Regulation of p42/p44 MAPK and p38 MAPK by the adenosine A(1) receptor in DDT1MF-2 cells

The mitogen-activated protein kinase (MAPK) family consists of the p42/p44 MAPKs and the stress-activated protein kinases, c-Jun N-terminal kinase (JN K) 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, DDT1MF-2 activates p42/p44 MAPK, JNK and p38 MAPK. The adenosine A(1) receptor agonist N-6-cyclopentyladenos ine stimulated time and concentration-dependent increases in p42/p44 MAPK a nd p38 MAPK phosphorylation in DDT1MF-2 cells. No increases in JNK phosphor ylation were observed following adenosine A(1) receptor activation. N-6-cyc lopentyladenosine-mediated increases in p42/p44 MAPK and p38 MAPK phosphory lation were blocked by the selective adenosine A(1) receptor antagonist 1,3 -dipropylcyclopentylxanthine and following pretreatment of cells with pertu ssis 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 SE 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyr idyl)1H-imidazole). The broad range protein tyrosine kinase inhibitors geni stein and tyrphostin A47 (alpha -cyano-(3,4-dihydroxy)thiocinnamide) did no t 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-chlorop henyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and the epidermal growth facto r receptor tyrosine kinase inhibitor AG1478 (4-(3-chloroanilino)-6,7-dimeth oxyquinazoline) also had no effect on adenosine A(1) receptor stimulation o f 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)-1H-i ndol-3-yl]3-(1H-indol-3-yl)-maleimide) were without effect on adenosine A(1 ) receptor-induced p42/p44 MAPK phosphorylation. In contrast, wortmannin an d LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), inhibitors of phosphatidylinositol 3-kinase, attenuated adenosine A(1) receptor stimul ation of p42/p44 MAPK phosphorylation. In conclusion, the adenosine A(1) re ceptor stimulates p42/p44 MAPK through a pathway which appears to be indepe ndent of tyrosine kinase activation but involves phosphatidylinositol 3-kin ase. Finally, adenosine A(1) receptor stimulation in DDT1MF-2 cells also ac tivated p38 MAPK but not JNK via a pertussis toxin-sensitive pathway. (C) 2 001 Elsevier Science B.V. All rights reserved.