JOURNAL OF EXPERIMENTAL ZOOLOGY 284:535–540 (1999)

© 1999 WILEY-LISS, INC.

Molecular Diversity, Localization, and BiologicalActions of Elasmobranch Tachykinins

J. MICHAEL CONLON*Regulatory Peptide Center, Department of Biomedical Sciences, CreightonUniversity Medical School, Omaha, Nebraska 68178-0405

The tachykinins are members of a family of re-lated peptides that are defined structurally by acommon amino acid sequence at the COOH-ter-mini of the molecules (Erspamer, ’81). This motifmay be represented as -Phe-Xaa-Gly-Leu-Met.NH2where Xaa is an aromatic or branched chain ali-phatic residue (Phe, Tyr, Val, Ile). The tachykininsare characterized functionally by a shared rangeof biological activities, such as an ability to stimu-late vascular, urogenital, airway, and gastrointes-tinal smooth muscle, and to excite spinal sensoryand motor neurons (Otsuka and Yoshioka, ’93).Tachykinins have been isolated from nervous and/or gastrointestinal tissues of all classes of verte-brates studied (from agnathans to mammals) aswell as in selected invertebrate species [reviewedin (Maggio and Mantyh, ’94; Waugh et al.,’95a;Conlon et al., ’97)]. Historically, in the light of thepioneering studies by the group of Erspamer,tachykinins terminating in the sequence with Xaa= Tyr are regarded as members of the physalaeminsub-family named after the peptide first isolatedfrom the skin of the leptodactylid frog, Phy-salaemus fuscomaculatus (Erspamer et al., ’64). Xaa= Val are members of the kassinin sub-family af-ter the peptide isolated from the skin of therhacophorid frog, Kassina senegalensis (Anastasiet al., ’77) and Xaa = Ile are members of the eledoi-sin sub-family after the peptide isolated from thesalivary glands of the Mediterranean octopus,Eledone moschata (Erspamer and Anastasi, ’62).

BIOSYNTHESIS OF TACHYKININSIN VERTEBRATES

The biosynthesis of tachykinins in mammals isrelatively well understood. Sequence analysis ofcloned cDNAs from several mammalian species hasidentified mRNAs directing the synthesis of fivebiosynthetic precursors of the tachykinins (α-, β-γ-, and δ-preprotachykinin A and preprotachykininB) [reviewed in (Nakanishi, ’86)]. α- and δ-Prepro-tachykinin A contain the sequence of substance Ponly (Nawa et al., ’84; Harmer et al., ’90). β-Pre-

protachykinin A contains substance P, neurokininA, and its 36 amino-acid-residue NH2-terminally ex-tended form, neuropeptide K (Nawa et al., ’84;Carter and Krause, ’90). γ-Preprotachykinin A con-tains the sequence of substance P, neurokinin A, andits 21 amino-acid-residue NH2-terminally extendedform, neuropeptide γ (Kawaguchi et al., ’86; Krauseet al., ’87). Preprotachykinin B is encoded by a sepa-rate gene and contains the sequence of neurokininB only (Kotani et al., ’86; Bonner et al., ’87).

At this time, the nucleotide sequence of a geneand/or cDNA encoding a preprotachykinin froman elasmobranch has not been described, but re-cently a teleost homologue of γ-preprotachykininA mRNA was characterized in the case of thegoldfish, Carassius auratus (Lin and Peter, ’97).The organization of the precursor is similar tothat of mammalian γ-preprotachykinins and con-tains the sequence of a substance P-related pep-tide and the 21 amino-acid tachykinin, carassin,which is analogous to mammalian neuropeptideγ (Conlon et al., ’91).

MOLECULAR DIVERSITY OFELASMOBRANCH TACHYKININS

As summarized in Fig. 1, tachykinins withstructural similarity to mammalian substance P,neurokinin A, and neuropeptide γ have been iso-lated from elasmobranch tissues and character-ized structurally. In addition, elasmobranchtissues have yielded the novel tachykinins, scy-liorhinin I, and scyliorhinin II for which mamma-lian homologues have not been described. Theamino acid sequences of the elasmobranch ta-chykinins are compared with the sequences of thecorresponding peptides from other vertebrate taxain Figs. 2–5. At this time, the biosynthetic rela-tionships between the elasmobranch tachykininsand the mammalian tachykinins and between the

*Correspondence to: J. Michael Conlon, Department of BiomedicalSciences, Creighton University Medical School, Omaha, NE 68178-0405. E-mail: jmconlon@creighton.edu

536 J.M. CONLON

elasmobranch peptides themselves are incom-pletely understood.

Peptides from gastrointestinal tissuesThe first tachykinins to be isolated from fish

tissues were scyliorhinin I and scyliorhinin II.These peptides were purified from an extract ofthe intestine of an elasmobranch fish, Scylior-hinus canicula (European common dogfish), onthe basis of their abilities to contract isolated lon-gitudinal smooth muscle from the guinea pigileum (Conlon et al., ’86). The 10 amino-acid-resi-due peptide scyliorhinin I terminates in the aminoacid sequence Phe-Tyr-Gly-Leu-Met. NH2 and somay be regarded as a member of the physalaeminsub-family of tachykinins (Fig. 2). The 18 amino-acid-residue peptide scyliorhinin II is the onlytachykinin yet described that contains a cystinebridge and shows structural similarity to kassininin its C-terminal region. The peptide des-Ser1,

Pro2-scyliorhinin II was subsequently isolatedfrom the gut of a second elasmobranch fish, theray Torpedo marmorata (Conlon and Thim, ’88)and may represent a metabolite formed by theaction of an enzyme with a specificity similar tothat of mammalian dipeptidylpeptidase IV (Con-lon and Sheehan, ’83).

A tachykinin with an identical amino acid se-quence to dogfish scyliorhinin I was isolated froman extract of the intestine of the hammerheadshark, Sphyrna lewini, using an antiserum di-rected against the COOH of substance P in radio-immunoassay to facilitate detection (Waugh et al.,’95a). Within the elasmobranch class, the ham-merhead shark and the spotted dogfish are placedin the same order (Galeomorpha) that is distinct

Fig. 1. Structurally characterized tachykinins isolatedfrom elasmobranch tissues.

Fig. 2. A comparison of the amino acid sequences of pep-tides related to scyliorhinin I and scyliorhinin II with thefrog skin peptides, physalaemin and kassinin. Dashes denoteresidue identity. <E denotes a pyroglutamyl residue.

Fig. 3. A comparison of the amino acid sequences of pep-tides related to substance P from different vertebrate taxa.Dashes denote residue identity.

Fig. 4. A comparison of the amino acid sequences of pep-tides related to neurokinin A from different vertebrate taxa.Dashes denote residue identity. In order to maximize sequencesimilarity, deletions denoted by the asterisks have been in-troduced into some sequences.

ELASMOBRANCH TACHYKININS 537

from that of the skates and rays (Batoidea).Scyliorhinin II was not detected in the extract ofthe hammerhead shark intestine using an anti-serum to neurokinin A that is able to detect thepeptide in an extract of dogfish gut. However, useof this antiserum led to the isolation from theshark gut of an N-terminal extended form of neu-rokinin A with 24 amino acid residues. This pep-tide shows only limited structural similarity tocarassin and to neuropeptide γ in the NH2-termi-nal region. The functionally important C-termi-nal region of the shark tachykinin containsunusual substitutions not previously seen in otherneurokinin A-related peptides. In particular, thestrongly conserved His1 and Ser5 in mammalianneurokinin A are replaced by Gln and Met, re-spectively (Fig. 5). Synthesis of scyliorhinins maynot be confined to elasmobranch tissues, as adecapeptide of the physalaemin sub-family withlimited structural similarity to scyliorhinin I wasisolated from an extract of the stomach of theprimitive Actinopterygian fish, the bowfin Amiacalva (Halecomorphi) (Waugh et al., ’95b) (Fig. 2).

Peptides from tissues of the CNSTwo peptides with substance P-like immunore-

activity were isolated in pure form from an ex-tract of the brain of the dogfish, S. canicula(Waugh et al., ’93). One peptide was identical toscyliorhinin I, previously identified in dogfish in-testine, and the second was a undecapeptide con-taining three amino acid substitutions compared,with substance P (Arg1 → Lys, Lys3 → Arg andGln5 → Gly) (Fig. 3). Neither Scyliorhinin II nora peptide analogous to mammalian neurokinin Awas detected in the extract. Conversely, [Lys1,Arg3,Gly5]substance P was not detected in an ex-tract of dogfish gut. The isolation from dogfishbrain of [Lys1,Arg3,Gly5] substance P in compa-rable amounts to scyliorhinin I suggests, as one

possibility, that scyliorhinin I is the elasmobranchequivalent of mammalian neurokinin A and is en-coded by the same gene as [Lys1,Arg3,Gly5]-substance P. However, in contradiction to thishypothesis, a scyliorhinin I-related peptide ([His3]-scyliorhinin I) was isolated from an extract of thebrain of the skate Raja rhina together with a pep-tide with much closer structural similarity tomammalian neurokinin A ([Leu3, Gly4]neurokininA) (Fig. 4). However, a tachykinin with structuralsimilarity to substance P was not detected (Waughet al., ’94). Clearly, nucleotide sequence analysisof cDNAs or genomic fragments encoding elasmo-branch preprotachykinins is required to clarifythis issue.

Neurokinin B has been isolated from the brainof the frog Rana ridibunda in a molecular formidentical to that of the mammalian peptide(O’Harte et al., ’91). At this time, however, neu-rokinin B has not been isolated from the tissuesof an elasmobranch or from any other non-tetra-pod species.

LOCALIZATION OF TACHYKININSIN ELASMOBRANCHS

In contrast to the situation in higher mammals,tachykinins are produced in elasmobranch tissuesin a dual location, occurring in both endocrine cellsand in neurons. In the spiny dogfish, Squalusacanthias, numerous endocrine cells with sub-stance P-like immunoreactivity are present in themucosa of both intestine and rectum, with a morescattered distribution in the stomach (El-Salhy,’84; Holmgren, ’85). Immunopositive nerve fibersare present in all layers of the gut wall and run-ning along blood vessels in the submucosa of thestomach. Nerve cell bodies were detected in thesubmucosal and myenteric plexi of the stomachand rectum. Among other species of elasmobranch,nerve fibers containing substance P-like immu-noreactivity were found sparsely distributed in thelamina propria, submucosa, and muscle layer ofthe stomach fundus of the dogfish, Scyliorhinusstellaris, and immunoreactive cells were observedin the mucosal epithelium (Cimini et al.,’85).Strong substance P-like immunoreactivity wasdetected in large fiber bundles and in the peri-karya of the ganglion cells of the sinus venosus ofthe heart of the dogfish, Scyliorhinus canicula(Munoz-Chapuli et al., ’94).

Using an antiserum raised against cod neuro-kinin A (Jensen and Conlon, ’92) that reactsstrongly with scyliorhinin II, but weakly withscyliorhinin I, numerous immunoreactive nerve

Fig. 5. A comparison of the amino acid sequences of pep-tides related to neuropeptide γ from different vertebrate taxa.Dashes denote residue identity and asterisks denote residuedeletions.

538 J.M. CONLON

fibers were visualized around the ventral aortaand following the celiac artery and the lienogastricartery of the spiny dogfish (Kågström et al., ’96).A dense perivascular plexus of varicose fibers wasobserved in the inner layer of the adventitia andthe adventitiomedial border of all the gut vessels.Consistent with the distribution of nerve fibersexpressing substance P-like immunoreactivity, theantiserum to cod neurokinin A visualized numer-ous fibers in the myenteric plexus and circularmuscle layer of the dogfish intestine. An innerva-tion of the intestinal, but not the gastric mucosa,was observed.

BIOLOGICAL ACTIVITIES OFELASMOBRANCH TACHYKININS

Receptor-binding propertiesMammalian tissues contain three well-charac-

terized, high-affinity binding sites for the ta-chykinins termed the NK-1, NK-2, and NK-3receptors, which differ in their ability to bind en-dogenous tachykinin ligands [reviewed in Regoliet al. (’89)]. Substance P is the most receptor-se-lective of the ligands and binds with highest af-finity to the NK-1 receptor, neurokinin A bindspreferentially to the NK-2 receptor, and neuroki-nin B to the NK-3 receptor. All three subtypesbelong to the G-protein-coupled receptor familyand their activation is associated with the involve-ment of the inositol polyphosphate-Ca2+ signal-ing pathway (Putney, ’94).

The binding affinity of some dogfish tachykininstoward mammalian tachykinin receptors havebeen studied using membrane preparations en-riched in NK-1 receptors (rat submandibulargland), NK-2 receptors (rat stomach fundus andhamster urinary bladder), and NK-3 receptors (ratcerebral cortex) (Mussap et al., ’93). Competitivebinding studies have shown that scyliorhinin I isa high affinity agonist for both the mammalianNK-1 and NK-2 tachykinin receptors, whereasscyliorhinin II is a selective agonist for the mam-malian NK3 receptor (Buck and Krstenansky, ’87).125I-labeled scyliorhinin II has found utility as aselective radioligand for investigation of the bind-ing properties of novel tachykinins at the NK-3receptor (Beaujouan et al., ’88; Mussap andBurcher, ’90). Synthetic dogfish substance P([Lys1,Arg3,Gly5]substance P) bound with high af-finity to the rat NK-1 receptor with an approxi-mately 3-fold lower KD value than mammaliansubstance P (Waugh et al., ’93). A study using Chi-nese hamster ovary cells expressing the cloned

rat NK-1, NK-2, and NK-3 receptors showed thatthe second proline residue (corresponding to po-sition 4 in substance P) is important in confer-ring selectivity toward NK1 receptors (Cascieriet al., ’92). It is noteworthy, therefore, that thisresidue has been retained in the dogfish sub-stance P-related peptide and, in general, has beenstrongly conserved during evolution of the verte-brates (Fig. 3).

The distribution and properties of tachykininreceptors in non-mammalian vertebrates have notbeen studied extensively. Two binding sites fortachykinins were identified in membranes pre-pared from S. canicula brain and stomach (VanGiersbergen et al., ’91). One site selectively boundradiolabeled substance P, and the rank order ofpotency of tachykinins for inhibiting binding sug-gested that this site was similar in ligand-bind-ing properties to a mammalian NK-1 receptor. Thesecond site selectively bound radiolabeled scy-liorhinin II, but the inability of neurokinin B todisplace binding clearly demonstrated that thepharmacological properties of this site were dis-tinct from the mammalian NK-3 receptor. Aninvestigation of the effect of GTP analogs on radio-ligand binding indicated that the substance P-binding site, but not the scyliorhinin II-bindingsite, was linked to a G-protein.

Cardiovascular actionsThe vasodilator action of tachykinins in mam-

mals is mediated primarily through interactionwith NK-1 receptors (Regoli et al., ’89). Consis-tent with their receptor-binding properties, bolusintravenous injections of [Lys1,Arg3,Gly5]substanceP (100 pmol) and scyliorhinin I (100 pmol) pro-duced appreciable (>30 mmHg) falls in arterialblood pressure in the rat. However, bolus intraar-terial injections of up to 5 nmol of the peptidesinto the unanesthetized and unrestrained dogfish,S. canicula, produced no change in arterial bloodpressure, pulse amplitude, or heart rate (Waughet al., ’93). Injections of greater amounts of thepeptides (10–50 nmol) produced a slight increase(3–5 mmHg) in blood pressure. The data suggestthat peripheral mammalian-type NK1 tachykininreceptors are not involved in cardiovascular regu-lation in this elasmobranch fish.

In a study carried out in the unanesthetizedspiny dogfish, Squalus acanthias, intraarterial in-jections of synthetic scyliorhinin I (S. caniculasequence; 0.3 nmol/kg body mass) produced a de-crease in vascular resistance that resulted in anincrease in blood flow through the gastrointesti-

ELASMOBRANCH TACHYKININS 539

nal vascular bed and a small decrease in arterialblood pressure (Kågström et al., ’96). ScyliorhininII, however, produced an initial hypertension in-duced by a general vasoconstriction followed by asubsequent vasodilation of the gastrointestinalbeds that again led to increased blood flow to thegut. Neither peptide affected heart rate. Despitethe similarity in trivial name, the spiny dogfish(Squalomorpha) and the spotted dogfish (Galeo-morpha) are not closely related phylogenetically.It is not known whether scyliorhinins are producedby S. acanthias tissues, and so the physiologicalsignificance of the data remains uncertain.

PHYSIOLOGICAL ROLE OFSCYLIORHININ II

The rectal gland is of fundamental importancein the control of salt and water balance in elas-mobranchs. After salt loading associated withfeeding and ingestion of seawater, the gland se-cretes a fluid that is isosmotic to seawater, but isapproximately two-fold enriched in sodium chlo-ride concentration relative to plasma (Burger andHess, ’60). Regulation of the activity of the rectalgland is incompletely understood and appears tobe species-dependent. An involvement of both neu-ral and endocrine factors has been proposed(Shuttleworth, ’83).

In the case of S. acanthias, vasoactive intesti-nal polypeptide is a potent stimulant of secretoryactivity (Stoff et al., ’79) but the rectal gland of S.canicula is not responsive to this peptide (Shuttle-worth, ’83). A putative peptide, termed rectin, thatis released by the intestine in response to feed-ing, was suggested to be the physiologically im-portant stimulatory factor for the rectal gland ofthe spotted dogfish (Shuttleworth and Thonrn-dyke, ’84), but the amino acid sequence of rectinwas not determined.

Using an isolated perfused S. canicula rectalgland preparation as a bioassay to monitor puri-fication, a factor present in an extract of the in-testine of this species was isolated in pure formusing reverse-phase high performance liquid chro-matography (Anderson et al., ’95). Characteriza-tion of this substance demonstrated that it was apeptide whose amino acid sequence was identicalto that of scyliorhinin II. Perfusion of the isolatedrectal gland with synthetic scyliorhinin II in-creased secretion rate in a concentration-depen-dent manner with a maximal response at 10–6 M(Fig. 6). The activity of the gland was not signifi-cantly affected by vasoactive intestinal polypep-tide (VIP). The molecular mass of scyliorhinin II

(1850 Daltons) is consistent with that previouslyreported for the putative stimulatory hormone,rectin—approximately 2000 Daltons (Shuttle-worth and Thorndyke,’84). Although release ofscyliorhinin II into plasma after feeding has notyet been demonstrated in this species, the dataare consistent with the hypothesis that scylior-hinin II may be the hormonal factor responsiblefor regulating the secretory activity of the rectalgland in the spotted dogfish, S. canicula.

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