Hazlerigg D G, Thompson M, Hastings M H, Morgan P J
Molecular Neuroendocrinology Group, Rowett Research Institute, Aberdeen, UK.
Endocrinology. 1996 Jan;137(1):210-8. doi: 10.1210/endo.137.1.8536615.
The pars tuberalis (PT) of the anterior pituitary is notable for the expression of levels of melatonin receptors that consistently exceed those in all other tissues in mammals. For this reason and because of its anatomical position, it has been suggested that the PT may play a role in seasonal reproductive responsiveness. However, no data have been forthcoming on the nature of the melatonin-responsive cells in this tissue or on the interaction of melatonin with other hormonal signals in the control of PT cells. A number of recent studies have reported that the tubero-infundibular region of the pituitary in several species contains binding sites for insulin-like growth factor-1 (IGF-1). The present study, therefore, sought to address the question of whether functional receptors for IGF-1 exist in the ovine PT (oPT). Primary cultures of cells from the oPT contained a widespread distribution of cells staining positively with a monoclonal antibody to the human IGF-1 receptor, with the strongest staining occurring over the small phase-bright cells that predominate in this culture system and are thought to constitute the melatonin-responsive cell type. As a functional assay for responsiveness to IGF-1, primary cultures of oPT cells were assayed for activation of mitogen-activated protein kinase (MAPK) using a previously validated phosphotransferase assay. Cytosolic extracts from PT cells treated with IGF-1 (100 pM-10 nM) caused a dose-dependent increase in the rate of phosphorylation of myelin basic protein; in contrast, treatment with melatonin had no significant effect on myelin basic protein phosphorylation. Immunostaining of Western blots of PT cell extracts with a pan-extracellular regulated kinase antibody demonstrated that both p42 and p44 MAPK are strongly expressed in this tissue. To confirm that the effects observed in the cytosol assay were indeed attributable to increased activation of p42/p44, gel renaturation assays of protein kinase activity were performed. These experiments revealed that IGF-1 (10 nM) and forskolin (1 microM) were both potent activators of 42- and 44-kDa moeities; however, neither of these agents had any significant effect on the phosphotransferase activity associated with several other higher molecular weight kinases also detected by the gel-renaturation assay procedure. Melatonin (10 nm) was consistently found to be a highly potent inhibitor of the activation of MAPK induced by forskolin; in contrast, melatonin did not inhibit the activation of MAPK induced by IGF-1.(ABSTRACT TRUNCATED AT 400 WORDS)
垂体前叶的结节部（PT）以表达褪黑素受体水平显著高于哺乳动物所有其他组织而闻名。鉴于此原因以及其解剖位置，有人提出PT可能在季节性繁殖反应中发挥作用。然而，关于该组织中褪黑素反应性细胞的性质，或者褪黑素与其他激素信号在控制PT细胞方面的相互作用，尚无数据报道。最近的一些研究报告称，几种物种垂体的结节 - 漏斗区域含有胰岛素样生长因子 - 1（IGF - 1）的结合位点。因此，本研究旨在探讨绵羊PT（oPT）中是否存在功能性IGF - 1受体这一问题。来自oPT的细胞原代培养物中，用抗人IGF - 1受体单克隆抗体染色呈阳性的细胞分布广泛，最强的染色出现在该培养系统中占主导的小的明亮相细胞上，这些细胞被认为构成了褪黑素反应性细胞类型。作为对IGF - 1反应性的功能测定，使用先前验证的磷酸转移酶测定法，对oPT细胞原代培养物进行有丝分裂原激活蛋白激酶（MAPK）激活的测定。用IGF - 1（100 pM - 10 nM）处理的PT细胞的胞质提取物导致髓鞘碱性蛋白磷酸化速率呈剂量依赖性增加；相比之下，用褪黑素处理对髓鞘碱性蛋白磷酸化没有显著影响。用泛细胞外调节激酶抗体对PT细胞提取物的蛋白质印迹进行免疫染色表明，p42和p44 MAPK在该组织中均强烈表达。为了证实胞质测定中观察到的效应确实归因于p42 / p44激活增加，进行了蛋白激酶活性的凝胶复性测定。这些实验表明，IGF - 1（10 nM）和福斯可林（1 microM）都是42 - 和44 - kDa部分的有效激活剂；然而，这些试剂对凝胶复性测定程序也检测到的其他几种高分子量激酶相关的磷酸转移酶活性均无显著影响。一直发现褪黑素（10 nM）是福斯可林诱导的MAPK激活的高效抑制剂；相比之下，褪黑素不抑制IGF - 1诱导的MAPK激活。（摘要截于400字）
