Dopamine D1-like and Angiotensin II Type 1 Receptors Counter-Regulate Autophagy and Cell Proliferation in Rat Embryonic Thoracic Vascular Smooth Muscle Cells

Figures

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  Figure 1. D1-like receptor-mediated increase in autophagy in VSMCs. (A) VSMCs were treated with Fen (1 µmol/L) at the indicated times. (B) VSMCs were treated with Fen for 6 h, at the indicated concentrations. (C) Effect of Sch22390 (Sch, 1.0 µmol/L), a D1-like receptor antagonist, on the Fen-mediated increase in LC3-II protein expression in VSMCs. VSMCs were treated as indicated for 6 h. (D) Effect of chloroquine (Chlo, 10 µmol/L), an autophagy inhibitor, on the Fen-mediated increase in LC3-II protein expression in VSMCs. The immunoblots (A–D) were quantified and expressed as the density ratio of LC3-II over loading control (α-tubulin or GAPDH), then normalized by the control (vehicle). Veh, vehicle; Fen, fenoldopam; Sch, Sch23390; Chlo, chloroquine; hr, hour. n = 4/Group, ** p < 0.01; *** p < 0.001. One-way ANOVA, Bonferroni multiple comparison test (A–D).

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  Figure 2. AT1R-mediated decrease in autophagy in VSMCs. (A) VSMCs were treated with Ang II (100 nmol/L) at the indicated times. (B) VSMCs were treated with Ang II for 6 h, at the indicated concentrations. (C) Effect of losartan (Los, 10 µmol/L), an AT1R antagonist, on the Ang II-mediated decrease in LC3-II protein expression in VSMCs. (D) Effect of chloroquine (Chlo, 10 µmol/L), an autophagy inhibitor, on the Ang II-mediated decrease in LC3-II protein expression in VSMCs. The immunoblots (A–D) were quantified and expressed as the density ratio of LC3-II over α-tubulin, then normalized by vehicle or control (0 h or 0 mol/L). Veh, vehicle; Ang, Ang II; Los, losartan; Chlo, chloroquine; hr, hour. n = 4–5/Group, ** p < 0.01, *** p < 0.001, ns, not significant. One-way ANOVA, Bonferroni multiple comparison test (A–D).

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  Figure 3. D1-like receptor-mediated increase in autophagy is dependent on cAMP/PKA signaling in VSMCs. (A) VSMCs were treated with vehicle (Veh), Fen (10−6 M), Rp-cAMPS (Rp, 50 µmol/L), or their combination (Fen + Rp) for 6 h. n = 4/Group, *** p < 0.001. One-way ANOVA, Bonferroni multiple comparison test. (B) The VSMCs were treated with vehicle (Veh), Ang II (10−7 M) (Ang), Rp-cAMPS (Rp, 50 µmol/L), or their combination (Ang + Rp) for 6 h. n = 4/Group, *** p < 0.001, one-way ANOVA, Bonferroni multiple comparison test. (C) The VSMCs were treated with Veh or Sp-cAMPS (Sp, 1.0 µmol/L) for 6 h. In the final 2 h of incubation, Veh or chloroquine (Chlo, 10 µmol/L) was added into the incubation medium. n = 4/Group, *** p < 0.001. One-way ANOVA, Bonferroni multiple comparison test. Veh, vehicle; Fen, fenoldopam; Rp, Rp-cAMPs; Sp, Sp-cAMPS; PBS, phosphate-buffered saline; Chlo, chloroquine.

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  Figure 4. Counter-regulatory effect of AT1R and D1-like receptors on cAMP production in VSMCs. (A) Pre-treatment of VSMCs with Ang II (Ang, 100 nmol/L, 15 min) prevented the Fen (1 μmol/L, 15 min)-mediated increase in cAMP production (Fen + Ang), that was restored by the addition of Los (10 µmol/L), an AT1R antagonist. (B) Pre-treatment of VSMCs with Fen (1 μmol/L, 15 min) prevented the Ang II (100 nmol/L, 15 min)-mediated decrease in cAMP production (Ang + Fen) in VSMCs, that was restored by the addition of Sch (1 µmol/L), D1-like receptor antagonist. (A,B) Fen, fenoldopam; Sch, Sch23390; Ang, Ang II; Los, losartan; Veh, vehicle. n = 6/Group, *** p < 0.001, ns, not significant. One-way ANOVA, Bonferroni multiple comparison test.

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  Figure 5. Fenoldopam (Fen) increases whereas Ang II decreases LC3-II puncta in VSMCs. (A) VSMCs were treated with Veh (vehicle), Fen (fenoldopam, 1.0 µmol/L, 12 h), Ang II (100 nmol/L, 12 h) or Fen + Ang II (combination of Fen and Ang II, 12 h), as indicated. Representative immunofluorescence images show endogenous LC3-II (green). Blue, nuclei (DAPI). Ang II, angiotensin II. Scale bars, 10 µm. (B) The number of LC3-II puncta was counted by ImageJ (Version 1.54d). The number of cells were counted manually by number of nuclei (cells on the edges were excluded for analysis). The number of LC3-II puncta were divided by the number of cells, and each group was normalized by the average number of the Veh group. Ang, angiotensin II. n = 5–6/Group, * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. One-way ANOVA, Bonferroni multiple comparison test.

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  Figure 6. Counter-regulatory effect of AT1R and D1-like receptors on LC3-II protein expression in VSMCs. (A) VSMCs were treated with Fen (1 μmol/L), Ang II (Ang) (100 nmol/L), losartan (Los, 10 μmol/L) or their combination, as indicated for 6 h. The ability of Ang II to prevent the Fen-mediated increase in LC3-II protein expression was abolished by pre-treatment with the AT1R antagonist, Losartan (Los). Los alone had no effect but Ang, by itself, decreased LC3-II expression. (B) The VSMCs were treated with Ang (100 nmol/L), Fen (1 μmol/L), Sch23390 (Sch,1 μmol/L) or their combination, as indicated for 6 h. The ability of Fen to prevent the Ang-mediated decrease in LC3-II protein expression was abolished by pre-treatment with the D1-like receptor antagonist, Sch. Sch alone had no effect but Fen, by itself, increased LC3-II protein expression, as in Figure 1A–D and Figure 4A. (A,B) Veh, vehicle; Fen, fenoldopam; Ang, angiotensin II; Sch, Sch23390. n = 4/Group, ** p < 0.01, *** p < 0.001, ns, not significant. One-way ANOVA, Bonferroni multiple comparison test.

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  Figure 7. Inhibition of mTOR by rapamycin prevented Fen- (A) and Ang II- (B) mediated regulation of autophagy in VSMCs. VSMCs were treated with vehicle (Veh), Fen (1 μmol/L) or Ang II (Ang, 100 nmol/L), or their combination with rapamycin (Rapa, 100 nmol/L), as indicated, for 6 h. Rapamycin, an mTOR inhibitor, prevented the Fen-mediated increase and Ang II-mediated decrease in LC3-II protein expression. α-tubulin is used for loading control. Rapa, rapamycin. Fen, fenoldopam; Ang II, angiotensin II. n = 4/Group, *** p < 0.001, ns, not significant. One-way ANOVA, Bonferroni multiple comparison test.

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  Figure 8. Effects of autophagy and cAMP signaling modulators on VSMC proliferation measured by BrdU incorporation assay. (A) VSMCs were treated with vehicle (Veh), L-690,330, an autophagy inducer (10 µmol/L), or spautin-1, an autophagy inhibitor (10 µmol/L), at the indicated times. (B) VSMCs were treated with vehicle (Veh), Sp-cAMPS, a PKA activator (1.0 µmol/L), forskolin, an adenylyl cyclase activator (10 µmol/L), or Rp-cAMPS, a PKA inhibitor (50 µmol/L), at the indicated times. (C) VSMCs were treated with vehicle (Veh), Fen (1 µmol/L), or Ang II (100 nmol/L) at the indicated times. Values (100% at 0 time) are shown, using Veh-treated cells at their initial time point (0 h) for normalization. (A–C) Veh, vehicle; Fen, fenoldopam; Ang, Ang II; BrdU, 5-bromo-2-deoxyuridine. Data are mean ± standard deviation. n = 3/Group, * p < 0.05 vs. Veh. Two-way ANOVA, Bonferroni multiple comparison test.

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  Figure 9. Counter-regulatory effects of AT1R and D1-like receptors on VSMC proliferation. (A) VSMCs were treated with Ang II (Ang,100 nmol/L), Fen (1 µmol/L) without or with Los (Los, 10 µmol/L), Sch (1 µmol/L) or their combination, as indicated, for 6 h. Fen attenuated the Ang II-induced cell proliferation in VSMCs. Los (AT1R antagonist) prevented the ability of Ang to counteract the ability of Fen to decrease cell proliferation, whereas the addition of Sch (D1-like receptor antagonist) restored the ability of Ang II to increase cell proliferation. (B) VSMCs were treated with Fen (1 µmol/L), Ang II (100 nmol/L) without or with Sch (1 µmol/L), and Los (10 µmol/L), as indicated, for 6 h. Ang II attenuated the Fen-mediated-decrease in cell proliferation. Sch restored the ability of Ang II to increase cell proliferation. By contrast, Los (Los, 10 µmol/L) restored the ability of Fen to decrease cell proliferation. Cell proliferation was quantified by BrdU incorporation, as described in the Section 4. Relative proliferation (%) is shown using the Veh group for normalization. (A,B) Veh, vehicle; Fen, fenoldopam; Sch, Sch23390; Ang or Ang II, angiotensin II; Los, losartan; BrdU,…