Ecophysiological and Environmental Toxicological Research Group, HUN-REN Balaton Limnological Research Institute, Tihany, 8237, Hungary.
Genomics and Bioinformatics Core Facilities, Szentágothai Research Centre, University of Pécs, Pécs, 7624, Hungary.
J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2024 Nov;210(6):909-925. doi: 10.1007/s00359-024-01706-5. Epub 2024 Jun 28.
Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail (Lymnaea stagnalis) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific.
有证据表明，脊椎动物垂体腺苷酸环化酶激活肽（PACAP）系统的某些元素在非脊索动物基因组中缺失，这与文献中的部分序列、免疫组织化学和生理学数据不一致。在大型圆田螺（Lymnaea stagnalis）上进行了多层次实验，以探索 PACAP 在无脊椎动物中的作用。对神经元转录组和基因组数据进行筛选并未发现与脊椎动物 PACAP 系统元件同源的物质。尽管如此，用抗人 PAC 受体抗体进行的免疫组织化学研究在心的神经元中产生了阳性信号。尽管从神经系统提取的蛋白质的 Western blot 发现了 PACAP-38 的相关带，但免疫沉淀和质谱分析并未揭示相应的肽片段。与在脊椎动物中报道的作用类似，PACAP-38 显著增加了心脏中的 cAMP 合成，并对心脏制剂具有正离子激动作用。此外，它还显著调节了 5-羟色胺和乙酰胆碱的作用。与脊椎动物 PACAP 受体、甲状旁腺激素受体和 GCGR 具有共同进化起源的 B 类受体成员的同源物被鉴定出来，并且表明它们不在心脏中表达，这并不支持它们在介导 PACAP 诱导的作用中的潜在作用。我们的发现支持了 PACAP 系统在原口动物和后口动物分化后出现的观点。再次强调，使用针对脊椎动物蛋白的抗体在无脊椎动物研究中几乎没有价值。原口动物中脊椎动物 PACAP 肽的生理作用，无论它们与脊椎动物中的作用多么相似，都应被视为非特异性的。
