Michael-Titus A T, Fernandes K, Setty H, Whelpton R
Neuroscience Section, Division of Biomedical Sciences, St. Bartholowmew's and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, London, UK.
Neuroscience. 2002;110(2):277-86. doi: 10.1016/s0306-4522(01)00530-9.
Neurons expressing the preprotachykinin A gene, which encodes the sequences of substance P, neurokinin A, neuropeptide gamma and neuropeptide K, exemplify peptide co-existence. Furthermore, there is also evidence that substance P fragments have biological activity. However, the relative contribution of each of these peptides to tachykinin signalling is still poorly understood. An important factor which will determine the characteristics of the signal mediated by co-localised peptides is their clearance from the extracellular space. The striatum, in which tachykinins are present and exert neuromodulatory roles, can be used as a model to investigate this aspect. Therefore, in this study we characterised in vivo in the striatum the metabolism and clearance of substance P and of the other three co-expressed peptides. After intrastriatal administration of 1 pmol, tritiated substance P disappeared too rapidly for metabolites to be detected. However, when 10 nmol substance P and 1 pmol tritiated substance P were co-injected, substance P(1-4) and substance P(1-7), which are biologically active, were detected as major metabolites. Under these conditions, the rate of decay of tritiated substance P was 0.2 nmol/min. The effects of the peptidase inhibitors thiorphan, bestatin and captopril suggested that neutral endopeptidase 24.11 and aminopeptidases were involved in primary substance P cleavages, whereas angiotensin-converting enzyme was involved in secondary cleavages. The monitoring of the decay of unlabelled substance P by high-performance liquid chromatography gave a rate of 0.16 nmol/min. Using high-performance liquid chromatography with capillary electrophoresis, the rates of decay of 10 nmol neurokinin A or neuropeptide gamma were five and seven times faster than that of substance P. In contrast, over the time course of the experiment, no significant decay of neuropeptide K was detected. These results show that substance P disappears rapidly from the extracellular space, and supports the formation in vivo of major N-terminal active substance P metabolites. Our study also highlights significant differences in the clearance of co-expressed tachykinins and suggests that certain species may disappear relatively slowly from the extracellular space, and thus may make a significant temporal and spatial contribution to signalling.
表达前速激肽原A基因的神经元可体现肽类共存现象，该基因编码P物质、神经激肽A、神经肽γ和神经肽K的序列。此外，也有证据表明P物质片段具有生物活性。然而，这些肽类各自对速激肽信号传导的相对贡献仍知之甚少。一个决定共定位肽介导信号特征的重要因素是它们从细胞外空间的清除情况。存在速激肽并发挥神经调节作用的纹状体可作为研究这一方面的模型。因此，在本研究中，我们在纹状体内对P物质以及其他三种共表达肽的代谢和清除进行了体内特征分析。在纹状体内注射1皮摩尔的氚标记P物质后，其消失速度太快，以至于无法检测到代谢产物。然而，当同时注射10纳摩尔P物质和1皮摩尔氚标记P物质时，具有生物活性的P物质（1-4）和P物质（1-7）被检测为主要代谢产物。在这些条件下，氚标记P物质的衰减速率为0.2纳摩尔/分钟。肽酶抑制剂硫磷酰胺、贝司他汀和卡托普利的作用表明，中性内肽酶24.11和氨肽酶参与了P物质的初级裂解，而血管紧张素转换酶参与了次级裂解。通过高效液相色谱法监测未标记P物质的衰减速率为0.16纳摩尔/分钟。使用高效液相色谱-毛细管电泳法，10纳摩尔神经激肽A或神经肽γ的衰减速率比P物质快五倍和七倍。相反，在实验过程中，未检测到神经肽K有明显衰减。这些结果表明P物质从细胞外空间迅速消失，并支持体内形成主要的N端活性P物质代谢产物。我们的研究还突出了共表达速激肽在清除方面的显著差异，并表明某些肽类可能从细胞外空间相对缓慢地消失，因此可能在信号传导中做出显著的时间和空间贡献。
