Camp.
Biochem.
Physiol.Vol. 103C, No. I, pp. 169-173, 1992
0306~4492/92 $5.00+ 0.00 0 1992Pergamon Press Ltd
Printed in Great Britain
IMMUNOCHEMICAL CHARACTERISATION OF TACHYKININ IMMUNOREACTIVITY IN THE NERVOUS SYSTEM OF THE GARDEN SNAIL, HELIX ASPERSA P. S.
LEUNG, C. SHAW, C. F. JOHNSTON
Schools of Clinical Medicine
and G. B.
IRVINE
and Biology and Biochemistry, The Queen’s University of Belfast, Northern Ireland
(Received 30 January 1992; accepted for publication 26 February 1992)
Abstract-l. Circumoesophageal ganglia and foot muscle of the garden snail, Helix aspersa, were subjected to immunocytochemistry using antisera to the tachykinins, substance P (SP), neurokinin A (NKA), kassinin (KAS) and eledoisin (ELE). 2. Immunoreactivity in neuronal somata and fibres was detected only with the SP antiserum. 3. SP and NKA radioimmunoassays were performed on extracts of circumoesophageal ganglia. In common with immunocytochemistry, immunoreactivity was only detected with the SP antiserum. 4. Gel permeation chromatography of extracts resolved a single peak of immunoreactivity eluting slightly later than synthetic mammalian SP. Reverse-phase HPLC of immunoreactive fractions resolved two immunoreactive peptides representing oxidised and reduced forms of a single peptide. 5. These data suggest that the nervous system of H. aspersa contains a single tachykinin with C-terminal structural characteristics similar to mammalian SP.
INTRODUCTION
Regulatory peptides are ubiquitous and diverse in the nervous systems of vertebrates (Krieger, 1983; Bertaccini, 1976) and even in the most simple invertebrates such as coelenterates (Schaller et al., 1984).
One of the most studied groups of regulatory peptides are the tachykinins, which are character&d by a common C-terminal Phe-X,-Gly-X,-Met-amide (X, and X, are variable) and similar spectra of biological activities (Erspamer and Melchiorri, 1980). The first tachykinin to be identified was mammalian substance P (SP) by von Euler and Gaddum in 1931, although its full primary structure was not determined until 1971 by Chang et al. Two additional mammalian tachykinins, designated neurokinin A (Kangawa et al., 1984) and neurokinin B (Minamino et al., 1984), have since been isolated. A number of novel tachykinins have also been isolated from phylogenetically-discrete groups. Amphibian skin has proven to be rich in tachykinins and the nomenclature of these peptides reflects either the generic or specific names of the source species. Amphibian skin tachykinins include physalaemin (Erspamer et al., 1964), phyllomedusin (Anastasi et at., 1970), uperolein (Anastasi et al., 1975) and kassinin (Anastasi et al., 1977). Moreover, the same nomenclature has been adopted for fish tachykinins which have been isolated to date, namely scyliorhinin I and II (Conlon et al., 1986) from the dogfish, Scyfiorhinus canicula, and carrasin from the goldfish, Carrasius auratus (Conlon et al., 1991). In the invertebrates, the first tachykinin was isolated in 1962 by Author for correspondence: Dr C. Shaw, Wellcome Research Laboratories, Mulhouse Building, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland.
Erspamer and Anastasi from the octopus, Eledone, and was named accordingly, eledoisin. Recently, a novel class of regulatory peptides were isolated from locust tissues and these have been named locustatachykinins (Schoofs et al., 1990a,b). Although analogous to tachykinins in the spectrum of biological activities displayed, these peptides are not structurallyanalogous to other known members of this family. However, data on the distribution and immunochemical characterisation of invertebrate tachykinins remains scanty. Indeed, little information exists on the presence of eledoisin or other tachykinins in the nervous system of molluscs, although SP-immunoreactivity has previously been demonstrated in Helix aspersa (Osborne and Dockray, 1982; Osborne et af., 1982). This species of gastropod mollusc, a widely-used model in invertebrate neurochemical research, was used in the present study to investigate the immunochemical characteristics and degree of heterogeneity of tachykinin immunoreactivity present within the nervous system, by employing immunocytochemistry and specific radioimmunoassays coupled to chromatographic fractionation.
MATERIALS
AND METHODS
Immunocytochemistry
Circumoesophageal ganglia and foot muscle tissues were excised and placed into 4% (w/v) paraforrnaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS), pH 7.4. After fixation for 4 hr, tissues were transferred to 5% (w/v) sucrose solution in PBS, stored overnight at 4°C followed by cryoprotection in PBS containing 30% (w/v) sucrose. Tissues were then frozen at -20°C in Cryo-m-bed and sections (12 pm) were cut on a Reichert Cryocut E cryostat and mounted onto gelatin-coated slides which were air-dried 169
P. S. &UNG et al.
170
for 30 min. Immunostaining employed the indirect immunofluorescence technique (Coons et al., 1955). Sections were immersed in PBS for 20 min at room temperature and then incubated overnight at 4°C in primary antisera (see Table 1 for details). Following a 20 min wash in PBS, sections were incubated for 30min at room temperature in fluoresceinconjugated swine anti-rabbit serum. After a final wash in PBS, specimens were mounted in PBS/glycerol (1: 9, v/v) and viewed on an MRC-500 confocal scanning laser microscope (Bio-Rad Lasersharp Ltd, Abingdon, Oxfordshire, U.K.). All antisera were diluted in PBS containing 0.1% (w/v) bovine serum albumin, 0.5% (v/v) Triton X-100 and 0.1% (w/v) sodium azide. Control of immunostaining was achieved by (i) omission of primary antiserum, (ii) omission of secondary antiserum and (iii) substitution of primary antiserum with non-immune rabbit serum. Preparation of tissue extracts
Two hundred circumoesophageal ganglia were excised, weighed and placed directly into ice-cold extraction medium consisting of ethanol/O.7 M HCl (3: 1, v/v, 8 vol/g tissue). Approximately 3 g wet weight of tissue was homogenised and the homogenate was stirred at 4°C for 24 hr. Following removal of tissue debris by centrifugation (3000g for 30 min), ethanol was removed from the decanted supernatant under reduced pressure. The ethanol-free extracts were acidified with trifluoroacetic acid (TFA) to a final concentration of 0.1% (v/v) and left at 4°C for 24 hr prior to recentrifugation (4000 g for 30 min). The supernatant was concentrated using 3 Waters Associates C-18 Sep-Pak cartridges connected in series. Sep-Pak cartridges were eluted using 0.1% acetonitrile (v/v) and the eluates were lyophilised.
TFA in
Radioimmunoassay
Antisera to synthetic SP and NKA were used in radioimmunoassay as described in detail previously (Maule et al., 1989). Brief details are given in Table 1. The assay systems consisted of 100~1 of diluted antiserum, 100~1 of iz51labelled tracer and 100 ~1 of peptide standard (3.9 to 500 pg/ assay tube) or sample. All samples were assayed in duplicate in serial dilution. The assay buffer used throughout was 40mM sodium phosphate buffer (pH 7.4), containing 140 mM sodium chloride and 0.2% (w/v) bovine serum albumin. All reactants were added simultaneously and assay tubes were incubated overnight at 4°C. Separation of antibody-bound and free peptide was achieved by addition of 1 ml of 0.5% (w/v) dextran-coated charcoal. Following centrifugation, charcoal pellets were counted on a Nuclear Enterprise NE 1600 gamma counter. Gel permeation chromatography The lyophilised extract was reconstituted in 2 ml of 2 M acetic acid and applied to a 90 x 1.6 cm column of Sephadex G-50 (fine) (Phannacia, Uppsala, Sweden) equilibrated in 2M acetic acid and eluted at a flow rate of lOml/hr. Fractions (2 ml) were collected and an aliquot (100 ~1) of
Table
each was removed, lyophilised and subjected to tachykinin radioimmunoassays. The column was previously calibrated with Blue dextran (V,) and potassium dichromate (V,) and was subsequently calibrated with synthetic SP. Reverse-phase HPLC Gel permeation chromatographic fractions containing tachykinin immunoreactivity were pumped directly onto the initial semi-preparative column and immunoreactivity was sequentially fractionated using a range of different column chemistries. The columns employed were; 1. Whatman Partisil ODS-3 (1 x 60cm) semi-preparative C-18 column, 2. Supelcosil LC-308 (0.46 x 25 cm) C-8 column and 3. Vydac 208TP54 (0.46 x 25 cm) C-8 column. Flow rate was 3 ml/min for the semi-preparative column and 1.5 ml/min for analytical columns. The equilibration solvent was trifluoroacetic acid/water, 0.1: 99.9, v/v and the elution solvent was trifluoroacetic acid/acetonitrile, 0.1: 99.9, v/v. Fractions were collected each minute and 10~1 was removed from each for tachykinin radioimmunoassays.
RESULTS Immunocytochemistry
Immunocytochemical staining results in the nervous system of H. aspersa are summarised in Table 1. Intense immunostaining of neuronal somata and fibres was obtained with the SP antiserum 152. NKA and kassinin antisera weakly immunostained the same structures as the SP antiserum and immunostaining with the eledoisin antiserum was consistently negative. SP immunoreactivity was present in a sub-population of small neurones situated peripherally around intensely-immunoreactive neuropiles (Fig. la-c) within the circumoesophageal ganglia. Immunoreactivity was also abundant in large fibre tracts and in fine anastomosing beaded fibres within the foot muscle (Fig. Id). All controls were consistently negative. Radioimmunoassay Only SP immunoreactivity (17 pmol/g) was detected in extracts of ganglia using the SP antiserum GSP 10, which is C-terminally directed. However, no immunoreactivity was detected with the SP antiserum 152. This antiserum is likewise C-terminally directed but requires an epitope in the middle of SP for full recognition and is thus highly SP specific. These data would suggest that the SP immunoreactive peptide in H. aspersa is not authentic SP but structurallyresembles mammalian SP in the far C-terminus. The NKA antiserum 570 did not detect any immunoreactivity.
I. Details of the tachykinin antisera employed in immunocytochemistry radioimmunoassay (RIA) and a summary of results Specificity
Working dilution
Code
lmmunoreactivity
SP NKA KAS ELE*
C-terminal C-terminal not known not known
I:500 1:200 I:200 I:200
152 570 RK7 RE2
+++ + + -
SP SP NKA
C-terminal C-terminal C-terminal
I :8000
GSPIO 152 570
Antiserum raised to ICC Synthetic Synthetic Synthetic Synthetic RIA Synthetic Synthetic Svnthetic
(ICC) and
1:BOOO
I :4o.ooo
17 pmol/g 0 0
Antiserum source: Department of Medicine, The Queen’s University of Belfast, Northern Ireland, U.K. and lDr E. Theodorsson, Karolinska Hospital, Stockholm, Sweden. Staining intensity: + + + strong, + + moderate, + weak, - negative.
Tachykinins in H. aspersa
171
Fig. 1. Immunofluorescence photomicrographs of SP immunoreactivity in H. aspersa. (a)Immunoreactive neurones of different morphology and staining intensity in the cerebral ganglion and associated dense neuropiles of immunoreactive fibres x 100. (b) Intensely immunoreactive neuronal soma and short axon beside a nerve trunk containing immunoreactive fibres x 200. (c) Immunoreactive fibre tracts within a nerve trunk x 200. (d) Immunoreactive fibres within nerve bundles innervating the foot muscle x 100. Chromatographic analyses Gel
permeation
chromatography
of
extracts
resolved a single peak of SP immunoreactivity eluting slightly later than synthetic SP standard, implying but not defining a lower molecular weight for the Helix peptide (Fig. 2). In reverse-phase HPLC, SP immunoreactivity was resolved into 2 fractions on the semi-
preparative column (Fig. 3). Both immunoreactive fractions were separately chromatographed on Supelcosil LC-308 and Vydac C8 analytical columns using the same gradients. These resolved the first SP immunoreactive fraction into a single fraction (Fig. 4), whereas the second immunoreactive fraction was resolved into two fractions. The first of these was
1
407
,,/, 80
F
0
10
20
30 40 50 Fraction number
60
70
1
8b
Fig. 2. Gel permeation chromatogram of SP immunoreactivity in a circumoesophageal ganglion extract. The arrow indicates the elution position of synthetic mammalian SP.
;
30
z : ?
20
7 %
IO
-L
0
./ ,,..” ,,..” ,_..‘~
,/ ..’
,./’ ,...I. 0
IO
20
30
Retention
---I’” 50
60
70
80’
time (min)
Fig. 3. Semi-preparative reverse phase HPLC chromatogram of SP immunoreactivity in pooled gel permeation chromatographic fractions of circumoesophageal extract. Two immunoreactive peptides were resolved.
P. S. LEuNG et al.
172
,.../ -40 w
s 15. c 0 rL & .z F %
//~" _._/" /,...."
IO5-
A -30 y$ s
/' ,./ ,../ ,,'
OJ 0
* lo
Fig. 4. Analytical
/,/~ ,,/"
-20 5 g -10
20 30 40 50 Retention time (min)
. -0 60
reverse phase HPLC (Vydac immunoreactive peptide 1.
C-8) of SP
of identical retention time to the previous fraction (Fig. 5). These data are consistent with the presence of a single SP immunoreactive peptide in Helix which, in common with mammalian SP, contains a methionine residue which is prone to oxidation. DISCUSSION
The present study has demonstrated that the nervous system of the garden snail, Helix aspersa, contains a single member of the tachykinin peptide family which exhibits similar immunochemical characteristics to mammalian SP in its C-terminal region. The lack of cross-reactivity of this peptide with an antiserum highly-specific for mammalian SP would however, preclude identity with the mammalian analogue. By means of immunocytochemistry, this SP-immunoreactive peptide has been localised to a sub-set of small neurones and associated dense and extensive fibres tracts in the circumoesophageal ganglia and in similar tracts and fibre networks within the foot muscle. This peptide appears to be restricted in distribution to the nervous system and might act as a neurotransmitter and/or neuromodulator, in common with tachykinins occurring in the nervous systems of vertebrates. Interpretation of the immunochemical data would suggest that this Helix tachykinin more closely resembles SP/physalaemin type tachykinins in its C-terminal region rather than neurokinimkassinin type tachykinins. The former group possess an aromatic residue (Phe or Tyr) in the variable X, position and the latter group possess a Val residue at this site. These single amino acid substitutions can differentiate both groups immuno-
g$
15
2 Y e
IO
F
5
% -1
,_.40 w 30 g
/....~~”// ,.*_,.,_._ . ..y..” /
0’ 0
IO
20
30
Retention
Fig. 5. Analytical
REFERENCES Anastasi A. and Falconieri Erspamer G. (1970) Occurrence of phyllomedusin, a physalaemin-like decapeptide in the skin of Phyllomedusa bicolor. Experientia 26, 866-867. Anastasi A., Erspamer V. and Endean R. (1975) Structure of uperolein, a physalaemin-like endecapeptide occurring in the skin of Uperoleia rugosa and Uperoleia marmorata. Experientia 31, 394-395. Anastasi A., Montecucchi P., Erspamer V. and Visser J. (1977) Amino acid composition and sequence of kassinin, a tachykinin dodecapeptide from the skin of the African frog Kassinia senegalensis. Experienfia 33, 857-858. Bertaccini G. (I 976) Active polypeptides of non-mammalian origin. Pharmacol. Rev. 28, 127-177. Chang M. M., Leeman S. E. and Niall H. D. (1971) Amino acid sequence of substance P. Nature New Biol. 232, 86-87. Conlon J. M., Deacon C. F., O’Toole L. and Thim L. (1986) Scyliorhinin I and II: two novel tachykinins from dogfish gut. FEBS Left. 200, 111-116. Cordon J. M.. O’Harte F.. Peter R. E. and Kah 0. (19911 Carassin: a tachykinin’ that is structurally-related-to neuropeptide gamma from the brain of the goldfish. J. Neurochem. 56, 1432-1436. Coons A. H., Leduc E. H. and Connolly J. M. (1955) Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J. exp. Med. 102, 49-60. Erspamer V. and Anastasi A. (1962) Structure and pharmacological actions of eledoisin, the active undecapeptide of
,_./ _./ /...~~ _../ ,L 50
L”
chemically by the radioimmunoassays employed. Moreover, the molluscan tachykinin eledoisin or eledoisin-like peptides seem to be absent in this species of mollusc as the specific eledoisin antiserum employed gave consistently negative results in immunocytochemistry. Eledoisin was originally isolated from octopus (Eledone) salivary glands and it has not been specifically-detected in other cephalopod or gastropod species. This tachykinin may therefore not perform a neurotransmitter/neuromodulator role within the octopus but rather may serve a role as a bioactive salivary toxin component. Reverse-phase HPLC of Helix tachykinin resolved two immunoreactive peptides, the first of which was generated in subsequent chromatographic runs of the second, implying that oxidation was responsible for this observation and this tachykinin contains a methionine residue in common with vertebrate tachykinins (Shaw et al., 1989). SP immunoreactivity has been previously demonstrated within the nervous systems of representatives from most major invertebrate phyla (Grimmelikhuijzen er al., 1981; Verhaert and De Loof, 1985; Goldberg et al., 1988) including the molluscs (Osborne and Dockray, 1982; Osborne et af., 1982). Recently, a novel group of peptides, termed locustatachykinins, have been isolated and sequenced from the locust, Locusta migratoria. These peptides terminate in a Phe-X-Gly-Val-Arg-amide and are devoid of methionine residues. This fact, coupled with the requirement of antiserum GSPlO for a C-terminal Leu-Met-amide would imply that the Helix tachykinin is not structurally-analogous to these insect peptides. Isolation and sequencing of Helix tachykinin is underway and will definitively elucidate its proposed C-terminal structural homology with vertebrate analogues.
,_...,. .”
40
2. 20 q IO 5
50
60
/ 0
time (min)
reverse phase HPLC (Vydac immunoreactive peptide 2.
C-8) of SP
Tachykinins in H. aspersa the posterior salivary glands of Eledone. Experientia 18, 58-59. Erspamer V. and Melchiorri P. (1980) Active polypeptides: from amuhibian skin to gastrointestinal tracts and brain of mammals. Trends Ph&macol. Sci. 1, 391-393. Erspamer V., Anastasi A., Bertaccini G. and Cei J. M. (1964) Structure and pharmacological actions of physalaemin, the main active polypeptide of the skin of Physalaemus fuscumaculatus. Experientia 20,489-490. Goldberg D., Nusbaum M. P. and Marder E. (1988) Substance P-like immunoreactivity in the stomatogastric nervous system of the crab Cancer borealis and the lobsters Panilurus interruptus and Homarus americanus. Cell Tissue Res. 252, 5155522.
Grimmelikhuijzen C. J. P., Balfe A., Emson P. C., Powell D. and Sundler F. (1981) Substance P-like immunoreactivity in the nervous system of Hydra. Histochemistry 71. 325-333.
Kangawa K., Minamino N., Fukuda A. and Matsou H. (1984) Neuromedin K: a novel mammalian tachykinin identified in porcine spinal cord. Eiochem. Biophys. Res. Commun. 114, 533-540. Krieger D. T. (1983) Brain peptides: What, where and why? Science,
Wash. 222, 975-985.
Maule A. G., Shaw C., Halton D. W., Johnston C. F., Fairweather I. and Buchanan K. D. (1989) Tachykinin immunoreactivitv in the parasitic flatworm Diclidophora merlangi and its fish host the whiting (Merlangius~merlangus): Radioimmunoassay and chromatographic characterisation using region-specific substance P and neurokinin A antisera. Comp. Biochem. Physiol. 94C, 533-541.
Minamino N., Kangawa K., Fukuda A. and Matsou H. (1984) Neuromedin L: a novel mammalian tachykinin identified in porcine spinal cord. Neuropeptides 4, 157-166.
173
Osborne N. N. and Dockray G. J. (1982) Bombesin-like immunoreactivity in specific neurones of the snail Helix aspersa and an example of the coexistence of substance P and serotonin in an invertebrate neurone. Neurochem. Intl. 4, 1755180. Osborne N. N., Cuello A. C. and Dockray G. J. (1982) Substance P and cholecystokinin-like peptides in Helix neurons and cholecystokinin and serotonin in a giant neuron. Science 216, 409-41 I. Schaller H. C., Hoffmeister S. and Bodenmuller H. (1984) Hormonal control of regeneration in Hydra. In Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones (Edited by Hoffman J. A. and Porchet M.),
pp. 5-9. Springer, Berlin. Schoofs L., Holman G. M., Hayes T. K., Nachman R. J. and De Loof A. (1990a) Locustatachykinin I and II: two novel insect neuropeptides with homology to peptides of the vertebrate tachykinin family. PEBS Lett. 261, 397-401. Schoofs L., Holman G. M., Hayes T. K., Kochansky J. P., Nachman R. J. and De Loof A. (1990b) Locustatachykinin III and IV: two additional insect neuropeptides with homology to peptides of the vertebrate tachykinin family. Regul. Pept. 31, 199-212.
Shaw C., Foy, W. L., Johnston C. F. and Buchanan K. D. (1989) Identification and characterization of multiple tachykinin immunoreactivities in bovine retina: evidence for the presence of a putative oxidative inactivation system for substance P. J. Neurochem. 53, 1547-1554. Verhaert P. and De Loof A. (1985) Substance P-like immunoreactivity in the central nervous system of the blattarian insect Periplaneta americana L. revealed by a monoclonal antibody. Histochemistry 83, 501-507. von Euler U. S. and Gaddum J. H. (1931) An unidentified depressor substance in certain tissue extracts. J. Physiol. Lond. 72, 74-87.
Biochem.
Physiol.Vol. 103C, No. I, pp. 169-173, 1992
0306~4492/92 $5.00+ 0.00 0 1992Pergamon Press Ltd
Printed in Great Britain
IMMUNOCHEMICAL CHARACTERISATION OF TACHYKININ IMMUNOREACTIVITY IN THE NERVOUS SYSTEM OF THE GARDEN SNAIL, HELIX ASPERSA P. S.
LEUNG, C. SHAW, C. F. JOHNSTON
Schools of Clinical Medicine
and G. B.
IRVINE
and Biology and Biochemistry, The Queen’s University of Belfast, Northern Ireland
(Received 30 January 1992; accepted for publication 26 February 1992)
Abstract-l. Circumoesophageal ganglia and foot muscle of the garden snail, Helix aspersa, were subjected to immunocytochemistry using antisera to the tachykinins, substance P (SP), neurokinin A (NKA), kassinin (KAS) and eledoisin (ELE). 2. Immunoreactivity in neuronal somata and fibres was detected only with the SP antiserum. 3. SP and NKA radioimmunoassays were performed on extracts of circumoesophageal ganglia. In common with immunocytochemistry, immunoreactivity was only detected with the SP antiserum. 4. Gel permeation chromatography of extracts resolved a single peak of immunoreactivity eluting slightly later than synthetic mammalian SP. Reverse-phase HPLC of immunoreactive fractions resolved two immunoreactive peptides representing oxidised and reduced forms of a single peptide. 5. These data suggest that the nervous system of H. aspersa contains a single tachykinin with C-terminal structural characteristics similar to mammalian SP.
INTRODUCTION
Regulatory peptides are ubiquitous and diverse in the nervous systems of vertebrates (Krieger, 1983; Bertaccini, 1976) and even in the most simple invertebrates such as coelenterates (Schaller et al., 1984).
One of the most studied groups of regulatory peptides are the tachykinins, which are character&d by a common C-terminal Phe-X,-Gly-X,-Met-amide (X, and X, are variable) and similar spectra of biological activities (Erspamer and Melchiorri, 1980). The first tachykinin to be identified was mammalian substance P (SP) by von Euler and Gaddum in 1931, although its full primary structure was not determined until 1971 by Chang et al. Two additional mammalian tachykinins, designated neurokinin A (Kangawa et al., 1984) and neurokinin B (Minamino et al., 1984), have since been isolated. A number of novel tachykinins have also been isolated from phylogenetically-discrete groups. Amphibian skin has proven to be rich in tachykinins and the nomenclature of these peptides reflects either the generic or specific names of the source species. Amphibian skin tachykinins include physalaemin (Erspamer et al., 1964), phyllomedusin (Anastasi et at., 1970), uperolein (Anastasi et al., 1975) and kassinin (Anastasi et al., 1977). Moreover, the same nomenclature has been adopted for fish tachykinins which have been isolated to date, namely scyliorhinin I and II (Conlon et al., 1986) from the dogfish, Scyfiorhinus canicula, and carrasin from the goldfish, Carrasius auratus (Conlon et al., 1991). In the invertebrates, the first tachykinin was isolated in 1962 by Author for correspondence: Dr C. Shaw, Wellcome Research Laboratories, Mulhouse Building, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland.
Erspamer and Anastasi from the octopus, Eledone, and was named accordingly, eledoisin. Recently, a novel class of regulatory peptides were isolated from locust tissues and these have been named locustatachykinins (Schoofs et al., 1990a,b). Although analogous to tachykinins in the spectrum of biological activities displayed, these peptides are not structurallyanalogous to other known members of this family. However, data on the distribution and immunochemical characterisation of invertebrate tachykinins remains scanty. Indeed, little information exists on the presence of eledoisin or other tachykinins in the nervous system of molluscs, although SP-immunoreactivity has previously been demonstrated in Helix aspersa (Osborne and Dockray, 1982; Osborne et af., 1982). This species of gastropod mollusc, a widely-used model in invertebrate neurochemical research, was used in the present study to investigate the immunochemical characteristics and degree of heterogeneity of tachykinin immunoreactivity present within the nervous system, by employing immunocytochemistry and specific radioimmunoassays coupled to chromatographic fractionation.
MATERIALS
AND METHODS
Immunocytochemistry
Circumoesophageal ganglia and foot muscle tissues were excised and placed into 4% (w/v) paraforrnaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS), pH 7.4. After fixation for 4 hr, tissues were transferred to 5% (w/v) sucrose solution in PBS, stored overnight at 4°C followed by cryoprotection in PBS containing 30% (w/v) sucrose. Tissues were then frozen at -20°C in Cryo-m-bed and sections (12 pm) were cut on a Reichert Cryocut E cryostat and mounted onto gelatin-coated slides which were air-dried 169
P. S. &UNG et al.
170
for 30 min. Immunostaining employed the indirect immunofluorescence technique (Coons et al., 1955). Sections were immersed in PBS for 20 min at room temperature and then incubated overnight at 4°C in primary antisera (see Table 1 for details). Following a 20 min wash in PBS, sections were incubated for 30min at room temperature in fluoresceinconjugated swine anti-rabbit serum. After a final wash in PBS, specimens were mounted in PBS/glycerol (1: 9, v/v) and viewed on an MRC-500 confocal scanning laser microscope (Bio-Rad Lasersharp Ltd, Abingdon, Oxfordshire, U.K.). All antisera were diluted in PBS containing 0.1% (w/v) bovine serum albumin, 0.5% (v/v) Triton X-100 and 0.1% (w/v) sodium azide. Control of immunostaining was achieved by (i) omission of primary antiserum, (ii) omission of secondary antiserum and (iii) substitution of primary antiserum with non-immune rabbit serum. Preparation of tissue extracts
Two hundred circumoesophageal ganglia were excised, weighed and placed directly into ice-cold extraction medium consisting of ethanol/O.7 M HCl (3: 1, v/v, 8 vol/g tissue). Approximately 3 g wet weight of tissue was homogenised and the homogenate was stirred at 4°C for 24 hr. Following removal of tissue debris by centrifugation (3000g for 30 min), ethanol was removed from the decanted supernatant under reduced pressure. The ethanol-free extracts were acidified with trifluoroacetic acid (TFA) to a final concentration of 0.1% (v/v) and left at 4°C for 24 hr prior to recentrifugation (4000 g for 30 min). The supernatant was concentrated using 3 Waters Associates C-18 Sep-Pak cartridges connected in series. Sep-Pak cartridges were eluted using 0.1% acetonitrile (v/v) and the eluates were lyophilised.
TFA in
Radioimmunoassay
Antisera to synthetic SP and NKA were used in radioimmunoassay as described in detail previously (Maule et al., 1989). Brief details are given in Table 1. The assay systems consisted of 100~1 of diluted antiserum, 100~1 of iz51labelled tracer and 100 ~1 of peptide standard (3.9 to 500 pg/ assay tube) or sample. All samples were assayed in duplicate in serial dilution. The assay buffer used throughout was 40mM sodium phosphate buffer (pH 7.4), containing 140 mM sodium chloride and 0.2% (w/v) bovine serum albumin. All reactants were added simultaneously and assay tubes were incubated overnight at 4°C. Separation of antibody-bound and free peptide was achieved by addition of 1 ml of 0.5% (w/v) dextran-coated charcoal. Following centrifugation, charcoal pellets were counted on a Nuclear Enterprise NE 1600 gamma counter. Gel permeation chromatography The lyophilised extract was reconstituted in 2 ml of 2 M acetic acid and applied to a 90 x 1.6 cm column of Sephadex G-50 (fine) (Phannacia, Uppsala, Sweden) equilibrated in 2M acetic acid and eluted at a flow rate of lOml/hr. Fractions (2 ml) were collected and an aliquot (100 ~1) of
Table
each was removed, lyophilised and subjected to tachykinin radioimmunoassays. The column was previously calibrated with Blue dextran (V,) and potassium dichromate (V,) and was subsequently calibrated with synthetic SP. Reverse-phase HPLC Gel permeation chromatographic fractions containing tachykinin immunoreactivity were pumped directly onto the initial semi-preparative column and immunoreactivity was sequentially fractionated using a range of different column chemistries. The columns employed were; 1. Whatman Partisil ODS-3 (1 x 60cm) semi-preparative C-18 column, 2. Supelcosil LC-308 (0.46 x 25 cm) C-8 column and 3. Vydac 208TP54 (0.46 x 25 cm) C-8 column. Flow rate was 3 ml/min for the semi-preparative column and 1.5 ml/min for analytical columns. The equilibration solvent was trifluoroacetic acid/water, 0.1: 99.9, v/v and the elution solvent was trifluoroacetic acid/acetonitrile, 0.1: 99.9, v/v. Fractions were collected each minute and 10~1 was removed from each for tachykinin radioimmunoassays.
RESULTS Immunocytochemistry
Immunocytochemical staining results in the nervous system of H. aspersa are summarised in Table 1. Intense immunostaining of neuronal somata and fibres was obtained with the SP antiserum 152. NKA and kassinin antisera weakly immunostained the same structures as the SP antiserum and immunostaining with the eledoisin antiserum was consistently negative. SP immunoreactivity was present in a sub-population of small neurones situated peripherally around intensely-immunoreactive neuropiles (Fig. la-c) within the circumoesophageal ganglia. Immunoreactivity was also abundant in large fibre tracts and in fine anastomosing beaded fibres within the foot muscle (Fig. Id). All controls were consistently negative. Radioimmunoassay Only SP immunoreactivity (17 pmol/g) was detected in extracts of ganglia using the SP antiserum GSP 10, which is C-terminally directed. However, no immunoreactivity was detected with the SP antiserum 152. This antiserum is likewise C-terminally directed but requires an epitope in the middle of SP for full recognition and is thus highly SP specific. These data would suggest that the SP immunoreactive peptide in H. aspersa is not authentic SP but structurallyresembles mammalian SP in the far C-terminus. The NKA antiserum 570 did not detect any immunoreactivity.
I. Details of the tachykinin antisera employed in immunocytochemistry radioimmunoassay (RIA) and a summary of results Specificity
Working dilution
Code
lmmunoreactivity
SP NKA KAS ELE*
C-terminal C-terminal not known not known
I:500 1:200 I:200 I:200
152 570 RK7 RE2
+++ + + -
SP SP NKA
C-terminal C-terminal C-terminal
I :8000
GSPIO 152 570
Antiserum raised to ICC Synthetic Synthetic Synthetic Synthetic RIA Synthetic Synthetic Svnthetic
(ICC) and
1:BOOO
I :4o.ooo
17 pmol/g 0 0
Antiserum source: Department of Medicine, The Queen’s University of Belfast, Northern Ireland, U.K. and lDr E. Theodorsson, Karolinska Hospital, Stockholm, Sweden. Staining intensity: + + + strong, + + moderate, + weak, - negative.
Tachykinins in H. aspersa
171
Fig. 1. Immunofluorescence photomicrographs of SP immunoreactivity in H. aspersa. (a)Immunoreactive neurones of different morphology and staining intensity in the cerebral ganglion and associated dense neuropiles of immunoreactive fibres x 100. (b) Intensely immunoreactive neuronal soma and short axon beside a nerve trunk containing immunoreactive fibres x 200. (c) Immunoreactive fibre tracts within a nerve trunk x 200. (d) Immunoreactive fibres within nerve bundles innervating the foot muscle x 100. Chromatographic analyses Gel
permeation
chromatography
of
extracts
resolved a single peak of SP immunoreactivity eluting slightly later than synthetic SP standard, implying but not defining a lower molecular weight for the Helix peptide (Fig. 2). In reverse-phase HPLC, SP immunoreactivity was resolved into 2 fractions on the semi-
preparative column (Fig. 3). Both immunoreactive fractions were separately chromatographed on Supelcosil LC-308 and Vydac C8 analytical columns using the same gradients. These resolved the first SP immunoreactive fraction into a single fraction (Fig. 4), whereas the second immunoreactive fraction was resolved into two fractions. The first of these was
1
407
,,/, 80
F
0
10
20
30 40 50 Fraction number
60
70
1
8b
Fig. 2. Gel permeation chromatogram of SP immunoreactivity in a circumoesophageal ganglion extract. The arrow indicates the elution position of synthetic mammalian SP.
;
30
z : ?
20
7 %
IO
-L
0
./ ,,..” ,,..” ,_..‘~
,/ ..’
,./’ ,...I. 0
IO
20
30
Retention
---I’” 50
60
70
80’
time (min)
Fig. 3. Semi-preparative reverse phase HPLC chromatogram of SP immunoreactivity in pooled gel permeation chromatographic fractions of circumoesophageal extract. Two immunoreactive peptides were resolved.
P. S. LEuNG et al.
172
,.../ -40 w
s 15. c 0 rL & .z F %
//~" _._/" /,...."
IO5-
A -30 y$ s
/' ,./ ,../ ,,'
OJ 0
* lo
Fig. 4. Analytical
/,/~ ,,/"
-20 5 g -10
20 30 40 50 Retention time (min)
. -0 60
reverse phase HPLC (Vydac immunoreactive peptide 1.
C-8) of SP
of identical retention time to the previous fraction (Fig. 5). These data are consistent with the presence of a single SP immunoreactive peptide in Helix which, in common with mammalian SP, contains a methionine residue which is prone to oxidation. DISCUSSION
The present study has demonstrated that the nervous system of the garden snail, Helix aspersa, contains a single member of the tachykinin peptide family which exhibits similar immunochemical characteristics to mammalian SP in its C-terminal region. The lack of cross-reactivity of this peptide with an antiserum highly-specific for mammalian SP would however, preclude identity with the mammalian analogue. By means of immunocytochemistry, this SP-immunoreactive peptide has been localised to a sub-set of small neurones and associated dense and extensive fibres tracts in the circumoesophageal ganglia and in similar tracts and fibre networks within the foot muscle. This peptide appears to be restricted in distribution to the nervous system and might act as a neurotransmitter and/or neuromodulator, in common with tachykinins occurring in the nervous systems of vertebrates. Interpretation of the immunochemical data would suggest that this Helix tachykinin more closely resembles SP/physalaemin type tachykinins in its C-terminal region rather than neurokinimkassinin type tachykinins. The former group possess an aromatic residue (Phe or Tyr) in the variable X, position and the latter group possess a Val residue at this site. These single amino acid substitutions can differentiate both groups immuno-
g$
15
2 Y e
IO
F
5
% -1
,_.40 w 30 g
/....~~”// ,.*_,.,_._ . ..y..” /
0’ 0
IO
20
30
Retention
Fig. 5. Analytical
REFERENCES Anastasi A. and Falconieri Erspamer G. (1970) Occurrence of phyllomedusin, a physalaemin-like decapeptide in the skin of Phyllomedusa bicolor. Experientia 26, 866-867. Anastasi A., Erspamer V. and Endean R. (1975) Structure of uperolein, a physalaemin-like endecapeptide occurring in the skin of Uperoleia rugosa and Uperoleia marmorata. Experientia 31, 394-395. Anastasi A., Montecucchi P., Erspamer V. and Visser J. (1977) Amino acid composition and sequence of kassinin, a tachykinin dodecapeptide from the skin of the African frog Kassinia senegalensis. Experienfia 33, 857-858. Bertaccini G. (I 976) Active polypeptides of non-mammalian origin. Pharmacol. Rev. 28, 127-177. Chang M. M., Leeman S. E. and Niall H. D. (1971) Amino acid sequence of substance P. Nature New Biol. 232, 86-87. Conlon J. M., Deacon C. F., O’Toole L. and Thim L. (1986) Scyliorhinin I and II: two novel tachykinins from dogfish gut. FEBS Left. 200, 111-116. Cordon J. M.. O’Harte F.. Peter R. E. and Kah 0. (19911 Carassin: a tachykinin’ that is structurally-related-to neuropeptide gamma from the brain of the goldfish. J. Neurochem. 56, 1432-1436. Coons A. H., Leduc E. H. and Connolly J. M. (1955) Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J. exp. Med. 102, 49-60. Erspamer V. and Anastasi A. (1962) Structure and pharmacological actions of eledoisin, the active undecapeptide of
,_./ _./ /...~~ _../ ,L 50
L”
chemically by the radioimmunoassays employed. Moreover, the molluscan tachykinin eledoisin or eledoisin-like peptides seem to be absent in this species of mollusc as the specific eledoisin antiserum employed gave consistently negative results in immunocytochemistry. Eledoisin was originally isolated from octopus (Eledone) salivary glands and it has not been specifically-detected in other cephalopod or gastropod species. This tachykinin may therefore not perform a neurotransmitter/neuromodulator role within the octopus but rather may serve a role as a bioactive salivary toxin component. Reverse-phase HPLC of Helix tachykinin resolved two immunoreactive peptides, the first of which was generated in subsequent chromatographic runs of the second, implying that oxidation was responsible for this observation and this tachykinin contains a methionine residue in common with vertebrate tachykinins (Shaw et al., 1989). SP immunoreactivity has been previously demonstrated within the nervous systems of representatives from most major invertebrate phyla (Grimmelikhuijzen er al., 1981; Verhaert and De Loof, 1985; Goldberg et al., 1988) including the molluscs (Osborne and Dockray, 1982; Osborne et af., 1982). Recently, a novel group of peptides, termed locustatachykinins, have been isolated and sequenced from the locust, Locusta migratoria. These peptides terminate in a Phe-X-Gly-Val-Arg-amide and are devoid of methionine residues. This fact, coupled with the requirement of antiserum GSPlO for a C-terminal Leu-Met-amide would imply that the Helix tachykinin is not structurally-analogous to these insect peptides. Isolation and sequencing of Helix tachykinin is underway and will definitively elucidate its proposed C-terminal structural homology with vertebrate analogues.
,_...,. .”
40
2. 20 q IO 5
50
60
/ 0
time (min)
reverse phase HPLC (Vydac immunoreactive peptide 2.
C-8) of SP
Tachykinins in H. aspersa the posterior salivary glands of Eledone. Experientia 18, 58-59. Erspamer V. and Melchiorri P. (1980) Active polypeptides: from amuhibian skin to gastrointestinal tracts and brain of mammals. Trends Ph&macol. Sci. 1, 391-393. Erspamer V., Anastasi A., Bertaccini G. and Cei J. M. (1964) Structure and pharmacological actions of physalaemin, the main active polypeptide of the skin of Physalaemus fuscumaculatus. Experientia 20,489-490. Goldberg D., Nusbaum M. P. and Marder E. (1988) Substance P-like immunoreactivity in the stomatogastric nervous system of the crab Cancer borealis and the lobsters Panilurus interruptus and Homarus americanus. Cell Tissue Res. 252, 5155522.
Grimmelikhuijzen C. J. P., Balfe A., Emson P. C., Powell D. and Sundler F. (1981) Substance P-like immunoreactivity in the nervous system of Hydra. Histochemistry 71. 325-333.
Kangawa K., Minamino N., Fukuda A. and Matsou H. (1984) Neuromedin K: a novel mammalian tachykinin identified in porcine spinal cord. Eiochem. Biophys. Res. Commun. 114, 533-540. Krieger D. T. (1983) Brain peptides: What, where and why? Science,
Wash. 222, 975-985.
Maule A. G., Shaw C., Halton D. W., Johnston C. F., Fairweather I. and Buchanan K. D. (1989) Tachykinin immunoreactivitv in the parasitic flatworm Diclidophora merlangi and its fish host the whiting (Merlangius~merlangus): Radioimmunoassay and chromatographic characterisation using region-specific substance P and neurokinin A antisera. Comp. Biochem. Physiol. 94C, 533-541.
Minamino N., Kangawa K., Fukuda A. and Matsou H. (1984) Neuromedin L: a novel mammalian tachykinin identified in porcine spinal cord. Neuropeptides 4, 157-166.
173
Osborne N. N. and Dockray G. J. (1982) Bombesin-like immunoreactivity in specific neurones of the snail Helix aspersa and an example of the coexistence of substance P and serotonin in an invertebrate neurone. Neurochem. Intl. 4, 1755180. Osborne N. N., Cuello A. C. and Dockray G. J. (1982) Substance P and cholecystokinin-like peptides in Helix neurons and cholecystokinin and serotonin in a giant neuron. Science 216, 409-41 I. Schaller H. C., Hoffmeister S. and Bodenmuller H. (1984) Hormonal control of regeneration in Hydra. In Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones (Edited by Hoffman J. A. and Porchet M.),
pp. 5-9. Springer, Berlin. Schoofs L., Holman G. M., Hayes T. K., Nachman R. J. and De Loof A. (1990a) Locustatachykinin I and II: two novel insect neuropeptides with homology to peptides of the vertebrate tachykinin family. PEBS Lett. 261, 397-401. Schoofs L., Holman G. M., Hayes T. K., Kochansky J. P., Nachman R. J. and De Loof A. (1990b) Locustatachykinin III and IV: two additional insect neuropeptides with homology to peptides of the vertebrate tachykinin family. Regul. Pept. 31, 199-212.
Shaw C., Foy, W. L., Johnston C. F. and Buchanan K. D. (1989) Identification and characterization of multiple tachykinin immunoreactivities in bovine retina: evidence for the presence of a putative oxidative inactivation system for substance P. J. Neurochem. 53, 1547-1554. Verhaert P. and De Loof A. (1985) Substance P-like immunoreactivity in the central nervous system of the blattarian insect Periplaneta americana L. revealed by a monoclonal antibody. Histochemistry 83, 501-507. von Euler U. S. and Gaddum J. H. (1931) An unidentified depressor substance in certain tissue extracts. J. Physiol. Lond. 72, 74-87.
