A(1) adenosine receptor-mediated PKC and p42/p44 MAPK signaling in mouse coronary artery smooth muscle cells

Abstract

The A(1) adenosine receptor (A(1)AR) is coupled to G(i)/G(o) proteins, but the downstream signaling pathways in smooth muscle cells are unclear. This study was performed in coronary artery smooth muscle cells (CASMCs) isolated from the mouse heart [A(1)AR wild type (A(1)WT) and A(1)AR knockout (A(1)KO)] to delineate A(1)AR signaling through the PKC pathway. In A(1)WT cells, treatment with (2S)-N(6)-(2-endo-norbornyl)adenosine (ENBA; 10(-5)M) increased A(1)AR expression by 150%, which was inhibited significantly by the A(1)AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (10(-6)M), but not in A(1)KO CASMCs. PKC isoforms were identified by Western blot analysis in the cytosolic and membrane fractions of cell homogenates of CASMCs. In A(1)WT and A(1)KO cells, significant levels of basal PKC-alpha were detected in the cytosolic fraction. Treatment with the A(1)AR agonist ENBA (10(-5)M) translocated PKC-alpha from the cytosolic to membrane fraction significantly in A(1)WT but not A(1)KO cells. Phospholipase C isoforms (betaI, betaIII, and gamma(1)) were analyzed using specific antibodies where ENBA treatment led to the increased expression of PLC-betaIII in A(1)WT CASMCs while having no effect in A(1)KO CASMCs. In A(1)WT cells, ENBA increased PKC-alpha expression and p42/p44 MAPK (ERK1/2) phospohorylation by 135% and 145%, respectively. These effects of ENBA were blocked by Gö-6976 (PKC-alpha inhibitor) and PD-98059 (p42/p44 MAPK inhibitor). We conclude that A(1)AR stimulation by ENBA activates the PKC-alpha signaling pathway, leading to p42/p44 MAPK phosphorylation in CASMCs.

Fig. 1.

Effect of the PKC-α inhibitor Gö-6976 on (2S)-N⁶-(2-endo-norbornyl) adenosine (ENBA)-induced contractile responses in A₁ adenosine receptor (A₁AR) wild-type (A₁WT) and A₁AR knockout (A₁KO) mouse mesenteric arteries. PE, phenylephine. Results are expressed as means ± SE; n = 4. *P < 0.05 compared with A₁WT (untreated) controls.

Fig. 2.

A₁AR protein expression in A₁WT and A₁KO coronary artery smooth muscle cells (CASMCs). A: Western blot analysis. DPCPX, 1,3-dipropyl-8-cyclopentylxanthine. B: densitometric analysis. ND, not detected. Data are presented as means ± SE; n = 5–7. *P < 0.05 compared with A₁WT (untreated) controls; #P < 0.05 compared with ENBA alone.

Fig. 3.

Phospholipase C (PLC) isoform (βI, βIII, and γ₁) expression in A₁WT CASMCs. A: Western blot analysis for PLC isoforms. B: densitometric analysis. data are presented as means ± SE; n = 3–4. *P < 0.05 compared with the respective controls.

Fig. 4.

Distribution of PKC isoforms (α, β, and γ) in the cytosolic fraction (C) and membrane fraction (M) with and without ENBA treatment of A₁WT CASMCs. A and C: Western blots analysis. B and D: densitometric analysis. Data are presented as means ± SE; n = 4–5. *P < 0.05 compared with the respective membrane fractions.

Fig. 5.

Effect of Gö-6976 (PKC-α inhibitor) on ENBA-induced PKC-α expression by Western blot analysis in A₁WT CASMCs. A: Western blot analysis. B: densitometric analysis. Data are presented as means ± SE; n = 6. *P < 0.05 compared with the untreated control in A₁WT CASMCs; #P < 0.05 compared with ENBA alone in A₁WT CASMCs.

Fig. 6.

PKC-α immunoreactivity in A₁WT and A₁KO CASMCs. A and D: representative immunofluorescence photomicrographs of control cells showing PKC-α with green staining in A₁WT (A) and A₁KO (D) CASMCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). The green color around the nucleus shows PKC-α immunoreactivity. Arrows show the translocation of PKC-α in A₁WT CASMCs toward the membrane after ENBA treatment (black empty area around the nucleus; B). ENBA-induced translocation toward the membrane was blocked by the PKC-α inhibitor Gö-6976 (C). Membrane translocation was not seen after ENBA treatment in A₁KO CASMCs (E and F).

Fig. 7.

Effect of PD-98059 (MAPK inhibitor) and Gö-6976 (PKC-α inhibitor) on ENBA-induced p42/p44 MAPK (ERK1/2) phosphorylation in A₁WT CASMCs. A: phosphorylated p42/p44 MAPK (p-ERK1/2). B: total ERK. C: densitometric analysis of p42/p44 MAPK (ERK1/2) phosphorylation. Data are presented as means ± SE; n = 6. *P < 0.05 compared with controls (untreated); #P < 0.05 compared with ENBA alone.

Fig. 8.

Putative signaling mechanism for A₁AR-mediated contraction in vascular smooth muscle. The activation of the A₁AR originates from the binding of an extracellular ligand (A₁AR agonist) and is followed by the activation of a G protein on the cytosolic side of the plasma membrane. The G protein, using GTP as an energy source, then activates PLC-βIII, leading to the activation of PKC-α via diacylglycerol (DAG). PKC-α then phosphorylates p42/p44 MAPK, leading to the contraction of smooth muscle. On the other hand, inositol (1,4,5)-trisphosphate (IP₃) also causes the contraction of smooth muscle through an increase in intracellular calcium. PIP2: phosphatidylinositol bisphosphate.