Development of the technique for expression of peptide receptors in the oocyte membrane

虽然肽受体细胞广泛分布于中枢和周围神经系统，但这些细胞太小，无法分析与受体激活相关的详细离子机制。本研究旨在分析直径约为1mm的爪蟾卵母细胞中表达的肽受体激活的离子机制。为此，将从大鼠脑或牛胃中分离的mRNA注射到卵母细胞中，并在孵育几天后在电压钳下记录注射的卵母细胞对多种肽的反应。虽然该技术可以表达5种不同肽的受体，但研究的重点是P物质受体。P物质是速激素家族的一员。目前，除了P物质外，已知哺乳动物神经系统中还存在两种速激素，即K物质和K神经质。根据快速激肽的存在，提出了三种快速激肽受体亚型的存在。通过不同速激素激动剂的效价排序，可以区分速激素受体的三种亚型。在此基础上，发现大鼠脑mRNA诱导的速激肽受体对应于SP-P速激肽受体，而牛胃mRNA诱导的速激肽受体对应于SP-K速激肽受体。因此，在相同的表达系统中可以诱导两种不同类型的速激肽受体，这取决于注入的外源mRNA的来源。这表明这两种受体是由不同的分子组成的。因此，两种受体之间的本质差异不能归因于可能的翻译后修饰单独发生在它们的目标组织中。另一方面，负责激活两种受体的离子机制都是基于<Cl^->电导的增加。这表明两种受体的激活使用相同的细胞内第二信使，而第二信使又激活卵母细胞膜上的<Cl^->离子通道。第二信使的性质可能在卵母细胞和哺乳动物神经元之间有所不同。因此，在卵母细胞中分析的离子机制不能推广到其他物种中存在的速激肽受体的激活。少

While peptide-receptive cells are widely distributed in both the central and peripheral nervous systems, these cells are too small to analyze detailed ionic mechanisms associated with activation of the receptors. The present study has been addressed to the analysis of ionic mechanisms underlying activation of the peptide receptors expressed in Xenopus oocytes which have a diameter of about 1 mm. For this purpose, mRNA isolated from the rat brain or the bovine stomach was injected into oocytes, and the response of the injected oocytes to a variety of peptides was recorded under voltage clamp following incubation for a few days. While the receptors to five different peptides could be expressed by this technique, the study was focused on the substance P receptor. Substance P is a member of the tachykinin family. At present, in addition to substance P, two other tachykinins, substance K and neuromedin K, are known to exist in the mammalian nervous system. In accord with the presence of mul … More tiple tachykinins, the existence of three subtypes of tachykinin receptors has been suggested. The three subtypes of tachykinin receptors could be distinguished by potency ranking of different tachykinin agonists. On this basis, the tachykinin receptor induced by rat brain mRNA was found to correspond to the SP-P tachykinin receptor, whereas that directed by bovine stomach mRNA corresponded to the SP-K tachykinin receptor. Thus, two different types of tachykinin receptors could be induced in the same expression system, depending upon the source of exogenous mRNA injected. This indicates that the two receptors are made of different molecules. Therefore, the difference in nature between the two receptors cannot be attributed to the possible posttranslational modification occurring at their destination tissue alone. On the other hand, the ionic mechanisms responsible for activation of the two receptors are both based on an increase in the <Cl^-> conductance. This suggests that activation of the two receptors uses the same intracellular second messenger, which in turn, activates <Cl^-> ionic channels in the oocyte membrane. The nature of the second messenger may be different between oocytes and mammalian neurons. Therefore, the ionic mechanisms analyzed in oocytes cannot be generalized to activation of tachykinin receptors present in other species. Less