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 potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8–37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8–37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [{ N-[2-(( p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride}] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin {[1 S-(1α,6bα,9aβ,11α,11bβ)]-11-(acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3 H-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-induced ERK and not P38 MAPK activation. Thus, these data suggest that activation of ERK by calcitonin gene-related peptide involves a H89-sensitive protein kinase A and a wortmannin-sensitive PI3-kinase while activation of p38 MAPK by calcitonin gene-related peptide involves only the H89 sensitive pathway and is independent of PI3 kinase. This also suggests that although both ERK and P38 can be activated by protein