Lelièvre V, Pineau N, Du J, Wen C H, Nguyen T, Janet T, Muller J M, Waschek J A
Department of Psychiatry, Mental Retardation Research Center, UCLA, Neuropsychiatric Institute, Los Angeles, CA 90024, USA.
J Biol Chem. 1998 Jul 31;273(31):19685-90. doi: 10.1074/jbc.273.31.19685.
The growth rate of rodent embryonic neuroblasts and human neuroblastoma cell lines is regulated in part by autocrine or paracrine actions of neuropeptides of the family that includes vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and pituitary adenylate cyclase-activating peptide (PACAP). These peptides act via seven transmembrane G-protein-linked receptors coupled to cAMP elevation, phospholipase C activation, intracellular Ca2+ release, and/or of mitogen-activated protein (MAP) kinase activation. Here we investigated the action of these peptides on the mouse neuroblastoma cell line Neuro2a. PHI and VIP inhibited proliferation at concentrations as low as 10(-13) M and 10(-10) M, respectively. In contrast, PACAP action was biphasic, with stimulation occurring at subnanomolar doses and inhibition at higher doses. Peptide actions were studied further by measuring cAMP and ERK1/2 MAP kinase activity and by assessing 3H-thymidine incorporation in conjunction with a panel of signal transduction pathways inhibitors. The data obtained indicated that the PHI-inhibitory and PACAP-stimulatory activities were mediated by corresponding changes in activity of the MAP kinase pathway and independent of protein kinase A (PKA) or protein kinase C (PKC). In contrast, the inhibitory actions of VIP and PACAP were specifically blocked by antagonists of PKA. Northern blot analysis revealed gene expression for only the PACAP-preferring (PAC1) receptor. However, binding experiments using 125I-labeled PACAP27, PHI, and VIP, demonstrated the presence of PACAP-preferring sites, bivalent VIP/PACAP sites, and PHI-binding sites that did not interact with VIP. The studies demonstrate potent regulatory actions of PACAP, PHI, and VIP on neuroblastoma cell proliferation which appear to be mediated by multiple subsets of receptors which differentially couple to MAP kinase and PKA signaling pathways.
啮齿动物胚胎神经母细胞和人类神经母细胞瘤细胞系的生长速率部分受包括血管活性肠肽（VIP）、肽组氨酸异亮氨酸（PHI）和垂体腺苷酸环化酶激活肽（PACAP）在内的该家族神经肽的自分泌或旁分泌作用调节。这些肽通过与cAMP升高、磷脂酶C激活、细胞内Ca2+释放和/或丝裂原活化蛋白（MAP）激酶激活偶联的七跨膜G蛋白偶联受体发挥作用。在此，我们研究了这些肽对小鼠神经母细胞瘤细胞系Neuro2a的作用。PHI和VIP分别在低至10^(-13)M和10^(-10)M的浓度下抑制增殖。相比之下，PACAP的作用是双相的，在亚纳摩尔剂量下刺激，在较高剂量下抑制。通过测量cAMP和ERK1/2 MAP激酶活性，并结合一组信号转导途径抑制剂评估3H-胸苷掺入，进一步研究了肽的作用。获得的数据表明，PHI的抑制活性和PACAP的刺激活性是由MAP激酶途径活性的相应变化介导的，且独立于蛋白激酶A（PKA）或蛋白激酶C（PKC）。相反，VIP和PACAP的抑制作用被PKA拮抗剂特异性阻断。Northern印迹分析仅显示了偏爱PACAP的（PAC1）受体的基因表达。然而，使用125I标记的PACAP27、PHI和VIP进行的结合实验表明，存在偏爱PACAP的位点、双价VIP/PACAP位点以及不与VIP相互作用的PHI结合位点。这些研究证明了PACAP、PHI和VIP对神经母细胞瘤细胞增殖具有强大的调节作用，这似乎是由与MAP激酶和PKA信号通路差异偶联的多个受体亚群介导的。
