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A1 adenosine receptor‐activated protein kinase C signaling in A1 knock‐out mice coronary artery smooth muscle cells
Author(s) -
Ansari Habib R.,
Teng Bunyen,
Nadeem Ahmed,
Schnermann J.,
Mustafa S. Jamal
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1152.11
Subject(s) - protein kinase c , phospholipase c , adenosine , medicine , endocrinology , gene isoform , signal transduction , chemistry , microbiology and biotechnology , receptor , cytosol , biology , biochemistry , enzyme , gene
A 1 adenosine receptor (A 1 AR) is coupled to Gi/Go proteins, inhibiting adenylate cyclase, but the downstream signaling pathway in smooth muscle cells is unclear. Present study was performed in coronary artery smooth muscle cells (CASMC) from WT and A 1 AR KO mice to determine the role of protein kinase C (PKC) pathway in A 1 AR signaling. We used CASMC isolation method from our lab (Teng et al 2006, Am J Physiol Heart Circ Physiol. 290:H1713–20). To detect PKC isoforms, cytosolic and membrane fractions were made from cell homogenates. PKC isoforms were identified by Western blotting. In both WT and A 1 AR KO, basal levels of PKC α, β, γ, δ, θ, were abundant in cytosolic, and of PKC μ, ε, λ in membrane fractions whereas PKCζ was equal in both fractions. In WT cells treated with A 1 AR agonist, ENBA (10 −5 M) increased A 1 AR expression by 162%, and this was inhibited by 55% compared with control by A 1 AR antagonist DPCPX (10 −6 M), whereas there was no effect in A 1 AR KO. In WT, ENBA activated PKCα expression by 190% over control, and it was inhibited by specific PKCα inhibitor Gö6976 (10 −7 M), but unchanged in A 1 AR KO (p<0.05). In organ bath experiments, Gö6976 (10 −7 M) inhibited ENBA‐induced aortic contraction by 45% in WT, but no effect was noted in A 1 AR KO. The phospholipase C (PLC) inhibitor U73122 (10 −6 M) reduced the expression of PLCγ and pERK1/2 by 54% and 51%, respectively (p<0.05). We conclude that A 1 AR activation by ENBA activates PLC, mainly through PKCα leading to ERK1/2 phosphorylation and contraction in CASMC. Supported by HL‐027339.