Inhibition of vascular smooth muscle G protein-coupled receptor kinase 2 enhances alpha1D-adrenergic receptor constriction

Abstract

G protein-coupled receptor kinase 2 (GRK2) is a serine/theorinine kinase that phosphorylates and desensitizes agonist-bound G protein-coupled receptors. GRK2 is increased in expression and activity in lymphocytes and vascular smooth muscle (VSM) in human hypertension and animal models of the disease. Inhibition of GRK2 using the carboxyl-terminal portion of the protein (GRK2ct) has been an effective tool to restore compromised beta-adrenergic receptor (AR) function in heart failure and improve outcome. A well-characterized dysfunction in hypertension is attenuation of betaAR-mediated vasodilation. Therefore, we tested the role of inhibition of GRK2 using GRK2ct or VSM-selective GRK2 gene ablation in a renal artery stenosis model of elevated blood pressure (BP) [the two-kidney, one-clip (2K1C) model]. Use of the 2K1C model resulted in a 30% increase in conscious BP, a threefold increase in plasma norepinephrine levels, and a 50% increase in VSM GRK2 mRNA levels. BP remained increased despite VSM-specific GRK2 inhibition by either GRK2 knockout (GRK2KO) or peptide inhibition (GRK2ct). Although betaAR-mediated dilation in vivo and in situ was enhanced, alpha(1)AR-mediated vasoconstriction was also increased. Further pharmacological experiments using alpha(1)AR antagonists revealed that GRK2 inhibition of expression (GRK2KO) or activity (GRK2ct) enhanced alpha(1D)AR vasoconstriction. This is the first study to suggest that VSM alpha(1D)ARs are a GRK2 substrate in vivo.

Fig. 1.

Vascular smooth muscle (VSM)-specific G protein-coupled receptor kinase 2 (GRK2) knockout (GRK2KO) and GRK2ct peptide inhibitor transgenic mouse lines. A: cartoon of Lox-P sites within the GRK2 floxed transgene and the frame shift that occurs when mating GRK2 floxed mice with smooth muscle Cre recombinase-expressing mice (sm-Cre-IRES-eGFP). Primers used for screening are indicated by arrows A–C. B: PCR genotyping of a heterozygous mouse confirming GRK2 deletion of exons 3–6 is specific to smooth muscle in the thoracic aorta (TA) and maintains a floxed GRK2 allele in the heart, liver, kidney, and lung compared with wild-type (wt) mice. C: quantitative real-time PCR with primers to the amino-terminal portion of GRK2, GRK5, and GRK3. GRK3 expression is too low to be visible on the y-axis used. VSM from the aorta of 8 mice were pooled and considered as n = 1. n = 6 for each group. *P < 0.05 vs. control by one-way ANOVA and Bonferroni post t-test. D: immunohistochemisty from a cross-sectional slice through the common carotid artery of control and VSM GRK2ct mice. The GRK2 antibody recognizes the carboxyl-terminal portion of GRK2 and therefore detects both GRK2ct and endogenous levels of GRK2. Bar = 50 μm.

Fig. 2.

Renal artery stenosis using the two-kidney, one-clip (2K1C) model increases mean arterial pressure (MAP), plasma norepinephrine (NOR), and VSM GRK2 expression. A: MAP in sham and 2K1C control mice. n = 8 for each. *P < 0.05 by an unpaired two-tailed Student's t-test. B: epinephrine (EPI) and norepinephrine levels were measured in plasma isolated from sham and 2K1C control mice. n = 5 for each. C: quantitative RT-PCR determined GRK2, GRK5, and GRK3 mRNA expression in sham and 2K1C control mice. GRK3 mRNA abundance was very low and not readily detectable on the y-axis scale used. n = 4 for each. *P < 0.05 vs. control by one-way ANOVA and Bonferroni's post t-test.

Fig. 3.

VSM GRK2 inhibition fails to rescue the 2K1C model of high blood pressure (BP). A: 4-wk postsurgical mice were assessed for conscious MAP for control (sham: n = 12 and 2K1C: n = 5), GRK2KO (sham: n = 10 and 2K1C: n = 6), and GRK2ct (sham: n = 10 and 2K1C: n = 6) mice. B: cardiac hypertrophy was measured as heart-to-body weight ratios for all groups. C: all three 2K1C mouse groups had increased right-to-left kidney weight ratios. *P < 0.05 vs. respective sham mice by one-way ANOVA and Bonferroni's post t-test.

Fig. 4.

Enhanced in vivo and in situ β-adrenergic receptor (AR) signaling in both GRK2KO and GRK2ct mice compared with controls. A: acute agonist infusion of isoproterenol (Iso) through the jugular vein with simultaneous anesthetized BP tracing was performed when heart rates rose above 400 beats/min in control (n = 5), GRK2KO (n = 5), and GRK2ct (n = 5) mice. MAP was normalized to the baseline reading, which was considered as 100%. B: in situ vascular reactivity experiments on TA segments in control (n = 5), GRK2KO (n = 5), and GRK2ct (n = 6) vessels. Tension was normalized (100%) to the maximal response of a concentration of 3 × 10⁻⁷ M phenylephrine (PE). Nitric oxide synthase activity was inhibited using N-nitro-l-arginine methyl ester (l-NAME) to prevent endothelial cell release of nitric oxide and verified with a lack of response to 10⁻⁵ M ACh. *P < 0.05 vs. GRK2KO by two-way ANOVA with respect to dose and control; †P < 0.05 vs. control by Bonferroni's post t-test.

Fig. 5.

Enhanced in vivo and in situ α₁AR and ANG II receptor signaling. A: acute agonist infusion revealed that ANG II-induced increases in BP were enhanced in GRK2ct mice, but not GRK2KO mice, compared with control animals. n = 5 each. B: vasoconstriction in vascular reactivity experiments in abdominal aorta segments from control (n = 5), GRK2KO (n = 5), and GRK2ct (n = 6) vessels. C: PE-induced elevations in BP in control (n = 5), GRK2KO (n = 5), and GRKct (n = 6) mice. D: PE-induced vasoconstriction in control (n = 11), GRK2KO (n = 17), and GRK2ct (n = 12) TA segments. Tension normalized to the 10⁻⁵ M response. *P < 0.05 vs. GRK2KO by two-way ANOVA with respect to dose and control; †P < 0.05 vs. control by Bonferroni's post t-test.

Fig. 6.

α₁AR subtype gene expression in VSM isolated from mouse TAs. Blood vessels were analyzed for α₁AR subtype expression via quantitative RT-PCR. Enzymatic removal of adventitia and mechanical scraping of endothelial cells isolated the VSM-only tunica media layer (8 aorta pooled and repeated 4 times).

Fig. 7.

α_1BAR inhibition does not alter enhanced in situ α₁AR signaling in GRK2-inhibited vessels. Responses to PE and α_1BAR antagonists in TAs were taken from control (A and D), GRK2KO (B and E), and GRK2ct (C and F) mice. All inhibitors were added 10 min prior to stimulation. A–C: control, GRK2KO, and GRK2ct vessels in the presence of the α_1BAR inhibitor AH11110A (1 μM). D–F: additional experiments with another α_1BAR inhibitor, chloroethylclonidine (CEC). n = 4–7 for all groups.

Fig. 8.

Vasoconstriction to PE in the presence of WB4101, an α_1AAR inhibitor. A–C: responses to PE in the TA taken from control (A), GRK2KO (B), and GRK2ct (C) mice. n = 4–7 for all groups.

Fig. 9.

BMY-7378, an α_1DAR inhibitor, restored normal α₁AR vasoconstriction in GRK2KO and GRK2ct TAs. A: PE constriction in the mouse TA isolated from control, GRK2KO, and GRK2ct mice in the presence of the α_1DAR antagonist BMY-7378 (1 μM). B–D: PE constriction in the mouse TA taken from control (B), GRK2KO (C), and GRK2ct (D) mice. n = 4–13 for all groups.