Study for the effects of neuroactive peptides newly isolated

本研究采用电生理方法综合研究了生物活性肽对非洲巨蜗牛（Achatina fulica Ferussac）可识别巨神经元的影响。Achatina巨神经元对P物质、脑啡肽、神经紧张素等哺乳动物神经递质不敏感，但对催产素不敏感。另一方面，这些神经元对从无脊椎动物中分离的神经活性肽敏感。这些发现不同于小分子假定的神经递质，如多巴胺、血清素和GABA。根据这些结果，认为一种多肽神经递质作用于动物物种的神经元的程度比小分子假定的神经递质要小。我们与三得利生物有机研究所和圣托马斯大学合作，从Achatina神经节中分离出Achatina - 1 (gly - d - ph - ala - asp)，对Achatina巨神经元进行了兴奋作用。APGW-amide （Ala-Pr . More o-Gly-Trp-NH_2）也从Achatina神经节中分离得到，对Achatina巨神经元有抑制作用。这两种多肽被认为是Achatina神经元的神经递质。此外，我们还证实了另一组从Achatina神经节分离的Achatina心脏兴奋肽-1 (ACEP-1) （ser - gly - gln - ser - trp - arg - pro - gln - gly - arg - ph - nh_2）对Achatina巨神经元有兴奋作用，另一组从Achatina神经节分离的fulicin （ph - d - asn - gln - ph - valh - nh_2）对Achatina巨神经元有抑制作用。我们还证实了从蛤神经节中分离的FMRFFamide (Phe-Met-Arg-Phe-NH_2)，从一种Ascaris中分离的FMRFamide的相关化合物AF1 （Lys-Asn-Glu-Phe-Ile-Arg-phe-NH_2）和从贻贝中分离的[Ser^2]-贻贝抑制肽（[Ser^2]-MIP） （gly -Ser- pro - met -phe- valh - nh_2）对这些神经元具有抑制作用。电生理学研究了细胞内信号转导系统的兴奋作用的achatin- 1和抑制作用的fulicin这些系统的抑制剂。H-89 （protein kinase (PK) A inhibitor）和W-7 （calmodulin inhibitor）抑制了achatin-I的作用，IBMX （cyclic AMP phosphodi酯酶inhibitor）促进了同样的作用，表明achatin-I通过环AMP- pka系统起作用，而calmodulin参与了该系统。另一方面，KT-5823 （PKG抑制剂）、calphostin C （PKC抑制剂）和W-7抑制了黄芩苷的抑制作用，表明黄芩苷通过环GMP-PKG系统和/或IP_3和DG-PKC系统起作用，钙调素参与了这些系统。IBMX抑制了抑制作用，其机制目前还不能解释。我们证明APGW-amide和achatin-I不仅作为神经递质，而且作为Achatina神经元的神经调节剂。少

This year, we studied comprehensively effects of biologically-active peptides on the identifiable giant neurones of an African giant snail (Achatina fulica Ferussac) by the electrophysiological methods. The Achatina giant neurones were insensitive to the mammalian neurotransmitter peptides, such as substance P,Met-enkephalin, neurotensin, etc., except for oxytocin. On the other hand, these neurones were sensitive to the neuroactive peptides isolated from invertebrates. These findings were different from those of small molecule putative neurotransmitters, such as dopamine, serotonin and GABA.With these results, it was considered that one peptide neurotransmitter acts on the neurones of the less extent of animal species than the small molecule putative neurotransmitter. Achatina giant neurones were excited by achatin-I (Gly-D-Phe-Ala-Asp), isolated from Achatina ganglia by the collaboration of us, Suntory Institute for Bioorganic Research and University of Santo Tomas. APGW-amide (Ala-Pr … More o-Gly-Trp-NH_2), isolated also from Achatina ganglia by us, were inhibited Achatina giant neurones. These two peptides were proposed to be neurotransmitters for Achatina neurones. Besides, we demonstrated that Achatina cardio-excitatory peptide-1 (ACEP-1) (Ser-Gly-Gln-Ser-Trp-Arg-Pro-Gln-Gly-Arg-Phe-NH_2) isolated from Achatina ganglia by another group showed excitatory effects on Achatina giant neurones, and that fulicin (Phe-D-Asn-Gln-Phe-Val-NH_2) isolated from Achatina ganglia by another group had inhibitory effects on these neurones. We demonstrated also that FMRFFamide (Phe-Met-Arg-Phe-NH_2) isolated from the clam ganglia, AF1 (Lys-Asn-Glu-Phe-Ile-Arg-phe-NH_2), a related compound of FMRFamide isolated from an Ascaris, and [Ser^2]-Mytilus inhibitory peptide ([Ser^2]-MIP) (Gly-Ser-Pro-Met-Phe-Val-NH_2) isolated from Mytilus, had inhibitory effects on these neurones.The intracellular signal transduction systems for the excitatory effects of achatin-I and the inhibitory effects of fulicin were studied electrophysiologically using the inhibitors for these systems. H-89 (protein kinase (PK) A inhibitor) and W-7 (calmodulin inhibitor) inhibited the achatin-I effects, and IBMX (cyclic AMP phosphodiesterase inhibitor) facilitated the same effects, indicating that achatin-I acted via cyclic AMP-PKA system, and calmodulin participated in this system. On the other hand, KT-5823 (PKG inhibitor), calphostin C (PKC inhibitor) and W-7 suppressed the inhibitory effects of fulicin, indicating that fulicin acted via cyclic GMP-PKG system and/or IP_3 and DG-PKC systems, and calmodulin participated in these systems. IBMX suppressed the inhibitory effects, the mechanism of which cannot be explained at present.We demonstrated that APGW-amide, as well as achatin-I,acted not only as a neurotransmitter but also as a neuromodulator for Achatina neurones. Less