the presence of L-DOPA for 1 minute. The arrowhead indicates predicted ADRA1B. (E) TIRF microscopic images of GPR143-EGFP (left), ADRA1B-mCherry (center), and merged images (right) as indicated by white arrowheads (n = 3, independent experiments). Scale bar: 3.5 μm. (F) FRET efficiency between Venus and CFP at the plasma membrane. *P < 0.05, **P < 0.01, 1-way ANOVA with Bonferroni’s multiple comparisons test (n = 36–59). n.s., not significant. (G) Displacement binding curve of [H3]-prazosin (0.4 nM) by phenylephrine in HEK293 cells expressing ADRA1B-Myc and GPR143-EGFP or ADRA1B-Myc and free-EGFP (F1,40 = 19.31, ***P < 0.001, n = 3). Two-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: The sections were incubated with anti-TH (#22941, ImmunoStar) antibody, anti-GPR143 antibody ( 12 ), or anti-ADRA1B (#AAR-018, Alomone Labs) antibody at 4°C for 24 hours.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Expressing

Journal: JCI Insight

Article Title: L-DOPA sensitizes vasomotor tone by modulating the vascular alpha1-adrenergic receptor

doi: 10.1172/jci.insight.90903

Figure Lengend Snippet: Expression of GPR143 (A) and ADRA1B (C) in the liver (arrowheads). No GPR143 signals were observed in the liver of Gpr143–/y mice (B). No ADRA1B signals were observed in sections incubated with the antigenic peptide–preadsorbed antibody (D). Scale bar: 100 μm. Endogenous interaction between GPR143 and ADRA1B. (E) The GPR143-ADRA1B interacting signals (green) were detected by in situ proximity ligation assay technology in WT liver (arrowheads). (F) No interacting signals were observed in Gpr143–/y liver (n = 2, independent experiments). The nucleus was counterstained by DAPI (blue). Scale bars: 5 μm.

Article Snippet: The sections were incubated with anti-TH (#22941, ImmunoStar) antibody, anti-GPR143 antibody ( 12 ), or anti-ADRA1B (#AAR-018, Alomone Labs) antibody at 4°C for 24 hours.

Techniques: Expressing, Incubation, In Situ, Proximity Ligation Assay