Regulation of p42/p44 MAPK and p38 MAPK by the adenosine A 1 receptor in DDT 1MF-2 cells

Abstract

The mitogen-activated protein kinase (MAPK) family consists of the p42/p44 MAPKs and the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 MAPK. We have previously reported that the human adenosine A 1 receptor stimulates p42/p44 MAPK in transfected Chinese hamster ovary cells. In this study, we have investigated whether the endogenous adenosine A 1 receptor in the smooth muscle cell line, DDT 1MF-2 activates p42/p44 MAPK, JNK and p38 MAPK. The adenosine A 1 receptor agonist N 6-cyclopentyladenosine stimulated time and concentration-dependent increases in p42/p44 MAPK and p38 MAPK phosphorylation in DDT 1MF-2 cells. No increases in JNK phosphorylation were observed following adenosine A 1 receptor activation. N 6-cyclopentyladenosine-mediated increases in p42/p44 MAPK and p38 MAPK phosphorylation were blocked by the selective adenosine A 1 receptor antagonist 1,3-dipropylcyclopentylxanthine and following pretreatment of cells with pertussis toxin. Furthermore, adenosine A 1 receptor-mediated increases in p42/p44 MAPK were sensitive to the MAPK kinase 1 inhibitor PD 98059 (2′-amino-3′-methoxyflavone), whereas p38 MAPK responses were blocked by the p38 MAPK inhibitor SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 H-imidazole). The broad range protein tyrosine kinase inhibitors genistein and tyrphostin A47 (α-cyano-(3,4-dihydroxy)thiocinnamide) did not block adenosine A 1 receptor stimulation of p42/p44 MAPK. For comparison, insulin-mediated increases in p42/p44 MAPK were blocked by genistein and tyrphostin A47. The Src tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine) and the epidermal growth factor receptor tyrosine kinase inhibitor AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline) also had no effect on adenosine A 1 receptor stimulation of p42/p44 MAPK. Furthermore, the protein kinase C inhibitors Ro 31-8220 (3-{1-[3-(2-isothioureido) propyl]indol-3-yl}-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione), chelerythrine and GF 109203X (2-[1-(3-dimethylaminopropyl)-1 H-indol-3-yl]-3-(1 H-indol-3-yl)-maleimide) were without effect on adenosine A 1 receptor-induced p42/p44 MAPK phosphorylation. In contrast, wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4 H-1-benzopyran-4-one), inhibitors of phosphatidylinositol 3-kinase, attenuated adenosine A 1 receptor stimulation of p42/p44 MAPK phosphorylation. In conclusion, the adenosine A 1 receptor stimulates p42/p44 MAPK through a pathway which appears to be independent of tyrosine kinase activation but involves phosphatidylinositol 3-kinase. Finally, adenosine A 1 receptor stimulation in DDT 1MF-2 cells also activated p38 MAPK but not JNK via a pertussis toxin-sensitive pathway.