Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor

Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a poten t vasodilator. Although calcitonin gene-related peptide has been shown to h ave a number of effects in a variety of systems, the mechanisms of action a nd the intracellular signaling pathways, especially the regulation of mitog en-activated protien kinase (MAPK) pathway, is not known. In the present st udy we investigated the role of calcitonin gene-related peptide in the regu lation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfecte d with a recombinant porcine calcitonin gene-related peptide-1 receptor. Ca lcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related pe ptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calci tonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen act ivated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activ ator of adenylyl cyclase also stimulated ERK and P38 activities in these ce lls suggesting the invovement of cAMP in this process. Calcitonin gene-rela ted peptide-stimulated ERK and P38 MAPK activities were inhibited significa ntly by calcitonin gene-related peptide receptor antagonist, calcitonin gen e related peptide-(8-37) suggesting the involvement of calcitonin gene-rela ted peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [{N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoqu inolinesulfonamide, hydrochloride}] inhibited calcitonin gene-related pepti de-mediated activation of ERK and p38 kinases. On the other hand, preincuba tion of the cells with wortmannin {[1S-(1 alpha,6b alpha,9a beta,11 alpha, 11b beta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxymethyl) -9a,11b-dimethyl-3H-furo[4,3,2-de]indeno[4,5-h]-2-benzopyran-3,6,9-trione}, a PI3-kinase inhibitor, attenuated only calcitonin gene-related peptide-in duced ERK and not P38 MAPK activation. Thus, these data suggest that activa tion of ERK by calcitonin gene-related peptide involves a H89-sensitive pro tein kinase A and a wortmannin-sensitive PI3-kinase while activation of p38 MAPK by calcitonin gene related peptide involves only the H89 sensitive pa thway and is independent of PI3 kinase. This also suggests that although bo th ERK and P38 can be activated by protein kinase A, the distal signaling c omponents to protein kinase A in the activation of these two kinases (ERK a nd P38) are different. (C) 2000 Elsevier Science B.V. All rights reserved.