Same same, but different: exploring the enigmatic role of the pituitary adenylate cyclase- activating polypeptide (PACAP) in invertebrate physiology

Abstract There is a long-standing debate about the presence and functionality of the pituitary adenylate cyclase-activating polypeptide (PACAP) in non-bilaterians, protostomes, and invertebrate deuterostomes. Evidence has been accumulating that homologous sequences to genes encoding PACAP peptides and their receptors in vertebrates are missing in invertebrate genomes. This is at odds, however, with the partial sequence-, immunohistochemical-, and physiological evidence in the literature. In this study, we first sequenced the neural transcriptome of the widely used invertebrate model species, the great pond snail ( Lymnaea stagnalis ), and then screened it for sequences homologous to the elements of the vertebrate PACAP system. Further, we performed in vitro and in vivo studies on the heart of L. stagnalis to explore the enigmatic role of vertebrate PACAP in invertebrate physiology. Our thorough screening failed to identify putative transcripts (or genes) to the vertebrate PACAP prepropeptides, active peptides, and their receptors. Despite the lack of the relevant sequences, our immunohistochemical investigations with an anti-human PAC 1 receptor antibody yielded a positive signal in the neuronal elements in the heart. Although gel electrophoretic separation, followed by immunostaining, of proteins extracted from the central nervous system found a relevant band for the vertebrate PACAP-38, mass spectrometric analysis of the band did not find any corresponding peptide sequences. Similarly to the effects reported in vertebrates, 10 µM synthetic PACAP-38 significantly increased the cAMP synthesis in the homogenate of the heart and had a positive ionotropic effect on isolated heart preparations. Moreover, it modulated significantly the effects of serotonin and acetylcholine. Our findings support the idea that elements of the PACAP system are absent in mollusks and emerged after the protostome-deuterostome divergence. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific. Further studies should be aimed at investigating the cellular and molecular underpinnings including the identification of the receptors to which the vertebrate PACAP peptides may bind non-specifically.