Functional characterization of a second pedal peptide/orcokinin-type neuropeptide signaling system in the starfish Asterias rubens

Abstract

Molluscan pedal peptides (PPs) and arthropod orcokinins (OKs) are prototypes of a family of neuropeptides that have been identified in several phyla. Recently, starfish myorelaxant peptide (SMP) was identified as a PP/OK-type neuropeptide in the starfish Patiria pectinifera (phylum Echinodermata). Furthermore, analysis of transcriptome sequence data from the starfish Asterias rubens revealed two PP/OK-type precursors: an SMP-type precursor (A. rubens PP-like neuropeptide precursor 1; ArPPLNP1) and a second precursor (ArPPLNP2). We reported previously a detailed analysis of ArPPLNP1 expression in A. rubens and here we report the first functional characterization ArPPLNP2-derived neuropeptides. Sequencing of a cDNA encoding ArPPLNP2 revealed that it comprises eleven related neuropeptides (ArPPLN2a-k), the structures of several of which were confirmed using mass spectrometry. Analysis of the expression of ArPPLNP2 and neuropeptides derived from this precursor using mRNA in situ hybridization and immunohistochemistry revealed a widespread distribution, including expression in radial nerve cords, circumoral nerve ring, digestive system, tube feet and innervation of interossicular muscles. In vitro pharmacology revealed that the ArPPLNP2-derived neuropeptide ArPPLN2h has no effect on the contractility of tube feet or the body wall-associated apical muscle, contrasting with the relaxing effect of ArPPLN1b (ArSMP) on these preparations. ArPPLN2h does, however, cause dose-dependent relaxation of cardiac stomach preparations, with greater potency/efficacy than ArPPLN1b and with similar potency/efficacy to the SALMFamide neuropeptide S2. In conclusion, there are similarities in the expression patterns of ArPPLNP1 and ArPPLNP2 but our data also indicate specialization in the roles of neuropeptides derived from these two PP/OK-type precursors in starfish.

Keywords: RRID: AB_2716765; deuterostome; echinoderm; neuropeptide; orcokinin; pedal peptide; starfish.

Figure 1

Sequence of ArPPLNP2 and alignments of predicted ArPPLNP2‐derived peptides. (a) Sequence of ArPPLNP2 with the predicted signal peptide marked in blue, predicted cleavage sites marked in green, and peptides ArPPLN2 a to k marked in red. (b) Alignment of ArPPLN2h, which occurs in triplicate in ArPPLNP2, with other peptides derived from ArPPLNP2, which occur singly in ArPPLNP2, as shown in (a). All of the peptides comprise nineteen residues, and residues that are identical to the corresponding residue in ArPPLN2h are shown with a black dot. Eight of the residues in ArPPLN2h are conserved in the ten other peptides (highlighted in yellow). (c) Manual alignment of ArPPLN2h, as a representative ArPPLNP2‐derived peptide, with ArPPLN1b and with PP/OK‐type peptides from other species. Hydrophobic residues (Phe, Leu or Met) are conserved at three positions (highlighted in yellow), one located in the N‐terminal region and two located in the C‐terminal region. Other residues that are conserved between peptides from at least one deuterostome (echinoderm) species and at least one protostome species are highlighted in gray. Citations and/or accession numbers for the peptide sequences included here are as follows: ArPPLN2h (this article; KT601720); ArPPLN1b (Kim et al., 2016; Lin, Egertová et al., 2017; KT870153); PpeSMPa (Kim et al., 2016; KT870152); SpPPLN1d (Rowe & Elphick, 2012; XP_785647); SpPPLN2h (Rowe & Elphick, 2012; XP_003727926); AjPPLN2a (Rowe et al., 2014; Isotig 17873); PP1‐peptide (Lloyd & Connolly, 1989; Moroz et al., 2006; NP_001191585); PP3‐peptide (Moroz et al., 2006; NP_001191625); NLP14‐peptide (Nathoo, Moeller, Westlund, & Hart, 2001; NP_001257067); NLP15 peptide (Nathoo et al., 2001; T20275); OK (Stangier et al., 1992; P37086)

Figure 2

Mass spectrometric identification of ArPPLN2 peptides in an extract of A. rubens radial nerve cords. (a–f) Annotated MS/MS spectra for ArPPLN2a, e, f, h, i, j, respectively. The b series of peptide fragment ions are shown in red, the y series in blue and additional identified peptide fragment ions in green. The amino acid sequence identified in the mass spectrum is highlighted at the top of the figures. MS/MS spectra are shown for (a) ArPPLN2a (Mascot score 31.30), (b) ArPPLN2e (Mascot score 38.01), (c) ArPPLN2f, (Mascot score 64.86), (d) ArPPLN2h, (Mascot score 67.8), (e) ArPPLN2i, (Mascot score 65.62) and (f) ArPPLN2j, (Mascot score 67.47)

Figure 3

Localization of ArPPLNP2 mRNA in the radial nerve cord and circumoral nerve ring of A. rubens using in situ hybridization. (a) Longitudinal parasagittal section of the radial nerve cords incubated with anti‐sense probes showing groups of stained cells interspersed along the length of the nerve cord in the hyponeural (rectangle and arrows) and ectoneural regions (rectangle and arrowheads). Higher magnification images of the boxed regions are shown in (b) and (c). The inset of panel (a) shows the absence of staining in a longitudinal parasagittal section of a radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with anti‐sense probes. (d) Transverse section of radial nerve cord showing groups of stained cells in the hyponeural (rectangle and arrow) and ectoneural regions (arrowheads); a higher magnification image of the boxed region is shown in (e). (f) Transverse section of circumoral nerve ring showing stained cells in the epithelium of the ectoneural region (arrowheads). A higher magnification image of the boxed region is shown in (g). Note that in (f) no hyponeural staining is evident because the hyponeural tissue is damaged (arrow). A high magnification image of stained cells in a detached hyponeural region of the circumoral nerve ring is shown in the inset of panel (g). CONR, circumoral nerve ring; Ec, ectoneural region of radial nerve cord; Hy, hyponeural region of radial nerve cord; RHS, radial hemal strand; RNC, radial nerve cord; TF, tube foot. Scale bars: 50 μm in (a), (a) inset, (d), (f); 10 μm in (b), (c), (e), (g), (g) inset

Figure 4

Localization of ArPPLNP2 mRNA in tube feet and the arm tip region of A. rubens using in situ hybridization. (a) Longitudinal section of a tube foot showing stained cells at the junction with an adjacent tube foot (arrow) and in the sub‐epithelial layer near to the junction with the adjacent radial nerve cord (rectangle), which is shown at higher magnification in (b). (c) Stained cells located near to the tube foot basal nerve ring. The inset shows the location of the stained cells (rectangle) in a lower magnification image of the tube foot. (d) Transverse cryostat section of an arm tip showing the pigmented optic cushion and terminal tentacle. Stained cells can be seen in the terminal tentacle external epithelium (rectangle with arrowhead) and in the body wall epithelium (rectangle with arrow). The boxed areas in (d) are shown at higher magnification in (e) and (f). (g) High magnification image of the photoreceptor cell layer of an optic cushion showing a stained cell (white arrowhead) between pigmented cells. BNR, basal nerve ring; CT, collagenous tissue; CuL, cuticle layer; Ep, epithelium; OC, optic cushion; RNC, radial nerve cord; Su, sucker; TF, tube foot; TT, terminal tentacle. Scale bars: 100 μm in (a), (c) inset; 10 μm in (b), (c), (e), (f), (g); 50 μm in (d)

Figure 5

Localization of ArPPLNP2 mRNA in the digestive system of A. rubens using in situ hybridization. (a) Transverse section of an arm showing stained cells (rectangle and arrowheads) in the coelomic epithelium of a cardiac stomach extrinsic retractor strand nodule and in mesenteries that attach the extrinsic retractor strands to the ambulacral ossicles. The boxed region is shown at higher magnification in (b), where stained cells can be seen in the coelomic epithelium of a mesentery (arrowheads) and in the coelomic epithelium of a nodule. (c) Section of a cardiac stomach showing stained cells (arrowheads) in the coelomic epithelium. (d) Section of pyloric stomach showing stained cells in the coelomic epithelium. (e, f) Transverse section of a pyloric duct showing stained cells (rectangle and arrowheads) in the coelomic epithelium. The boxed region in (e) is shown at higher magnification in panel (f). (g, h) Transverse section of a pyloric caecum diverticulum showing stained cells (rectangle and arrowheads) in the coelomic epithelium layer. The boxed region in (g) is shown at higher magnification in panel (h). AMO, ambulacral ossicle; CE, coelomic epithelium; CT, collagenous tissue; Lu, lumen; Me, mesentery; Mu, mucosa; No, nodule. Scale bars: 100 μm in (a), (g); 20 μm in (b), (c), (d); 50 μm in (e); 10 μm in (f), (h)

Figure 6

Localization of ArPPLN2h‐immunoreactivity (ArPPLN2h‐ir) in the radial nerve cord, the circumoral nerve ring and marginal nerve of A. rubens. (a) Transverse section of a radial nerve cord showing ArPPLN2h‐ir in both the ectoneural and hyponeural regions. The inset of (a) shows absence of immunostaining in a radial nerve cord section incubated with ArPPLN1b antiserum pre‐absorbed with the antigen peptide (ArPPLN2h‐ag), demonstrating the specificity of immunostaining observed with the ArPPLN2 antiserum. (b) Immunostaining in a transverse section of the circumoral nerve ring; here immunostained processes can also be seen here in the peristomial membrane, an adjacent oral tube foot and in a Tiedemann's body. A high magnification image of the boxed region can be seen in (d). (c) High magnification image of the ectoneural region of the radial nerve cord showing immunostained bipolar cells in the sub‐cuticular epithelium (arrowheads) and densely packed immunostained processes (asterisks) in the underlying neuropile region. (d). Immunostained monopolar shaped cells (black arrowheads) in the hyponeural region of the circumoral nerve ring, with stained processes (white arrowhead) projecting into a fiber layer that is adjacent to the unstained collagenous tissue layer (arrow). The intensely stained ectoneural neuropile is labeled here with an asterisk. (e) The continuity of immunostaining in the ectoneural region of the radial nerve and in the basiepithelial nerve plexus of an adjacent tube foot (asterisks) can be seen here. The stained process(es) (arrowhead) of a hyponeural neuron(s) can be seen projecting over the roof of the peri‐hemal canal in close association with the transverse infra‐ambulacral muscle (arrow). (f) Immunostaining in the marginal nerve (arrow) and in axonal processes of the lateral motor nerve (arrowheads). CONR, circumoral nerve ring; CuL, cuticle layer; Ec, ectoneural region; Hy, hyponeural region; PM, peristomial membrane; RHS, radial hemal strand; TB, Tiedemann's body; TF, tube foot. Scale bars: 50 μm in (a), (f); 200 μm in (a) inset; 10 μm in (b), (d); 100 μm in (c); 20 μm in (e)

Figure 7

Localization of ArPPLN2h immunoreactivity in tube feet and ampullae of A. rubens. (a) Longitudinal section of a tube foot showing immunostaining in the sub‐epithelial nerve plexus, basal nerve ring and sucker. The boxed regions are shown at higher magnification in panels (b, c). (b) Immunostaining in the sub‐epithelial nerve plexus and in processes projecting into epithelial folds of the contracted tube foot (arrowheads). (c) Immunostaining in the basal nerve ring and in processes projecting into the tube foot sucker (arrowheads). (d) Transverse section of a tube foot showing immunostaining in the sub‐epithelial nerve plexus and basal nerve ring. (e, f) Immunostaining in the sub‐epithelial nerve plexus of an ampulla shown at low (e; arrowheads) and high (f; arrowhead) magnification. Am, ampulla; BNR, basal nerve ring; CT, collagenous tissue; Ep, epithelium; ML, muscle layer; SNP, sub‐epithelial nerve plexus; Su, sucker. Scale bars: 100 μm in (a), (e); 20 μm in (b), (c), (d); 10 μm in (f)

Figure 8

Localization of ArPPLN2h immunoreactivity in the terminal tentacle and associated structures in the arm tip of A. rubens. (a) Immunostaining in a transverse section at the base of the terminal tentacle. Stained cells and processes can be seen here in the body wall epithelium, the terminal tentacle and associated lateral lappets and the optic cushion. (b) A high magnification image of arm tip epithelium showing immunostained bipolar cells in the epithelium (arrowhead) and in a dense meshwork of fibers in the underlying basiepithelial plexus (asterisks). (c) High magnification image of the terminal tentacle showing immunostained bipolar cells in the epithelium (arrowheads) with stained processes projecting into a dense meshwork of fibers in the underlying basiepithelial plexus (asterisks). CuL, cuticle layer; Ep, epithelium; LL, lateral lappet; TT, terminal tentacle; OC, optic cushion. Scale bars: 100 μm in (a); 10 μm in (b, c)

Figure 9

Localization of ArPPLN2h immunoreactivity in the digestive system of A. rubens. (a) Transverse section of the central disk showing immunostaining in the peristomial membrane, the esophagus and the cardiac stomach. The intensely stained circumoral nerve ring can also be seen here. (b) High magnification transverse section of the esophagus showing immunostained cells and processes in the coelomic lining (arrowheads) and dense immunostaining in the basiepithelial plexus beneath the lumenal epithelial lining of the esophagus. (c) Immunostaining in the cardiac stomach and in the nodule that links the cardiac stomach to extrinsic retractor strands; a high‐magnification image of the boxed area is shown in (d). (d) Immunostained processes can be seen in the basiepithelial plexus beneath the coelomic lining of the nodule (arrowhead) and in the core of the nodule (asterisk). (e) High magnification image of a cardiac stomach showing immunostained cells in the mucosa (arrowhead) and immunostained processes in the basiepithelial nerve plexus beneath the mucosa and in the visceral nerve plexus beneath the coelomic epithelium. (f) High magnification image of the pyloric stomach showing immunostained cells in the mucosa (arrowheads) and immunostained processes in the basiepithelial nerve plexus (black arrow). (g) Immunostaining in a horizontal section of a pyloric duct and pyloric caecum. (h) High magnification image of a pyloric caecum showing immunostaining in mucosal cell bodies (arrowhead), in the basiepithelial nerve plexus (black arrow) and in the visceral nerve plexus (white arrow). BNP, basiepithelial nerve plexus; CE, coelomic epithelium; CONR, circumoral nerve ring; CS, cardiac stomach; CT, collagenous tissue; Es, esophagus; Lu, lumen; Mu, mucosa; No, nodule; PC, pyloric caeca; PD, pyloric duct; PM, peristomial membrane; VNP, visceral nerve plexus. Scale bars: 200 μm in (a); 20 μm in (b), (d), (e), (f), (h); 100 μm in (c), (g)

Figure 10

Localization of ArPPLN2h immunoreactivity in the body wall, body wall appendages and interossicular muscles of A. rubens. (a) High magnification image of a transverse section of an arm showing the coelomic lining of the body wall, with stained processes in the coelomic basiepithelial nerve plexus (white arrowhead) and in the nerve plexus associated with the circular muscle layer (black arrowheads). (b) Transverse section of an arm showing immunostaining in the sub‐epidermal nerve plexus of the body wall and in papulae. The boxed area is shown at higher magnification in (c), which shows immunostaining in the sub‐epidermal nerve plexus of a papula and adjacent body wall. (d) High magnification image showing immunostained processes associated with adductor muscles (arrowheads) in a pedicellaria. (e, f) Immunostaining in the body wall at the junction between two arms in a horizontal section of a juvenile starfish. Immunostained fibers can be seen associated with muscles that link adambulacral ossicles (arrowheads). Stained fibers are also evident in thickenings of the sub‐epithelial nerve plexus of the body wall (arrows) and in the tube feet. A high magnification image of the boxed region is shown in (f), which shows immunostained processes (arrowheads) in muscles that link adambulacral ossicles. BW, body wall; CBNP, coelomic basiepithelial nerve plexus; CMLNP, circular muscle layer nerve plexus; CT, collagenous tissue; Os, ossicle; Pa, papulae; SNP, sub‐epidermal nerve plexus; TF, tube foot. Scale bars: 20 μm in (a), (c), (d), (f); 100 μm in (b), (e)

Figure 11

ArPPLN2h causes dose‐dependent relaxation of in vitro preparations of the cardiac stomach from A. rubens. (a) Representative recording showing the dose‐dependent relaxing effect of ArPPLN2h (10⁻¹⁰ to 10⁻⁶ M) on a cardiac stomach preparation pre‐contracted with artificial seawater containing 30 mM added KCl (leftward arrow). Following tests with ArPPLN2h, the preparation was washed with artificial seawater (downward arrow). (b) Graph showing concentration‐dependent relaxing effect of ArPPLN2h (green) on cardiac stomach preparations. Each point represents the mean ± SEM from at least four separate experiments, with the effect calculated as the percentage reversal of contraction induced by KCl. The effectiveness of ArPPLN2h as a cardiac stomach relaxant is much greater than that of the ArPPLN1b (red) but similar to that of the SALMFamide neuropeptide S2 (blue)