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 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. 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 microM) 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 (IC(50) 10.3+/-0.6 nM) and LY 294002 (IC(50) 10.3+/-1.2 microM) attenuated the phosphorylation of PKB elicited by CPA (1 microM) in a concentration-dependent manner. Wortmannin (30 nM) and LY 294002 (30 microM) also blocked responses to insulin (100 nM). 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 microM), tyrosine kinase (genistein; 100 microM), mitogen-activated protein (MAP) kinase kinase (PD 98059; 50 microM) and p38 MAPK (SB 203580; 20 microM) 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.
Germack
R
R
Department of Life Sciences, Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS.
Dickenson
J M
JM
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009025:Morpholines / Q000494:pharmacology; D037342:Pertussis Toxin; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D011725:Pyridines / Q000494:pharmacology; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000077191:Wortmannin; D014970:Xanthines / Q000494:pharmacology
D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D007211:Indoles; D007328:Insulin; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D011725:Pyridines; D018047:Receptors, Purinergic P1; D010566:Virulence Factors, Bordetella; D014970:Xanthines; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C048599:N(6)-cyclopentyladenosine; C051360:1,3-dipropyl-8-cyclopentylxanthine; D019833:Genistein; D037342:Pertussis Toxin; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D000241:Adenosine; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium; C064758:Ro 31-8220; D000077191:Wortmannin