Biochemical, Anatomical, and Pharmacological Characterization of Calcitonin-Type Neuropeptides in Starfish: Discovery of an Ancient Role as Muscle Relaxants.

Weigang Cai,Chan-Hee Kim,Hye-Jin Go,Maurice R Elphick,Alexandra M Jones,Nam Gyu Park,Cleidiane G Zampronio,Michaela Egertová

doi:10.3389/fnins.2018.00382

Abstract

Calcitonin (CT) is a peptide hormone released by the thyroid gland that regulates blood Ca2+ levels in mammals. The CT gene is alternatively spliced, with one transcript encoding CT and another transcript encoding the CT-like neuropeptide calcitonin-gene related peptide (α-CGRP), which is a powerful vasodilator. Other CT-related peptides in vertebrates include adrenomedullin, amylin, and intermedin, which also act as smooth muscle relaxants. The evolutionary origin of CT-type peptides has been traced to the bilaterian common ancestor of protostomes and deuterostomes and a CT-like peptide (DH31) has been identified as a diuretic hormone in some insect species. However, little is known about the physiological roles of CT-type peptides in other invertebrates. Here we characterized a CT-type neuropeptide in a deuterostomian invertebrate—the starfish Asterias rubens (Phylum Echinodermata). A CT-type precursor cDNA (ArCTP) was sequenced and the predicted structure of the peptide (ArCT) derived from ArCTP was confirmed using mass spectrometry. The distribution of ArCTP mRNA and the ArCT peptide was investigated using in situ hybridization and immunohistochemistry, respectively, revealing stained cells/processes in the nervous system, digestive system, and muscular organs, including the apical muscle and tube feet. Investigation of the effects of synthetic ArCT on in vitro preparations of the apical muscle and tube feet revealed that it acts as a relaxant, causing dose-dependent reversal of acetylcholine-induced contraction. Furthermore, a muscle relaxant present in whole-animal extracts of another starfish species, Patiria pectinifera, was identified as an ortholog of ArCT and named PpCT. Consistent with the expression pattern of ArCTP in A. rubens, RT-qPCR revealed that in P. pectinifera the PpCT precursor transcript is more abundant in the radial nerve cords than in other tissues/organs analyzed. In conclusion, our findings indicate that the physiological action of CT-related peptides as muscle relaxants in vertebrates may reflect an evolutionarily ancient role of CT-type neuropeptides that can be traced back to the common ancestor of deuterostomes.

Highlights

The thyroid hormone calcitonin was discovered in mammals as a regulator of blood calcium levels (Copp and Cameron, 1961; Copp et al, 1962) and identified as 32-residue C-terminally amidated peptide with an N-terminal disulfide bond (Potts et al, 1968; Niall et al, 1969)
Comparison of the sequence of ArCT with CT-type peptides from other species reveals conservation of several structural features that are characteristic of this family of neuropeptides, including two N-terminal cysteine residues that form a disulfide bond followed by an amphipathic alpha-helix and C-terminal amidation
Comparative analysis of the sequence of the ArCT precursor (ArCTP) with CT-type precursors in other species revealed that, consistent with the phylogenetic position of A. rubens as an ambulacrarian deuterostomian invertebrate, A. rubens calcitonin-type precursor in (ArCTP) clusters with other deuterostomian CT-type precursors and is positioned in a clade comprising CT-type precursors from other ambulacrarians that is distinct from a clade comprising CT-type precursors from chordates

Summary

Introduction

The thyroid hormone calcitonin was discovered in mammals as a regulator of blood calcium levels (Copp and Cameron, 1961; Copp et al, 1962) and identified as 32-residue C-terminally amidated peptide with an N-terminal disulfide bond (Potts et al, 1968; Niall et al, 1969). Genes encoding calcitonin-like peptides and calcitonin receptor-like proteins have been identified in deuterostomian invertebrates, including the urochordate Ciona intestinalis (Sekiguchi et al, 2009), the cephalochordate Branchiostoma floridae (Sekiguchi et al, 2016), and the echinoderm Strongylocentrotus purpuratus (Rowe and Elphick, 2012), and in protostomian invertebrates, including insects [e.g., Diploptera punctata (Furuya et al, 2000; Zandawala, 2012)] and the mollusk Lottia gigantea (Veenstra, 2010) It appears that a gene duplication in a common ancestor of the protostomes gave rise to two types of calcitonin-related peptides. DH31-type peptides are present in annelids but they appear to have been lost in mollusks and nematodes (Conzelmann et al, 2013; Veenstra, 2014)

Objectives

Methods

Results

Conclusion