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

1 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 DDT1MF-2. Increases in PKB phosphorylation were determined by Western blotting using an antibody that detects PKB phosphorylation at Ser(473). 2 Insulin, a recognized activator of PKB, stimulated a concentration-dependent increase in PKB phosphorylation in DDT1MF-2 cells (EC50 5+/-1 pM). 3 The selective A(1)-adenosine receptor agonist N-6-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 A(1)-adenosine receptor selective antagonist 1,3-dipropylcyclopentylxanthine (DPCPX) yielding an apparent K-D value of 2.3 nM. 4 Pre-treatment of DDT1MF-2 cells with pertussis toxin (PTX, 100 ng ml(-1) for 16 h), to block G(i)/ G(o)-dependent pathways, abolished CPA (1 mu M) induced phosphorylation of PKB. In contrast, responses to insulin (100 nM) were resistant to PTX pre-treatment. 5 The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin (IC50 10.3+/-0.6 nM) and LY 294002 (IC50 10.3 +/- 1.2 mu M) attenuated the phosphorylation of PKB elicited by CPA (1 mu M) in a concentration-dependent manner. Wortmannin (30 nM) and LY 294002 (30 mu M) also blocked responses to insulin (100 nM). 6 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 mu M), tyrosine kinase (genistein; 100 mu M), mitogen-activated protein (MAP) kinase kinase (PD 98059; 50 mu M) and p38 MAPK (SB 203580; 20 mu M) had no significant effect on CPA-induced PKB phosphorylation. 7 In conclusion, these data demonstrate that A(1)-adenosine receptor stimulation in DDT1MF-2 cells increases PKB phosphorylation through a PTX and PI-3K-sensitive pathway.