Santama N, Wheeler C H, Skingsley D R, Yeoman M S, Bright K, Kaye I, Burke J F, Benjamin P R
Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Brighton, UK.
Eur J Neurosci. 1995 Feb 1;7(2):234-46. doi: 10.1111/j.1460-9568.1995.tb01059.x.
We are interested in analysing the detailed modulation of defined neuronal systems by multiple neuropeptides encoded in the FMRFamide locus of the snail Lymnaea. Cloning of the FMRFamide gene has predicted the existence of two novel peptides previously unknown from biochemical analysis, the pentapeptides EFLRlamide and QFYRlamide. These peptides may form part of a new family of peptides sharing the sequence motif -FXRlamide. In this paper we adopt a novel approach to first identify and characterize -FXRlamide-like peptides in extracts from the central nervous system of Lymnaea. By a combination of high-performance liquid chromatography (HPLC) and continuous-flow fast atom bombardment mass spectrometry, we identify three novel peptides: EFLRlamide, pQFYRlamide and pQFLRlamide. The first two are those predicted in exon II of the FMRFamide locus whereas the last is, interestingly, a product which cannot be derived from post-translational modification of the predicted peptides but must be encoded by as yet unidentified nucleotide sequences. A specific antibody raised to EFLRlamide, and immunoreactive to all three peptides, revealed EFLRlamide-like expression throughout the central nervous system in the same cells where exon II is transcribed and the peptide SEEPLY (a post-translational product of exon II) was localized. Additional cells, however, were also identified. Immunoreactivity was mapped in a number of identified neurons in the central nervous system, including two heart cardioexcitatory motoneurons, the Ehe cells (E heart excitors of the visceral ganglion) and penial motoneurons in the right cerebral ganglion. The peripheral tissues (heart and penial complex) that these respective classes of neurons innervate also exhibited EFLRlamide immunoreactivity. The central and peripheral localization of EFLRlamide-like immunoreactivity suggested that EFLRlamide/pQFYRlamide may have an important physiological role in both these peripheral systems as well as in the central nervous system. This was confirmed by physiological experiments that showed that EFLRlamide and pQFYRlamide inhibited many central neurons and in particular the Bgp neurons in the right parietal ganglion. EFLRlamide had complex biphasic effects on the frequency of heart-beat: an initial inhibitory response was followed by a long-lasting increase in the rate of beating. Taken together with earlier work, this study now completes the analysis and localization of the full set of post-translational products of the FMRFamide precursor in Lymnaea and supplies further evidence towards the characterization of the physiological systems which such peptides may modulate in concert.
我们感兴趣的是分析由蜗牛椎实螺FMRF酰胺基因座中编码的多种神经肽对特定神经元系统的详细调节作用。FMRF酰胺基因的克隆预测了两种以前生化分析未知的新型肽的存在，即五肽EFLR酰胺和QFYR酰胺。这些肽可能构成了一个共享序列基序-FXR酰胺的新肽家族的一部分。在本文中，我们采用一种新方法，首先在椎实螺中枢神经系统提取物中鉴定和表征-FXR酰胺样肽。通过高效液相色谱（HPLC）和连续流快原子轰击质谱联用，我们鉴定出三种新型肽：EFLR酰胺、pQFYR酰胺和pQFLR酰胺。前两种是FMRF酰胺基因座外显子II中预测的肽，而有趣的是，最后一种是一种产物，它不能从预测肽的翻译后修饰中产生，而必须由尚未鉴定的核苷酸序列编码。针对EFLR酰胺产生的一种特异性抗体，对所有三种肽都有免疫反应，在整个中枢神经系统中，在外显子II转录的同一细胞以及肽SEEPLY（外显子II的翻译后产物）定位的细胞中显示出EFLR酰胺样表达。然而，也发现了其他细胞。免疫反应性定位在中枢神经系统的一些已鉴定神经元中，包括两个心脏兴奋运动神经元、Ehe细胞（内脏神经节的E心脏兴奋神经元）和右脑神经节中的阴茎运动神经元。这些不同类型神经元所支配的外周组织（心脏和阴茎复合体）也表现出EFLR酰胺免疫反应性。EFLR酰胺样免疫反应性在中枢和外周的定位表明，EFLR酰胺/pQFYR酰胺可能在这些外周系统以及中枢神经系统中都具有重要的生理作用。生理实验证实了这一点，实验表明EFLR酰胺和pQFYR酰胺抑制许多中枢神经元，特别是右顶神经节中的Bgp神经元。EFLR酰胺对心跳频率有复杂的双相作用：最初的抑制反应之后是心跳速率的长期增加。结合早期的工作，这项研究现在完成了对椎实螺FMRF酰胺前体全套翻译后产物的分析和定位，并为表征这些肽可能协同调节的生理系统提供了进一步的证据。
