Parameswaran N, Disa J, Spielman W S, Brooks D P, Nambi P, Aiyar N
Department of Physiology, Michigan State University, East Lansing, MI, USA.
Eur J Pharmacol. 2000 Feb 18;389(2-3):125-30. doi: 10.1016/s0014-2999(99)00874-2.
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 ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-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-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 kinase A, the distal signaling components to protein kinase A in the activation of these two kinases (ERK and P38) are different.
降钙素基因相关肽是一种由37个氨基酸组成的神经肽，是一种强效血管舒张剂。尽管降钙素基因相关肽已被证明在多种系统中具有多种作用，但其作用机制和细胞内信号通路，尤其是丝裂原活化蛋白激酶（MAPK）通路的调节尚不清楚。在本研究中，我们研究了降钙素基因相关肽在稳定转染重组猪降钙素基因相关肽-1受体的人胚肾（HEK）293细胞中对MAPK的调节作用。降钙素基因相关肽引起cAMP反应显著的剂量依赖性增加，且该效应被降钙素基因相关肽（8-37）（降钙素基因相关肽受体拮抗剂）抑制。降钙素基因相关肽还引起细胞外信号调节激酶（ERK）和P38丝裂原活化蛋白激酶（P38 MAPK）活性的时间和浓度依赖性增加，而c-jun氨基末端激酶（JNK）活性显然没有显著变化。佛司可林（一种腺苷酸环化酶的直接激活剂）也刺激了这些细胞中的ERK和P38活性，表明cAMP参与了这一过程。降钙素基因相关肽受体拮抗剂降钙素基因相关肽（8-37）显著抑制了降钙素基因相关肽刺激的ERK和P38 MAPK活性，表明降钙素基因相关肽-1受体参与其中。用cAMP依赖性蛋白激酶抑制剂H89[盐酸N-[2-（（对溴肉桂基）氨基）乙基]-5-异喹啉磺酰胺]预孵育细胞可抑制降钙素基因相关肽介导的ERK和p38激酶的激活。另一方面，用渥曼青霉素[1S-（1α，6βα，9αβ，11α，11ββ）]-11-（乙酰氧基）-1，6β，7，8，9α，10，11，11β-八氢-1-（甲氧基甲基）-9α，11β-二甲基-3H-呋喃[4，3，2-de]茚并[4，5-h]-2-苯并吡喃-3，6，9-三酮]（一种PI3激酶抑制剂）预孵育细胞仅减弱降钙素基因相关肽诱导的ERK激活，而不影响P38 MAPK激活。因此，这些数据表明，降钙素基因相关肽对ERK的激活涉及H89敏感的蛋白激酶A和渥曼青霉素敏感的PI3激酶；而降钙素基因相关肽对p38 MAPK的激活仅涉及H89敏感的途径，且不依赖于PI3激酶。这也表明，尽管ERK和P38都可以被蛋白激酶A激活，但在这两种激酶（ERK和P38）激活过程中，蛋白激酶A下游的信号成分是不同的。
