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 (2
S
)-
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-α were detected in the cytosolic fraction. Treatment with the A
1
AR agonist ENBA (10
−5
M) translocated PKC-α from the cytosolic to membrane fraction significantly in A
1
WT but not A
1
KO cells. Phospholipase C isoforms (βI, βIII, and γ
1
) were analyzed using specific antibodies where ENBA treatment led to the increased expression of PLC-βIII in A
1
WT CASMCs while having no effect in A
1
KO CASMCs. In A
1
WT cells, ENBA increased PKC-α expression and p42/p44 MAPK (ERK1/2) phospohorylation by 135% and 145%, respectively. These effects of ENBA were blocked by Gö-6976 (PKC-α inhibitor) and PD-98059 (p42/p44 MAPK inhibitor). We conclude that A
1
AR stimulation by ENBA activates the PKC-α signaling pathway, leading to p42/p44 MAPK phosphorylation in CASMCs.