Activation of protein kinase B by the A 1 ‐adenosine receptor in DDT 1 MF‐2 cells

In this study the effect of insulin and A 1 ‐adenosine receptor stimulation on protein kinase B (PKB) activation has been investigated in the hamster vas deferens smooth muscle cell line DDT 1 MF‐2. Increases in PKB phosphorylation were determined by Western blotting using an antibody that detects PKB phosphorylation at Ser 473 . Insulin, a recognized activator of PKB, stimulated a concentration‐dependent increase in PKB phosphorylation in DDT 1 MF‐2 cells (EC 50 5±1 pM). The selective A 1 ‐adenosine receptor agonist N 6 ‐cyclopentyladenosine (CPA) stimulated time and concentration‐dependent increases in PKB phosphorylation in DDT 1 MF‐2 cells (EC 50 1.3±0.5 n M ). CPA‐mediated increases in PKB phosphorylation were antagonized by the A 1 ‐adenosine receptor selective antagonist 1,3‐dipropylcyclopentylxanthine (DPCPX) yielding an apparent K D value of 2.3 n M . Pre‐treatment of DDT 1 MF‐2 cells with pertussis toxin (PTX, 100 ng ml −1 for 16 h), to block G i /G o ‐dependent pathways, abolished CPA (1 μ M ) induced phosphorylation of PKB. In contrast, responses to insulin (100 n M ) were resistant to PTX pre‐treatment. The phosphatidylinositol 3‐kinase (PI‐3K) inhibitors wortmannin (IC 50 10.3±0.6 n M ) and LY 294002 (IC 50 10.3±1.2 μ M ) attenuated the phosphorylation of PKB elicited by CPA (1 μ M ) in a concentration‐dependent manner. Wortmannin (30 n M ) and LY 294002 (30 μ M ) also blocked responses to insulin (100 n M ). Removal of extracellular Ca 2+ and chelation of intracellular Ca 2+ 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 μ M ), tyrosine kinase (genistein; 100 μ M ), mitogen‐activated protein (MAP) kinase kinase (PD 98059; 50 μ M ) and p38 MAPK (SB 203580; 20 μ M ) had no significant effect on CPA‐induced PKB phosphorylation. In conclusion, these data demonstrate that A 1 ‐adenosine receptor stimulation in DDT 1 MF‐2 cells increases PKB phosphorylation through a PTX and PI‐3K‐sensitive pathway.British Journal of Pharmacology (2000) 130 , 867–874; doi: 10.1038/sj.bjp.0703396