Activation of protein kinase B by the A1‐adenosine receptor in DDT1MF‐2 cells

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Abstract
In this study the effect of insulin and A1-adenosine receptor stimulation on protein kinase B (PKB) activation has been investigated in the hamster vas deferens smooth muscle cell line DDT1MF-2. Increases in PKB phosphorylation were determined by Western blotting using an antibody that detects PKB phosphorylation at Ser473.Insulin, a recognized activator of PKB, stimulated a concentration-dependent increase in PKB phosphorylation in DDT1MF-2 cells (EC50 5±1 pM).The selective A1-adenosine receptor agonist N6-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in PKB phosphorylation in DDT1MF-2 cells (EC50 1.3±0.5 nM). CPA-mediated increases in PKB phosphorylation were antagonized by the A1-adenosine receptor selective antagonist 1,3-dipropylcyclopentylxanthine (DPCPX) yielding an apparent KD value of 2.3 nM.Pre-treatment of DDT1MF-2 cells with pertussis toxin (PTX, 100 ng ml−1 for 16 h), to block Gi/Go-dependent pathways, abolished CPA (1 μM) induced phosphorylation of PKB. In contrast, responses to insulin (100 nM) were resistant to PTX pre-treatment.The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin (IC50 10.3±0.6 nM) and LY 294002 (IC50 10.3±1.2 μM) attenuated the phosphorylation of PKB elicited by CPA (1 μM) in a concentration-dependent manner. Wortmannin (30 nM) and LY 294002 (30 μM) also blocked responses to insulin (100 nM).Removal of extracellular Ca2+ and chelation of intracellular Ca2+ with BAPTA had no significant effect on CPA-induced PKB phosphorylation. Similarly, pretreatment (30 min) with inhibitors of protein kinase C (Ro 31-8220; 10 μM), tyrosine kinase (genistein; 100 μM), mitogen-activated protein (MAP) kinase kinase (PD 98059; 50 μM) and p38 MAPK (SB 203580; 20 μM) had no significant effect on CPA-induced PKB phosphorylation.In conclusion, these data demonstrate that A1-adenosine receptor stimulation in DDT1MF-2 cells increases PKB phosphorylation through a PTX and PI-3K-sensitive pathway.