Pedal peptide/orcokinin-type neuropeptide signaling in a deuterostome: The anatomy and pharmacology of starfish myorelaxant peptide in Asterias rubens

Abstract

Pedal peptide (PP) and orcokinin (OK) are related neuropeptides that were discovered in protostomian invertebrates (mollusks, arthropods). However, analysis of genome/transcriptome sequence data has revealed that PP/OK-type neuropeptides also occur in a deuterostomian phylum-the echinoderms. Furthermore, a PP/OK-type neuropeptide (starfish myorelaxant peptide, SMP) was recently identified as a muscle relaxant in the starfish Patiria pectinifera. Here mass spectrometry was used to identify five neuropeptides (ArPPLN1a-e) derived from the SMP precursor (PP-like neuropeptide precursor 1; ArPPLNP1) in the starfish Asterias rubens. Analysis of the expression of ArPPLNP1 and neuropeptides derived from this precursor in A. rubens using mRNA in situ hybridization and immunohistochemistry revealed a widespread pattern of expression, with labeled cells and/or processes present in the radial nerve cords, circumoral nerve ring, digestive system (e.g., cardiac stomach) and body wall-associated muscles (e.g., apical muscle) and appendages (e.g., tube feet and papulae). Furthermore, our data provide the first evidence that neuropeptides are present in the lateral motor nerves and in nerve processes innervating interossicular muscles. In vitro pharmacological tests with SMP (ArPPLN1b) revealed that it causes dose-dependent relaxation of apical muscle, tube foot and cardiac stomach preparations from A. rubens. Collectively, these anatomical and pharmacological data indicate that neuropeptides derived from ArPPLNP1 act as inhibitory neuromuscular transmitters in starfish, which contrasts with the myoexcitatory actions of PP/OK-type neuropeptides in protostomian invertebrates. Thus, the divergence of deuterostomes and protostomes may have been accompanied by an inhibitory-excitatory transition in the roles of PP/OK-type neuropeptides as regulators of muscle activity.

Keywords: deuterostome; echinoderm; neuropeptide; orcokinin; pedal peptide; starfish myorelaxant peptide.

Figure 1

Mass spectrometric identification of peptides derived from ArPPLNP1 in an extract of A. rubens radial nerve cords. (a) Amino acid sequence of ArPPLNP1 with the predicted signal peptide shown in blue, predicted cleavage sites shown in green and peptides derived from the precursor shown in orange (ArPPLN1a), red (ArPPLN1b), brown (ArPPLN1c), pink (ArPPLN1d) and purple (ArPPLN1e). (b–f) annotated MS/MS spectra for ArPPLN1a‐e, 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 each panel and M + 16 represents oxidized methionine. (b) MS/MS spectrum for the ArPPLN1a (GFGMGAYDPLSAGFTD) observed at 811.35 m/z, 2+ ion, with precursor mass error −0.40 ppm (Mascot score 84). (c) MS/MS spectrum for ArPPLN1b (FGGKGAFDPLSAGFTD) observed at 793.88 m/z, 2+ ion, with precursor mass error 0.99 ppm (Mascot score 61). (d) MS/MS spectrum for ArPPLN1c (FGGSRGAFDPLSAGFTD) observed at 851.40 m/z, 2+ ion, with precursor mass error −0.019 ppm (Mascot score 60). (e) MS/MS spectrum for ArPPLN1d (SFVHGDFDPLSTGFVDGD) observed at 956.42 m/z, 2+ ion, with precursor mass error −0.23 ppm (Mascot score 69). (f) MS/MS spectrum for ArPPLN1e (AGFMNGVFHPLVA) observed at 680.35 m/z, 2+ ion, with precursor mass error 1.0 ppm (Mascot score 42)

Figure 2

Trichrome stained sections of A. rubens. (a) This section runs transversely across the middle of the central disk region and parasagittally through two arms. (b) Transverse section of an arm. (c–e) Horizontal sections through the central disk and arms of a juvenile specimen, with (c) at the level of the esophagus, (d) at the level of the circumoral nerve ring and (e) at the level of the highly folded pouches of the cardiac stomach. Abbreviations: ADO, adambulacral ossicle; AM, apical muscle; Am, ampulla; AMO, ambulacral ossicle; CE, coelomic epithelium; CO, carinal ossicle; CONR, circumoral nerve ring; CS, cardiac stomach; Es, esophagus; MO, marginal ossicles; Pa, papulae; PC, pyloric caeca; PD, pyloric duct; Pe, pedicellariae; PM, peristomial membrane; PS, pyloric stomach; RC, rectal caeca; Re, rectum; RNC, radial nerve cord; RO, reticular ossicles; Sp, spine; TF, tube feet. Scale bars: 500 μm in (a), (b); 200 μm in (c), (d), (e)

Figure 3

Localization of ArPPLNP1 mRNA in the radial nerve cord and circumoral nerve ring of A. rubens using in situ hybridization. (a) Longitudinal parasagittal section of a radial nerve cord incubated with antisense probes showing stained cells in the ectoneural (arrowheads) and hyponeural (arrows) regions. Higher magnification images of the boxed regions are shown in (b) and (c). The inset of panel (a) shows absence of staining in a longitudinal parasagittal section of radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. (b) High magnification image showing stained cells in the hyponeural region of the radial nerve cord. (c) High magnification image showing stained cells in the ectoneural region of the radial nerve cord. (d) Transverse section of a radial nerve cord showing stained cells in the ectoneural (arrowheads) and hyponeural (arrows) regions. Higher magnification images of the boxed regions are shown in (e) and (f). (e) Stained cells at the junction between the ectoneural region of the radial nerve cord and an adjacent tube foot. (f) Stained cells in the ectoneural (arrowheads) and hyponeural (arrows) regions of the nerve cord. (g) Transverse section of the circumoral nerve ring showing stained cells in the ectoneural (arrowheads) and hyponeural (arrow) regions. Stained cells can also be seen in the coelomic epithelial lining of the peristomial membrane (white arrowheads). (h) High magnification image of the circumoral nerve ring showing stained cells in the ectoneural and hyponeural regions. Abbreviations: CONR, circumoral nerve ring; CuL, cuticular layer; Ec, ectoneural region; Hy, hyponeural region; PM, peristomial membrane; RHS, radial hemal strand; RNC, radial nerve cord; TF, tube foot. Scale bars: 50 μm in (a), (a) inset, (d), (g); 10 μm in (b), (c), (e), (f), (h)

Figure 4

Localization of ArPPLNP1 mRNA in tube feet of A. rubens using in situ hybridization. (a, b) Longitudinal section of a tube foot showing stained cells (arrowheads) in the sub‐epithelial layer of the tube foot stem. The boxed region in panel (a) is shown at higher magnification in (b). (c) Stained cells located in the sub‐epithelial layer at the junction between adjacent tube feet. (d) Stained cells (arrowheads) in the sub‐epithelial layer just above the tube foot sucker. The boxed region is shown at higher magnification in (e). (e) Stained cells located near to the tube foot basal nerve ring. Abbreviations: BNR, basal nerve ring; CT, collagenous tissue; Ep, epithelium; ML, muscle layer; Su, sucker; TF: tube foot. Scale bars: 100 μm in (a); 20 μm in (b), (c); 50 μm in (d); 10 μm in (e)

Figure 5

Localization of ArPPLNP1 mRNA in the arm tip region of A. rubens using in situ hybridization. (a, b) Transverse section of a paraffin wax embedded arm tip showing the terminal tentacle cut obliquely. Stained cells can be seen in the external epithelial layer of the terminal tentacle (arrowheads) and in the body wall epithelium (arrows) that surrounds the terminal tentacle. The boxed region in (a) is shown at higher magnification in panel (b). (c) 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 (black arrowheads) and in the lateral lappets (white arrowheads). (d) A high magnification image of an arm tip showing stained cells in the terminal tentacle and lateral lappet. Abbreviations: Ep, epithelium; LL, lateral lappet; OC, optic cushion; TT, terminal tentacle. Scale bars: 50 μm in (a), (c); 10 μm in (b); 20 μm in (d)

Figure 6

Localization of ArPPLNP1 mRNA in the digestive system of A. rubens using in situ hybridization. (a) Transverse section of the peristomial membrane showing stained cells (arrowheads) in the coelomic epithelium. (b) Longitudinal section of the esophagus showing stained cells in the coelomic epithelium. (c, d) Transverse section of the central disk showing stained cells in the cardiac stomach. The boxed region in (c) is shown at higher magnification in panel (d), where stained cells can be seen in mucosal layer. (e, f) Transverse section of the central disk showing stained cells in the cardiac stomach and pyloric stomach. The boxed region of the pyloric stomach in (e) is shown at higher magnification in panel (f), where stained cells can be seen in the mucosal layer. Abbreviations: CE, coelomic epithelium; CS, cardiac stomach; CT, collagenous tissue; Es, esophagus; Mu, mucosa; PM, peristomial membrane; PS, pyloric stomach; VM, visceral muscle. Scale bars: 20 μm in (a), (b), (d), (f); 100 μm in (c), (e)

Figure 7

Localization of ArPPLNP1 mRNA in the pyloric ducts and pyloric caeca of A. rubens using in situ hybridization. (a, b) Transverse section of a pyloric duct showing stained cells (arrowheads) that are concentrated in the oral (lower) domain of the duct. The boxed region in (a) is shown at higher magnification in panel (b), where stained cells can be seen in the mucosal layer. (c, d) Transverse section of a pyloric caecum showing stained cells (arrowheads) that are concentrated in the oral (lower) domain of a pyloric caecum diverticulum. The boxed region in (c) is shown at higher magnification in panel (d), where stained cells can be seen in the mucosal layer. Abbreviations: Lu, lumen; PC, pyloric caeca; PD, pyloric duct. Scale bars: 50 μm in (a); 10 μm in (b), (d); 100 μm in (c)

Figure 8

Localization of ArPPLNP1 mRNA in the apical muscle and coelomic lining of the body wall in A. rubens using in situ hybridization. (a, b) Transverse section of apical muscle showing stained cells (arrowheads) in the coelomic epithelium. The boxed region in (a) is shown at higher magnification in panel (b). (c, d) Longitudinal section of apical muscle showing stained cells (arrowheads) in the coelomic epithelium. The boxed region in (c) is shown at higher magnification in panel (d). (e, f) Transverse section of an arm showing stained cells (arrowheads) in the coelomic epithelium at the base of a papula. The boxed region in (e) is shown at higher magnification in panel (f). Abbreviations: AM, apical muscle; CE, coelomic epithelium; Pa, papulae. Scale bars: 50 μm in (a), (c), (e); 10 μm in (b), (d), (f)

Figure 9

Characterization of rabbit antiserum to ArPPLN1b using an enzyme‐linked immunosorbent assay (ELISA). (a) Incubation of antiserum (red) and pre‐immune serum (blue) at dilutions between 10⁻³ and 10⁻⁸ with 0.1 nmol of antigen peptide (ArPPLN1‐ag) per well reveals no immunoreaction with pre‐immune serum, whereas with the antiserum the antigen is detected at well above the background optical density (OD) with dilutions from 10⁻³ to 10⁻⁵. (b) Incubation of antiserum (red) and pre‐immune serum (blue) at 10⁻⁴ dilution with between 10⁻¹⁵ and 10⁻¹⁰ moles of antigen peptide (ArPPLN1‐ag) per well reveals no immunoreaction with pre‐immune serum, whereas with the antiserum the antigen is detected at well above the background OD with 10⁻¹² to 10⁻¹⁰ moles per well. All data points are mean values from two separate experiments performed in duplicate

Figure 10

Localization of ArPPLN1b‐immunoreactivity (ArPPLN1b‐ir) in the radial nerve cord of A. rubens. (a) Tranvserse section of a radial nerve cord showing ArPPLN1b‐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 (ArPPLN1b‐ag), demonstrating the specificity of immunostaining observed with the ArPPLN1b antiserum. (b) High magnification image of the ectoneural region of the radial nerve cord showing immunostained bipolar shaped cells in the sub‐cuticular epithelium (arrowhead) and densely packed immunostained processes in the underlying neuropile region. (c) High magnification image of the ectoneural region at the tip of the V‐shaped radial nerve cord showing layer‐specific variation in the density of immunostained processes. (d) High magnification image showing immunostained monopolar shaped cells (arrowheads) and their stained processes (arrow) in the hyponeural region of the radial nerve cord. The unstained collagenous tissue layer (white arrowhead) that separates the hyponeural region from the densely stained ectoneural neuropile can also be seen here. (e, f) High magnification images showing immunostaining at the junction between the radial nerve cord and an adjacent tube foot. The continuity of immunostaining in the ectoneural region of the radial nerve and in the basiepithelial nerve plexus of the tube foot (asterisks) can be seen here. In (e) the stained process(es) (arrowhead) of a hyponeural neuron(s) can be seen projecting over the roof of the perihemal canal in close association with the transverse infra‐ambulacral muscle. In (f) the stained processes (arrowhead) of hyponeural neurons can be seen projecting to the base of the adjacent tube foot. Abbreviations: CuL, cuticle layer; Ec, ectoneural region; Hy, hyponeural region; RHS, radial hemal strand; TF, tube foot. Scale bars: 50 μm in (a); 200 μm in (a) inset; 10 μm in (b), (d), (e); 20 μm in (c), (f)

Figure 11

Localization of ArPPLN1b‐immunoreactivity in the circumoral nerve ring and marginal nerve of A. rubens. (a) ArPPLN1b‐ir in a transverse section of the circumoral nerve ring; here immunostained processes can also be seen in the peristomial membrane, in an adjacent oral tube foot and in a Tiedemann's body. (b) A high magnification image of the circumoral nerve ring showing immunostained bipolar shaped cells in the sub‐cuticular epithelium (arrowheads) of the ectoneural region and densely packed immunostained processes in the underlying neuropile. In the hyponeural region immunostained monopolar shaped cells (white arrowhead) can be seen with their stained processes (arrow) adjacent to the unstained collagenous tissue layer (white arrow). (c) High magnification image of the ectoneural region of the circumoral nerve ring showing layer‐specific variation in the density of immunostained processes. (d) Immunostaining in a sub‐epithelial thickening of the basiepithelial plexus known as the marginal nerve (arrow), which is located at the junction between the outer row of tube feet and the adjacent body wall. Internal to the marginal nerve, separated by a thin layer of collagenous tissue, can be seen stained axonal processes that coalesce to form the lateral motor nerve (arrowhead). Individual stained axonal processes derived from the lateral motor nerve can be seen here innervating an adjacent interossicular muscle (white arrowhead). Abbreviations: CONR, circumoral nerve ring; Ec, ectoneural region; Hy, hyponeural region; PM, peristomial membrane; TB, Tiedemann's body; TF, tube foot. Scale bars: 100 μm in (a); 20 μm in (b), (c); 50 μm in (d)

Figure 12

Localization ArPPLN1b immunoreactivity in the hemal system and Tiedemann's bodies of A. rubens. (a) Immunostained processes (arrowheads) are present in the radial hemal strand, which is connected to the intensely stained radial nerve cord. (b) Immunostained processes (white arrowheads) are present in the oral hemal ring, which is connected to the circumoral nerve ring (out of focus at bottom left). Immunostained processes can also be seen here in the coelomic epithelium (arrows). (c, d) Immunostained processes in one of the ten Tiedemann's bodies, which are located aboral to the circumoral nerve ring in the central disk. Immunostained processes can also be seen on the right side of the image, where they are associated with interossicular muscles of the body wall (see Figure 18). The boxed region in (c) is shown at higher magnification in panel (d), where immunostained processes can be seen in the Tiedemann's body (black arrowheads), the coelomic epithelium (arrow) and the oral hemal ring (white arrowheads). Abbreviations: CE, coelomic epithelium; CONR, circumoral nerve ring; Ec, ectoneural region; Hy, hyponeural region; OHR, oral hemal ring; Os, ossicle; RHS, radial hemal strand; TB, Tiedemann's body. Scale bars: 20 μm in (a), (b), (d); 100 μm in (c)

Figure 13

Localization of ArPPLN1b 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) and (c). (b) High magnification image showing immunostaining in the sub‐epithelial nerve plexus and in processes projecting into epithelial folds of a contracted tube foot (arrowheads). (c) High magnification image showing immunostaining in the basal nerve ring and in processes projecting into the tube foot sucker (arrowheads). (d) Immunostaining in a 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 (arrowheads). The boxed region in panel (e) is shown at higher magnification in panel (f). Abbreviations: 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 14

Localization of ArPPLN1b immunoreactivity in the terminal tentacle and associated structures in the arm tip of A. rubens. (a) Immunostaining in a transverse section of a terminal tentacle and in the surrounding body wall epithelium. The boxed regions are shown at higher magnification in panel (b) and (c). (b) A high magnification image of arm tip body wall epithelium showing immunostained bipolar shaped cells (arrowhead) and in a dense meshwork of fibers in the underlying basiepithelial nerve plexus (asterisks). (c) High magnification image of the terminal tentacle showing immunostained bipolar shaped cells in the epithelium (arrowheads) and in a dense meshwork of fibers in the underlying basiepithelial nerve plexus (asterisks). (d) Immunostaining in a transverse section at the base of the terminal tentacle. Immunostained cells and processes can be seen here in the body wall epithelium, the terminal tentacle and associated lateral lappets and the optic cushion. Abbreviations: CuL, cuticle layer; Ep, epithelium; LL, lateral lappet; OC, optic cushion; TT, terminal tentacle. Scale bars: 50 μm in (a); 10 μm in (b), (c); 100 μm in (d)

Figure 15

Localization of ArPPLN1b immunoreactivity in the peristomial membrane and esophagus of A. rubens. (a) An immunostained bipolar shaped cell can be seen here in the external epithelial layer of the peristomial membrane (arrowhead) and in processes located in the underlying basiepithelial nerve plexus. Immunostaining is also present in cells and processes located beneath the coelomic epithelium, which is separated from the basiepithelial nerve plexus by a layer of collagenous tissue. (b) Transverse section of the central disk showing immunostaining in the peristomial membrane, the esophagus and the cardiac stomach. The boxed region is shown at higher magnification in panel (c). (c) Longitudinal section of the esophagus (in a transverse section of the central disk) showing immunostained cells (arrowheads) and processes (asterisks) beneath the coelomic epithelium and stained processes (arrows) in the basiepithelial nerve plexus beneath the epithelial layer that forms the external lining of the esophagus. (d) Horizontal section of the central disk at the level of the junction between the esophagus and the peristomial membrane showing immunostained cells (arrowheads) and processes (arrows). (e) High magnification transverse section of the esophagus (in a horizontal section of the central disk) showing immunostained cells and processes beneath the folded coelomic epithelium and dense immunostaining in the basiepithelial nerve plexus beneath the epithelial lining of the eosophagus lumen. Abbreviations: BNP, basiepithelial nerve plexus; CE, coelomic epithelium; CS, cardiac stomach; CT, collagenous tissue; Es, esophagus; Mo, mouth; PM, peristomial membrane. Scale bars: 20 μm in (a), (c), (e); 100 μm in (b); 50 μm in (d)

Figure 16

Localization of ArPPLN1b immunoreactivity in the cardiac stomach, pyloric stomach, pyloric duct and pyloric caeca of A. rubens. (a) Transverse section of the central disk region showing immunostaining in the cardiac stomach and pyloric stomach. (b) High magnification image of the cardiac stomach showing immunostaining in bipolar‐shaped cells in the mucosa and in the underlying basiepithelial nerve plexus. (c) High magnification image of the pyloric stomach showing immunostaining in bipolar‐shaped cells in the mucosa and in the underlying basiepithelial nerve plexus. (d) Horizontal section of the arm of a juvenile starfish showing immunostaining in the pyloric duct and pyloric caeca. (e) High magnification image of a transverse section of pyloric duct showing immunostaining in bipolar shaped mucosal cells and in the underlying basiepithelial nerve plexus. (f) High magnification image of a transverse section through a lobe of a pyloric caecum showing immunostaining in bipolar shaped mucosal cells and in the underlying basiepithelial nerve plexus (arrowheads). Abbreviations: BNP, basiepithelial nerve plexus; CE, coelomic epithelium; CS, cardiac stomach; CT, collagenous tissue; Lu, lumen; Mu, mucosa; PC, pyloric caeca; PD, pyloric duct; PS, pyloric stomach. Scale bars: 200 μm in (a); 20 μm in (b), (c), (d); 100 μm in (e); 50 μm in (f)

Figure 17

Localization of ArPPLN1b immunoreactivity in the body wall, apical muscle and body wall appendages of A. rubens. (a) Transverse section of aboral body wall showing immunostaining in the coelomic lining of the body wall (arrowheads), the coelomic lining of papulae and in the sub‐epithelial plexus (arrow) of the external body wall epithelium. (b) High magnification image of the coelomic lining of the body wall showing stained cells in the coelomic epithelium (arrowheads) and in the underlying basiepithelial nerve plexus. Immunostaining is also present in a nerve plexus (arrow) that is closely associated with a layer of circularly orientated muscle. (c) Immunostained axon profiles (arrowheads) in a transverse section of the apical muscle. Immunostained processes (arrow) can also be seen associated with a layer of circularly orientated muscle. (d) High magnification view of a longitudinal section of a papula showing immunostaining in the coelomic lining (arrowheads). (e) High magnification of the body wall external epithelium showing immunostaining in the sub‐epithelial nerve plexus. Abbreviations: CBNP, coelomic basiepithelial nerve plexus; CMLNP, circular muscle layer nerve plexus; CT, collagenous tissue; Os, ossicle; Pa, papulae; SNP, sub‐epithelial nerve plexus. Scale bars: 100 μm in (a); 20 μm in (b), (d), (e); 50 μm in (c)

Figure 18

Localization of ArPPLN1b immunoreactivity in the innervation of interossicular muscles in A. rubens. (a) Transverse section of the ambulacrum showing immunostaining associated with muscles linking the ambulacral ossicles, which include the TSM, TIM, and LSM. The intensely stained radial nerve cord (arrowhead) can also be seen in this image. The boxed regions are shown at higher magnification in panels (b) and (c). (b) Immunostained nerve fibers (arrowheads) in a longitudinal section through the TSM. (c) Immunostained profiles of nerve fibers in a transverse section through the LSM. (d, e) Immunostaining in the body wall at the junction between two arms in 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 (e). (f) Trichrome stained section of the body wall showing an interossicular muscle (white asterisks) and collagenous tissue (area bounded by black dots) linking adjacent ossicles. (g) Immunostained section adjacent to the section shown in panel (f). By comparing the immunostaining with the trichrome staining it can be seen that the immunostained fibers are associated with the interossicular muscle but not with the collagenous tissue (area bounded by black dots). Abbreviations: LSM, longitudinal supra‐ambulacral muscle; TF, tube foot; TIM, transverse infra‐ambulacral muscle; TSM, transverse supra‐ambulacral muscle. Scale bars: 200 μm in (a); 20 μm in (b), (c), (e), (f), (g); 100 μm in (d)

Figure 19

Graphs comparing the concentration‐dependent relaxing effects of ArPPLN1b and the SALMFamide neuropeptide S2 on in vitro apical muscle, tube foot and cardiac stomach preparations from A. rubens. Each point represents the mean ± SEM from at least four separate experiments, with the effect calculated as the percentage reversal of contraction induced by application of 10 µM acetylcholine (apical muscle and tube foot) or 30 mM KCl (stomach). (a) Apical muscle, where ArPPLN1b is more effective as a relaxant than S2 when tested at 10⁻⁶ M (p = .004; t‐test). (b) Tube foot, where ArPPLN1b is more effective as a relaxant than S2 when tested at 10⁻⁶ M (p = .007; t‐test) and 10⁻⁵ M (p = .001; t‐test). (c) Cardiac stomach, where ArPPLN1b is less effective as a relaxant than S2 when tested at 10⁻⁷ M (p = .0002; t‐test) and 10⁻⁶ M (p = .00002; t‐test)