Inhibitory effect of D1-like and D3 dopamine receptors on norepinephrine-induced proliferation in vascular smooth muscle cells

Abstract

The sympathetic nervous system plays an important role in the regulation of blood pressure. There is increasing evidence for positive and negative interactions between dopamine and adrenergic receptors; the activation of the alpha-adrenergic receptor induces vasoconstriction, whereas the activation of dopamine receptor induces vasorelaxation. We hypothesize that the D1-like receptor and/or D3 receptor also inhibit alpha1-adrenergic receptor-mediated proliferation in vascular smooth muscle cells (VSMCs). In this study, VSMC proliferation was determined by measuring [3H]thymidine incorporation, cell number, and uptake of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT). Norepinephrine increased VSMC number and MTT uptake, as well as [3H]thymidine incorporation via the alpha1-adrenergic receptor in aortic VSMCs from Sprague-Dawley rats. The proliferative effects of norepinephrine were attenuated by the activation of D1-like receptors or D3 receptors, although a D1-like receptor agonist, fenoldopam, and a D3 receptor agonist, PD-128907, by themselves, at low concentrations, had no effect on VSMC proliferation. Simultaneous stimulation of both D1-like and D3 receptors had an additive inhibitory effect. The inhibitory effect of D3 receptor was via protein kinase A, whereas the D1-like receptor effect was via protein kinase C-zeta. The interaction between alpha1-adrenergic and dopamine receptors, especially D1-like and D3 receptors in VSMCs, could be involved in the pathogenesis of hypertension.

Fig. 1

Effect of the α₁-adrenergic receptor antagonist prazosin on norepinephrine (NE)-mediated proliferation of aortic vascular smooth muscle cells (VSMCs) from Sprague-Dawley (SD) rats. VSMC proliferation was determined by [³H]thymidine incorporation after incubation with the indicated concentrations of NE (10⁻⁶ M) with or without the presence of α₁-adrenergic receptor antagonist prazosin (10⁻⁶ M). Results are expressed as counts per minute (cpm) per mg protein (n = 5 experiments, *P < 0.05 vs. others; ANOVA, Duncan's test).

Fig. 2

Effect of dopamine D₁-like receptor on NE-mediated proliferation of aortic VSMCs from SD rats. A: effect of D₁-like receptor agonist fenoldopam (Fen) on NE-mediated proliferation of aortic VSMCs from SD rats. VSMC proliferation was determined by [³H]thymidine incorporation after incubation with the indicated concentrations of NE (10⁻⁶ M) with or without the presence of Fen (10⁻¹²-10⁻⁵ M). B: effect of a D₁-like receptor agonist Fen and D₁-like receptor antagonist SCH-23390 (SCH) on NE-mediated proliferation of aortic VSMCs from SD rats. The cells were incubated with the indicated reagents: Fen (10⁻⁷ M) and SCH (10⁻⁷ M) for 24 h. VSMC proliferation was determined by [³H]thymidine incorporation. C: effect of PKC inhibitor, peptide 19–31 (PKCI), on the inhibitory effect of D₁-like receptor in aortic VSMCs from SD rats. The cells were incubated with the indicated reagents: NE (10⁻⁶ M), Fen (10⁻⁷ M), and PKCI (10⁻⁶ M) for 24 h. VSMC proliferation was determined by [³H]thymidine incorporation. D: effect of PKC-ζ inhibitor (PKCzI) on the inhibitory effect of D₁-like receptor in aortic VSMCs from SD rats. The cells were incubated with the indicated reagents: NE (10⁻⁶ M), Fen (10⁻⁷ M), and PKCzI (10⁻⁵ M) for 24 h. VSMC proliferation was determined by [³H]thymidine incorporation. E: effect of PKC activator, PMA and PKC inhibitor, PKCI on NE-mediated proliferation of aortic VSMCs from SD rats. The cells were incubated with the indicated reagents: NE (10⁻⁶ M), PKCI (10⁻⁶ M), and PMA (10⁻⁷ M) for 24 h. VSMC proliferation was determined by [³H]thymidine incorporation. All results are expressed as cpm/mg protein [n = 10 (A), n = 5 (B), n = 10 (C), n = 6 (D), and n = 6 (E) experiments]. *P < 0.05 vs. control; #P < 0.05 vs. NE (ANOVA, Duncan's test).

Fig. 3

Effect of D₃ receptor on NE-mediated proliferation of aortic VSMCs from SD rats. A: effect of D₃ receptor agonist PD-128907 (PD) on NE-mediated proliferation of aortic VSMCs from SD rats. VSMC proliferation was determined by [³H]thymidine incorporation after incubation with the indicated concentrations of NE (10⁻⁶ M) with or without the presence of PD (10⁻¹²-10⁻⁷ M). B: effect of a D₃ receptor agonist PD and D₃ receptor antagonist U-99194A (U) on NE-mediated proliferation of aortic VSMCs from SD rats. The cells were incubated with the indicated reagents: PD (10⁻⁷ M) and U (10⁻⁷ M) for 24 h. VSMC proliferation was determined by [³H]thymidine incorporation. C: effect of PKA inhibitor, protein kinase A inhibitor 14–22 (PKAI), on the inhibitory effect of D₃ receptor in aortic VSMCs from SD rats. The cells were incubated with the indicated reagents: NE (10⁻⁶ M), PD (10⁻⁷ M), and PKAI (10⁻⁶ M) for 24 h. VSMC proliferation was determined by [³H]thymidine incorporation. D: effect of PKA activator 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer sodium salt (Sp-cAMP[S]; PKAa) and PKAI on NE-mediated proliferation of aortic VSMCs from SD rats. The cells were incubated with the indicated reagents: NE (10⁻⁶ M), PKAI (10⁻⁶ M), and Sp-cAMP[S] (10⁻⁷ M) for 24 h. VSMC proliferation was determined by [³H]thymidine incorporation. All results are expressed as cpm/mg protein [n = 9 (A), n = 5 (B), n = 8 (C), and n = 4 (D) experiments]. *P < 0.05 vs. control; **P < 0.01 vs. control; #P < 0.05 vs. NE (ANOVA, Duncan's test).

Fig. 4

A: Effect of D₁-like and D₃ receptors on NE-mediated proliferation of aortic VSMCs from SD rats. A: VSMC proliferation was determined by [³H]thymidine incorporation after incubation with the indicated concentrations of NE (10⁻⁶ M) with or without the presence of PD (10⁻⁷ M) and Fen (10⁻⁷ M). Results are expressed as cpm/mg protein (n = 8 experiments). B: VSMC proliferation was determined by cell number after incubation with the indicated concentrations of NE (10⁻⁶ M) with or without the presence of PD (10⁻⁷ M) and Fen (10⁻⁷ M). Results are expressed as cell number per well (n = 4 experiments). C: VSMC proliferation was determined by the 3-(4,5-dimethyl-thiazol-2-yl)-diphenyltetrazolium bromide (MTT) method after incubation with the indicated concentrations of NE (10⁻⁶ M) with or without the presence of PD (10⁻⁷ M) and Fen (10⁻⁷ M). Results are expressed as MTT optical density (OD) = 490 nm (n = 14 experiments). *P < 0.01 vs. control; #P < 0.05 vs. NE, &P < 0.05 vs. NE + Fen or NE + PD (ANOVA, Duncan's test).