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• W2023159529 abstract "Cells immunoreactive to an antiserum specifically directed against vertebrate α-Neo-endorphin (α-NE) were detected in the internal wall of anterior and posterior suckers of the rhynchobdellid leech Theromyzon tessulatum. These cells have morphological and ultrastructural characteristics close to the “releasing gland cells” of adhesive organs. The epitope recognized by anti-α-NE was contained in granules having a diameter of 0.2-0.3 µm. Previous works involving the brain of this leech demonstrate the existence of ~14 neurons immunoreactive to the anti-α-NE. Following an extensive purification including high pressure gel permeation and reversed-phase high performance liquid chromatography, epitopes contained in both suckers and central nervous system were isolated. Purity of the isolated peptides was controlled by capillary electrophoresis. Their sequences were determined by a combination of automated Edman degradation, electrospray mass spectrometry measurement, and coelution experiments in reversed-phase high performance liquid chromatography with synthetic α-NE. The results demonstrate that epitopes recognized by the anti-α-NE in the suckers and the central nervous system are identical to vertebrate α-NE (YGGFLRKYPK). This finding constitutes the first biochemical characterization of a prodynorphin-derived peptide in invertebrates. Moreover the isolation of this peptide in the annelida establishes the very ancient phylogenetic origin of α-NE as well as its conservation in evolution. Cells immunoreactive to an antiserum specifically directed against vertebrate α-Neo-endorphin (α-NE) were detected in the internal wall of anterior and posterior suckers of the rhynchobdellid leech Theromyzon tessulatum. These cells have morphological and ultrastructural characteristics close to the “releasing gland cells” of adhesive organs. The epitope recognized by anti-α-NE was contained in granules having a diameter of 0.2-0.3 µm. Previous works involving the brain of this leech demonstrate the existence of ~14 neurons immunoreactive to the anti-α-NE. Following an extensive purification including high pressure gel permeation and reversed-phase high performance liquid chromatography, epitopes contained in both suckers and central nervous system were isolated. Purity of the isolated peptides was controlled by capillary electrophoresis. Their sequences were determined by a combination of automated Edman degradation, electrospray mass spectrometry measurement, and coelution experiments in reversed-phase high performance liquid chromatography with synthetic α-NE. The results demonstrate that epitopes recognized by the anti-α-NE in the suckers and the central nervous system are identical to vertebrate α-NE (YGGFLRKYPK). This finding constitutes the first biochemical characterization of a prodynorphin-derived peptide in invertebrates. Moreover the isolation of this peptide in the annelida establishes the very ancient phylogenetic origin of α-NE as well as its conservation in evolution. In vertebrates, all known opioids peptides are cleavage products of three different precursors, i.e. proopiomelanocortin (POMC), 1The abbreviations used are: POMCproopiomelanocortinanti-α-NEanti-α-Neo-endorphinDIAdot immunobinding assayHPGPChigh pressure gel permeationHPLChigh performance liquid chromatographyMSHmelanocyte-stimulating hormoneα-NEα-Neo-endorphinRTretention timeCNScentral nervous systemELISAenzyme-linked immunosorbent assay. proenkephalin, and prodynorphin (1). Processing of the prodynorphin yields a number of bioactive peptides including leucine-enkephalin, Neo-endorphins (α and β), and dynorphins (A and B) (26Patey G. Rossier J. Ann. Endocrinol. 1986; 47: 71-87PubMed Google Scholar). Among these peptides, α-Neo-endorphin (α-NE) has been isolated from all vertebrates phyla (36Sei C.A. Richard R. Dores R.M. Brain Res. 1989; 479: 162-166Crossref PubMed Scopus (23) Google Scholar; 6Dores R.M. McDonald L.K. Goldsmith A. Deviche P. Rubin D.A. Cell. Physiol. Biochem. 1993; 3 (a): 231-244Crossref Scopus (30) Google Scholar; 16Goldsmith L.A. Physiology, Biochemistry, and Molecular Biology of the Skin. Oxford University Press, New York1992Google Scholar). The α-NE isolated from different tetrapods revealed by reversed-phase HPLC similar biochemical properties, reflecting the great conservation of this peptide in tetrapods (36Sei C.A. Richard R. Dores R.M. Brain Res. 1989; 479: 162-166Crossref PubMed Scopus (23) Google Scholar, 16Goldsmith L.A. Physiology, Biochemistry, and Molecular Biology of the Skin. Oxford University Press, New York1992Google Scholar). NE immunocytochemical probes in cyclostomes, holostean and teleostean fish, proved negative (4Dores R.M. Gorbman A. Gen. Comp. Endocrinol. 1989; 77: 489-499Crossref Scopus (23) Google Scholar; 7Dores R.M. McDonald L.K. Purdom L.C. Sei C.A. Brain Behav. Evol. 1993; 42 (b): 69-76Crossref PubMed Scopus (4) Google Scholar; 5Dores R.M. McDonald L.K. Gen. Comp. Endocrinol. 1992; 88: 292-297Crossref PubMed Scopus (13) Google Scholar). This result may be due to the following: 1) α-NE sequence changes that render this opioid undetectable to heterologous mammalian antisera, 2) a unique set of posttranslational processing reactions in which α-NE is not liberated from fragments of the prodynorphin precursor, or 3) the possibility that during evolution the prodynorphin precursor may be absent in fishes (see 7Dores R.M. McDonald L.K. Purdom L.C. Sei C.A. Brain Behav. Evol. 1993; 42 (b): 69-76Crossref PubMed Scopus (4) Google Scholar). proopiomelanocortin anti-α-Neo-endorphin dot immunobinding assay high pressure gel permeation high performance liquid chromatography melanocyte-stimulating hormone α-Neo-endorphin retention time central nervous system enzyme-linked immunosorbent assay. If these three genes encoding opioid peptides are related (17Gubler U. Habener J.F. Molecular Cloning of Hormone Genes. Humana Press, Clifton, NJ1987: 229Crossref Google Scholar), one of the central questions is in what order did these genes evolve from a hypothetical ancestral gene? In this context research on different opioid peptides in invertebrates is essential (38Stefano G.B. Adv. Neuroimmunol. 1991; 1: 71-82Abstract Full Text PDF Scopus (27) Google Scholar). For example, enkephalin peptides have been isolated in crustacea (23Luschen W. Buck F. Willig A. Jarros P.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8671-8675Crossref PubMed Scopus (59) Google Scholar), in mollusks (22Leung M.K. Stefano G.B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 955-956Crossref PubMed Scopus (104) Google Scholar), and in annelids (29Salzet M. Bulet P. Verger-Bocquet M. Malecha J. FEBS Lett. 1995; 357 (a): 187-191Crossref PubMed Scopus (28) Google Scholar). Moreover, a peptide derived from POMC maturation in vertebrates, γ-MSH, has also been characterized in the leech Theromyzon tessulatum (32Salzet M. Wattez C. Bulet P. Malecha J. FEBS Lett. 1994; 348: 102-106Crossref PubMed Scopus (17) Google Scholar). Given this amount of information in invertebrates concerning opioid peptides, little is known about α-NE in these animals. This peptide was not detected by immunocytochemistry in the mollusk Lymnaea stagnalis (1Boer H.H. van Minnen J. Peptides. 1985; 6: 459-463Crossref PubMed Scopus (10) Google Scholar). By contrast, immunoreactive material was present in annelids i.e. polychaeta (3Dhainaut-Courtois N. Tramu G. Beauvillain J.C. Masson M. Neurochem. Int. 1986; 8: 327-338Crossref PubMed Scopus (13) Google Scholar) and achaeta (43Verger-Bocquet M. Malecha J. Tramu G. Comp. Endocrinol. 1987; 6 (a): 195-199Google Scholar, 44Verger-Bocquet M. Malecha J. Tramu G. Cell Tissue Res. 1987; 250 (b): 63-71Crossref PubMed Scopus (11) Google Scholar, 45Verger-Bocquet M. Malecha J. Wattez C. Tramu G. Comp. Endocrinol. 1988; 7: 21-27Google Scholar). In the leech T. tessulatum, a great number of cells immunoreactive to the antiserum raised against α-NE were found in the proboscis (43Verger-Bocquet M. Malecha J. Tramu G. Comp. Endocrinol. 1987; 6 (a): 195-199Google Scholar, 45Verger-Bocquet M. Malecha J. Wattez C. Tramu G. Comp. Endocrinol. 1988; 7: 21-27Google Scholar) and in the brain (6-14 neurons) (43Verger-Bocquet M. Malecha J. Tramu G. Comp. Endocrinol. 1987; 6 (a): 195-199Google Scholar, 44Verger-Bocquet M. Malecha J. Tramu G. Cell Tissue Res. 1987; 250 (b): 63-71Crossref PubMed Scopus (11) Google Scholar). The aim of the present study is to fully characterized the α-NE-like peptide(s) in the leech T. tessulatum. Here we report for the first time that such material is present in the central nervous system and in a novel type of cell in the suckers. Mature specimens of the rhynchobdellid leech T. tessulatum were reared under controlled laboratory conditions as described in detail by 24Malecha J. Verger-Bocquet M.A. Tramu G. Can. J. Zool. 1989; 67: 636-640Crossref Google Scholar. After anesthesia with chloretone, animals were pinned flat, ventral side up in leech saline solution (25Muller K.J. Nicholls J.G. Stent G.S. Neurobiology of the Leech. Cold Spring Harbor Laboratory Cold Spring Harbor, NY1981: 255Google Scholar). Central nervous system (CNS: brain and nerve cord) and anterior and posterior suckers were excised. Except for the purpose of immunocytochemistry, in which case they were fixed, suckers and CNS were immediately frozen in liquid nitrogen and stored at −70°C for storage. Polyclonal antiserum anti-α-Neo-endorphin (anti-α-NE) was raised in rabbits and provided by Dr. G. Tramu (Laboratoire de Neurocytochimie fonctionnelle, Université de Bordeaux I, Talence, France). Anti-α-NE specificity was determined by radioimmunoassay as noted elsewhere (12Fallon J.H. Ciofi P. Björklund A. Hökfelt T. Handbook of Chemical Neuroanatomy, Neuropeptides in the CNS, Part II. Elsevier Science Publishers B.V., Amsterdam1990: 1Google Scholar). No significant cross-reactivity was found with methionine-enkephalin, leucine-enkephalin, dynorphin 1-17 and dynorphin 1-8, or α or β endorphin. Anterior and posterior parts of T. tessulatum (including suckers) were fixed overnight at 4°C in Bouin-Hollande fixative (+10% HgCl2 saturated solution). They were then embedded in paraffin and serially sectioned at 7 µm. After removal of paraffin with toluene, the sections were successively treated with the anti-α-NE diluted 1:200 and with goat anti-rabbit IgG conjugated to horseradish peroxidase as described elsewhere (46Verger-Bocquet M. Wattez C. Salzet M. Malecha J. Can. J. Zool. 1992; 70: 856-865Crossref Google Scholar). The specificity of the antiserum was tested on consecutive sections mounted on different slides by preadsorbing the antiserum overnight at 4°C with the homologous antigen (synthetic α-NE, Sigma) at a concentration of 500 µg/ml pure antiserum. Anterior and posterior suckers were fixed for 2 h at 4°C in a mixture of 4% paraformaldehyde, 0.2% picric acid, and 0.1% glutaraldehyde in 0.1 M phosphate buffer. The tissue were post-fixed in 1% OsO4 2 h and were dehydrated before embedding in Epon. Immunostaining was performed directly on ultrathin sections of suckers collected on nickel grids and treated according to the following procedure: 1) 10% H2O2, 8 min; 2) distilled water, 10 min; 3) Coons buffer, pH 7.4, containing 1% normal goat serum (NGS), 10 min; 4) anti-α-NE diluted 1:1200 in Coons buffer containing 1% NGS and 1% bovine serum albumin, 24 h, 4°C; 5) Tris 0.1 M, pH 7.6, containing 0.5 M NaCl, 1% bovine serum albumin, and 1% NGS, for two 10-min periods; 6) 5 nM colloidal gold-labeled goat anti-rabbit IgG (Janssen, Belgium) diluted 1:50 in the precedent buffer, 1.5 h; and 7) washing in the buffer (twice for 10 min each) and distilled water (twice for 10 min each). The sections were then stained with uranyl acetate and lead citrate and then examined with a Jeol JEM 100 CX electron microscope. Enzyme-linked immunosorbent assays (ELISA) and dot immunobinding assay (DIA) were conducted according to 33Salzet M. Wattez C. Slomianny M.C. Léü B. Siegert K.J. Comp. Biochem. Physiol. 1992; 102C: 483-487Google Scholar, 28Salzet M. Bulet P. Van Dorsselaer A. Malecha J. Eur. J. Biochem. 1993; 217 (a): 897-903Crossref PubMed Scopus (79) Google Scholar with anti-α-NE employed at a dilution of 1:1000. As a control, preadsorption of α-NE was carried out using homologous peptide. Prior to ELISA and DIA, anti-α-NE, at its working dilution was incubated overnight at 4°C with synthetic α-NE (Sigma) (300 µg/ml undiluted anti-α-NE). A four-step procedure was employed (Table I).Table IPurification of the α-Neo-endorphin related peptide in the brain of T. tessulatumStepColumn materialGradient of acetonitrileYield in brain%%1. PrepurificationWaters Sep-Pak C18501002. HPGPCSec2000, 7.5 × 300 mm30783. Reversed-Phase HPLCVydac C18, 4.8 × 250 mm0-15 (10 min)7015-45 (30 min)4. Reversed-Phase HPLCVydac C18, 4.8 × 250 mm0-15 (10 min)6915-45 (40 min)3. Final purificationODS C18, 2 × 250 mm0-60 (60 min)65 Open table in a new tab Batches of 200 CNS were homogenized and extracted with 200 µl of 1 M acetic acid at 4°C. After centrifugation at 12,000 rpm for 30 min at 4°C, the pellet was reextracted once. The two supernatants were combined and loaded onto Sep-Pak C18 cartridges (500 µl of extract/cartridge; Waters). After washing the cartridges with 5 ml of 1 M acetic acid, elution was performed with 5 ml of 50% acetonitrile in water acidified with 0.1% trifluoroacetic acid (Pierce). The eluted fractions were reduced 20-fold in a vacuum centrifuge (Savant) to remove acid and organic solvent. The total amount of anti-α-NE-like material was quantified using ELISA. The 50% eluted fraction was taken up to a volume of 250 µl with acidified water (0.1% trifluoroacetic acid) and fractionated on a HPGPC column (Ultraspherogel, 7, 5 × 300 mm, Sec2000, Beckman) associated to a precolumn (Ultraspherogel, 7, 5 × 40 mm, Beckman). Samples were eluted with 30% acetonitrile in acidified water at a flow rate of 500 µl/min. Fractions immunoreactive in DIA to anti-α-NE were concentrated before separated on a C18-peptide protein column (250 mm × 4.6 mm; Vydac) equilibrated with acidified water (0.1% trifluoroacetic acid). Elution was performed with a discontinuous linear gradient of acetonitrile in acidified water over 0-15% for 10 min and over 15-45% for 30 min at a flow rate of 1 ml/min. The column effluent was monitored by absorbance at 226 nm and the presence of α-NE-like material detected in aliquots of each fraction by DIA. Fractions that contained the immunoreactive material were applied on the same column with a shallower gradient. Elution was performed with a discontinuous linear gradient of acetonitrile in acidified water over 0-15% for 10 min and over 15-45% for 40 min at a flow rate of 1 ml/min. After a 20-fold concentration by freeze-drying, fraction aliquots of 0.5 µl were tested by DIA. The α-NE-like material was finally purified on an ODS C18 reversed-phase column (Ultrasphere, 250 mm × 2 mm; Beckman) developed with a linear gradient of 0-60% acetonitrile in acidified water for 60 min at a flow rate of 50 µl/min. The column effluent was monitored by absorbance at 226 nm and the immunoreactive material detected as above. Suckers underwent same purification procedure, except an additional prepurification step was added. The supernatant obtained after acidic extraction contained green and yellow pigments as well as mucus. They were removed early on the procedure by precipitation with water/acetone (20/80, v/v). The acetonic fraction was reduced 20-fold before subjected to the Sep-Pack prepurification step procedure. All HPLC purifications were performed with a Beckman Gold HPLC system equipped with a photodiode array detector (Beckman 168). A comparison between first derivatives of absorbance of scan profiles obtained from synthetic peptides and from the endogenous immunoreactive peptides, at identical concentration (100 pmol) was performed as noted in detail elsewhere (31Salzet M. Wattez C. Baert J.L. Malecha J. Brain Res. 1993; 631 (b): 247-255Crossref PubMed Scopus (32) Google Scholar). Prior to microsequencing, the purity of the peptide was controlled by capillary electrophoresis. Separation was performed on an Applied Biosystems model 270A-HT capillary electrophoresis system. Silica capillary (72 cm length) was used. Under these conditions, separation was achieved from anode to cathode in a citrate buffer (20 mM) at pH 2.5. Detection was at 200 nm, temperature was 30°C, and the volume injected was 2 nl. Automated Edman degradation of the purified peptide and detection of phenylthiohydantoin-derivatives were performed on a pulse liquid automatic sequenator (Applied Biosystems, model 473A). The purified peptide was dissolved in water/methanol (50/50, v/v) containing 1% acetic acid and analyzed on a VG Biotech BioQ mass spectrometer (Manchester, UK). Details of the method have been described elsewhere (28Salzet M. Bulet P. Van Dorsselaer A. Malecha J. Eur. J. Biochem. 1993; 217 (a): 897-903Crossref PubMed Scopus (79) Google Scholar). In addition to the cells previously described in the proboscis (43Verger-Bocquet M. Malecha J. Tramu G. Comp. Endocrinol. 1987; 6 (a): 195-199Google Scholar) and in the brain (44Verger-Bocquet M. Malecha J. Tramu G. Cell Tissue Res. 1987; 250 (b): 63-71Crossref PubMed Scopus (11) Google Scholar, 45Verger-Bocquet M. Malecha J. Wattez C. Tramu G. Comp. Endocrinol. 1988; 7: 21-27Google Scholar) of T. tessulatum, numerous α-NE immunoreactive cells are observed in the epithelium of anterior and posterior suckers (Figs. 1, panels 1 and 2). In T. tessulatum, the α-NE-like material exhibited subepidermal bodies located among muscular and mucous gland cells. These small cells, 6-10 µm in diameter, possess a large nucleus (3-5 µm) for their size (Fig. 1, panels 4 and 5). They bear elongated processes that terminate immediately beneath the cuticle (Fig. 1, panel 3) and are abundant (>12,000/mm2). None of the processes had pores through the cuticle (Fig. 1, panel 7). At the ultrastructural level, numerous granules can be noted in the cell body and in the processes. These ovoid or somewhat crescent-shaped granules (0.2-0.3 µm) are characterized by a homogenous electron-dense material (Fig. 1, panel 7). After immunogold labeling, numerous colloidal gold particles are observed in these granules (Fig. 1, panels 6 and 8). Central nervous systems (1000) or suckers (400) were subjected to a peptide extraction in 1 M acetic acid, pH 2. ELISA revealed in crude extract 6.32 ± 1.8 pmol of α-NE-like material/CNS and 15.45 ± 4.6 pmol of α-NE-like material/sucker (after acetonic precipitation). Crude extracts were prepurified using Sep-Pak C18 cartridges. The fraction eluted by 50% of acetonitrile was reduced 20-fold by freeze-drying and applied to a HPGPC column. Eluted fractions tested in DIA revealed a single immunoreactive zone in the two cases (CNS or suckers) corresponding to peptides with molecular mass of ~1-4.5 kDa (data not shown). An amount of 5.45 ± 0.75 pmol of α-NE-like material/CNS (recovery of ~87%) and of 13.44 ± 2.25 pmol of α-NE-like material/sucker (recovery of ~82%) was obtained. Results obtained after preadsorption of the antiserum by synthetic α-NE established the specificity of the immunodetection. Each immunoreactive fraction was then concentrated 20-fold and applied to a reversed-phase HPLC. In a first step of reversed-phase HPLC on a Vydac C18 column, α-NE-like substances (suckers or CNS) eluted at a same retention time (RT) comprised between 21-22 (corresponding to 26-27% of acetonitrile) (Fig. 2). In these conditions the vertebrate α-NE eluted at a RT of 21.85 min. Total amount of α-NE-like material determined by ELISA at this step of purification was 4.85 ± 0.92 pmol of α-NE-like material/CNS (recovery of ~80%) and of 12.63 ± 4.25 pmol of α-NE-like material/sucker (recovery of ~81%). The immunoreactive zone containing this material (suckers or CNS) was analyzed on the same column with a shallower gradient. A peak immunoreactive to anti-α-NE, at a RT comprised between 25 and 25.3 min (corresponding to 26.25-26.48% of acetonitrile), was resolved in both cases. At this step of purification, quantification by ELISA indicated an amount of 4.15 ± 0.75 pmol of α-NE-like material/CNS (recovery of ~66%) and of 10.25 ± 3.75 pmol of α-NE-like material/sucker (recovery of ~66%). In both cases (suckers or CNS), a peak was then purified to homogeneity on an ODS C18 reversed-phase column and gave in each case a single peak at a RT of 28.3 min. Purity of the immunoreactive material (suckers, CNS) was established by capillary electrophoresis (Fig. 3). Quantification by ELISA at this step of purification indicated 3.75 ± 0.86 pmol of α-NE-like material/CNS (recovery of ~60%) and of 9.86 ± 2.62 pmol of α-NE-like material/sucker (recovery of ~64%).Fig. 3Capillary electrophoresis of purified α-NE-like peptide from T. tessulatum CNS. Analyses were performed on a 72-cm capillary in 20 mM citrate buffer at pH 2.5.View Large Image Figure ViewerDownload (PPT) In order to ensure that the α-NE-like peptide purified either from CNS or from suckers were the same compound, a coinjection on ODS C18 reversed-phase HPLC column of the two purified peptides was performed. A single peak was eluted at a RT of 28.3 min. Moreover, a comparison of the first derivatives of absorbance of the purified α-NE-like peptide from CNS and from suckers at a same concentration revealed a total spectral overlapping between 190 and 300 nm and a ratio equal to 1, reflecting a very similar homology. After the final purification step, a fraction aliquot of the immunoreactive material was evaluated by Edman degradation. The sequence, established on 139 pmol of purified α-NE-like peptide with a sequencing yield of 95%, was Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys (Table II). The molecular mass of the CNS α-NE-like peptide measured by electrospray mass spectrometry (m/z = 1228.04 ± 0.06 Da) is in agreement with the calculated isotopic mass (1228.68 Da) of the α-NE (Fig. 4). The same result was obtained with the α-NE-like peptide isolated from suckers (m/z = 1228.3 ± 0.35 versus 1228.68). Moreover, coinjection on ODS C18 reversed-phase HPLC column of each purified peptides (CNS, suckers) and synthetic α-NE revealed a single peak at a RT of 28.5 min. This procedure established that the primary structure of T. tessulatum α-NE-like peptide is fully superposable on that of vertebrate α-NE peptide. Its amount was estimated to 2.45 ± 1.15 pmol/CNS and 8.95 ± 2.15 pmol/sucker.Table IIAutomated Edman degradation of 139 pmol of the α-neoendorphin-related peptide, with central nervous system of the leech T. tessulatumCycle no.PTH-XaaYield (pmol)1Tyr100.462Gly121.243Gly141.174Phe124.65Leu1556Arg43.957Lys100.488Tyr99.529Pro63.6410Lys62.95 Open table in a new tab The present study demonstrates that to the peptide α-NE isolated from the CNS and suckers of the leech T. tessulatum is structurally identical with the one identified in vertebrates (YGGFLRKYPK). The characterization of α-NE in the leech constitutes the first report of the presence of a prodynorphin-derived peptide in an invertebrate. The same true α-NE was isolated and identified both from CNS and suckers of mature T. tessulatum. α-Neoendorphin immunoreactivity is expressed by specific cells in CNS (44Verger-Bocquet M. Malecha J. Tramu G. Cell Tissue Res. 1987; 250 (b): 63-71Crossref PubMed Scopus (11) Google Scholar, 45Verger-Bocquet M. Malecha J. Wattez C. Tramu G. Comp. Endocrinol. 1988; 7: 21-27Google Scholar), neuroendocrine cells of the foregut (43Verger-Bocquet M. Malecha J. Tramu G. Comp. Endocrinol. 1987; 6 (a): 195-199Google Scholar), and by epithelial cells at the level of the suckers in this animal. Interestingly, this is not a novel observation in that in leeches certain monoclonal antibodies reacted with cells in the CNS and with epithelial cells (18Hogg N. Flaster M. Zipser B. J. Neurosci. Res. 1983; 9: 445-457Crossref PubMed Scopus (23) Google Scholar). Such an observation of a common localization among neurons and epithelial cells is very frequent in vertebrates. The present study also shows that α-NE immunoreactive cells in T. tessulatum suckers have morphological and ultrastructural characteristics close to the “releasing gland cells” of adhesive organs of the Branchiobdellids (13Farnesi R.M. Marinelli M. Tei S. Vagnetti D. J. Morphol. 1981; 170: 195-205Crossref PubMed Scopus (7) Google Scholar; 18Hogg N. Flaster M. Zipser B. J. Neurosci. Res. 1983; 9: 445-457Crossref PubMed Scopus (23) Google Scholar; 15Gelder S.R. Rowe J.P. Can. J. Zool. 1988; 66: 2057-2064Crossref Google Scholar; 47Weigl A.M. Trans. Am. Microsc. Soc. 1994; 113: 276-301Crossref Google Scholar). These cells might constitute with the viscid gland cells a duo-gland adhesive organ (42Tyler N. Zoomorphology. 1976; 84: 1-76Crossref Scopus (127) Google Scholar). In this regard, the sticky secretion of the viscid cells is used for substrate attachment and the secretion of the releasing cells may assist in detachment. However, we never found pores for these cells or releasing granules on the surface of the leech suckers. The same observation was made by 47Weigl A.M. Trans. Am. Microsc. Soc. 1994; 113: 276-301Crossref Google Scholar in Branchiobdellids. The function of these releasing cells is currently not understood. Interestingly, they may be derived from nerve cells (42Tyler N. Zoomorphology. 1976; 84: 1-76Crossref Scopus (127) Google Scholar; 15Gelder S.R. Rowe J.P. Can. J. Zool. 1988; 66: 2057-2064Crossref Google Scholar), an observation that is based on similarities in structure and staining properties between them. In Branchiobdellids, the interpretation of 13Farnesi R.M. Marinelli M. Tei S. Vagnetti D. J. Morphol. 1981; 170: 195-205Crossref PubMed Scopus (7) Google Scholar is that these cells are neurons and the granules might be considered neurosecretory granules. 13Farnesi R.M. Marinelli M. Tei S. Vagnetti D. J. Morphol. 1981; 170: 195-205Crossref PubMed Scopus (7) Google Scholar suggest that the releasing cells are actually neurons with junctions on the viscid adhesive cell ducts to control the release of the viscid secretion granules. In T. tessulatum, the hypothesis of the nerve cell nature of these releasing cells is sustained by our observations showing that their intracytoplasmic granules (0.2-0.3 µm) contain α-NE, identical to the one present in specific neurons. It is known that in leeches, neurons and epithelial cells are derived from the same blastomeres (48Weisblat D.A. Harper G. Stent G.S. Sawyer R.T. Dev. Biol. 1980; 76: 58-78Crossref PubMed Scopus (93) Google Scholar). In Hirudinea, 41Stewart R.R. Macagno E.R. Zipser B. Brain Res. 1985; 332: 150-157Crossref PubMed Scopus (12) Google Scholar suggest that the epidermal cells recognized by monoclonal antibodies specific to the CNS are indeed peripheral neurons. Their study with the monoclonal antibody Lan 3-6 shows that during the embryonic development the same labeled epithelial cells possess an apical dendrite and a basal process; therefore, they are neurons. 41Stewart R.R. Macagno E.R. Zipser B. Brain Res. 1985; 332: 150-157Crossref PubMed Scopus (12) Google Scholar suggest that the expression of the antigens in the axon, is either absent or below the level detectable with these technique in this cell. To answer this question anatomical experiences, e.g. anterograde transport, could be conducted. In T. tessulatum, as in Branchiobdellids (47Weigl A.M. Trans. Am. Microsc. Soc. 1994; 113: 276-301Crossref Google Scholar), continuity between these cells and necks of the viscid cells are scarce, which reinforces the improbable nervous control as proposed by 13Farnesi R.M. Marinelli M. Tei S. Vagnetti D. J. Morphol. 1981; 170: 195-205Crossref PubMed Scopus (7) Google Scholar. If the nerve nature of these α-NE positive sucker cells is confirmed and considering their abundance, we could propose that they exert an important role either in the control of the adhesivity or in one of the multiple sensorial functions of the suckers. The existence of secretory granules suggests that α-NE can be released in the intracellular spaces and acts via a paracrine mechanism. Although that relationship between Branchiobdellids and the other clitellates remains unclear (19Holt P.C. Hydrobiologia. 1989; 180: 1-5Crossref Scopus (15) Google Scholar; 2Brinkhurst R.O. Gelder S.R. Hydrobiologia. 1989; 180: 7-15Crossref Scopus (18) Google Scholar), the discovery in T. tessulatum of cells resembling the releasing cells of Branchiobdellids argues in favor of a link between adhesive organs of Branchiobdellids and suckers of Hirudinea. Furthermore, in the CNS of the leech, the α-NE secretion of neurons into the circulatory system at the neurohemal site suggests a hormonal role. Alternatively, an action as neurotransmitter or neuromodulator at the level of nerve endings located in the dorsal commissure or the neuropile is also possible, as in the presence of both mechanisms in these animals. The fact that α-NE is highly conserved in course of evolution from annelids to vertebrates suggests an essential function for this peptide. Actually we know that peptides conserved during evolution appear to keep an action on a same physiological function. This argument is sustained by two neuropeptides, acting on the osmoregulation, isolated in both T. tessulatum and vertebrates, i.e. the lysine-conopressin (28Salzet M. Bulet P. Van Dorsselaer A. Malecha J. Eur. J. Biochem. 1993; 217 (a): 897-903Crossref PubMed Scopus (79) Google Scholar) and the angiotensin II (30Salzet M. Bulet P. Wattez C. Verger-Bocquet M. Malecha J. J. Biol. Chem. 1995; 270 (b): 1575-1582Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In vertebrates, it is known that opioids are involved in variety of physiological functions and interact with endocrine system. The endogenous opioids peptides appear to have a role in the interaction between the CNS and the immune system (35Scharrer B. Am. Zool. 1990; 30: 887-895Crossref Scopus (21) Google Scholar; 34Scharrer Smith E.M. Stefano G.B. Neuropeptides in Neuroimmunology. Springer, Heidelberg Germany1994Google Scholar). In invertebrates, until now, by contrast, nothing is known about the role of α-NE. Consequently, the leech provides for a good model system in which to study this conserved peptide as well as the presence of a possibly conserved precursor, i.e. prodynorphin. The existence of an ancestral proenkephalin gene is supported by the isolation of enkephalin peptides in invertebrate taxa, i.e. the crustacea Carcinus maenas (23Luschen W. Buck F. Willig A. Jarros P.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8671-8675Crossref PubMed Scopus (59) Google Scholar), in the mollusk Mytilus edulis (22Leung M.K. Stefano G.B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 955-956Crossref PubMed Scopus (104) Google Scholar), and in the leech T. tessulatum (32Salzet M. Wattez C. Bulet P. Malecha J. FEBS Lett. 1994; 348: 102-106Crossref PubMed Scopus (17) Google Scholar). However, the ratio of Leu-enkephalin and Met-enkephalin in C. maenas and T. tessulatum is 3:1 and 2:1, respectively, whereas in vertebrates and Mytilus edulis Met-enkephalin is the major opioid peptide. Moreover, immunocytochemical studies performed at the level of T. tessulatum brains revealed that Leu-enkephalin and Met-enkephalin are not detected in the same cells (44Verger-Bocquet M. Malecha J. Tramu G. Cell Tissue Res. 1987; 250 (b): 63-71Crossref PubMed Scopus (11) Google Scholar). From these observations several hypothesis can be given. First, unlike in vertebrates, there are two separated genes, one coding for Met-enkephalin and the other for Leu-enkephalin. Second, the two pentapeptides come from a unique ancestral proenkephalin precursor and the expression of Met- or Leu-enkephalin is due to different posttranslation processing mechanisms; Leu-enkephalin is not expressed in cells expressing Met-enkephalin and vice versa. If we consider the first hypothesis, the question that is raised is: what can be the second opioid precursor, i.e. a prodynorphin or a POMC-like precursor? Peptides yielded from POMC processing have been identified by immunocytochemistry and radioimmunoassay in invertebrates, i.e. ACTH and β endorphin in the mollusk Planorbarius corneus (14Franchesi C. Ottaviani E. J. Immunol. Res. 1992; 4: 53-55Google Scholar) and in insects (11Duve H. Thorpe A. Cell Tissue Res. 1988; 251: 399-415Crossref PubMed Scopus (31) Google Scholar), β endorphin in the flatworms (27Reuter M. Gustafsson M. Arch. Histol. Cytol. 1989; 52: 253-263Crossref PubMed Scopus (35) Google Scholar) and MSH, and endorphins in annelids (3Dhainaut-Courtois N. Tramu G. Beauvillain J.C. Masson M. Neurochem. Int. 1986; 8: 327-338Crossref PubMed Scopus (13) Google Scholar; 43Verger-Bocquet M. Malecha J. Tramu G. Comp. Endocrinol. 1987; 6 (a): 195-199Google Scholar, 44Verger-Bocquet M. Malecha J. Tramu G. Cell Tissue Res. 1987; 250 (b): 63-71Crossref PubMed Scopus (11) Google Scholar, 45Verger-Bocquet M. Malecha J. Wattez C. Tramu G. Comp. Endocrinol. 1988; 7: 21-27Google Scholar). These results envisage the existence of a POMC-like precursor in invertebrates. Complete molecular studies have been performed in the trematod Schistosoma mansoni, where a gene related to the vertebrate POMC has been cloned (10Duvaux-Miret O. Dissous C. Gautran J.P. Pattou E. Kordon C. Capron A. New Biol. 1992; 2: 93-99Google Scholar; 9Duvaux-Miret O. Capron A. Ann. N. Y. Acad. Sci. 1992; 650: 245-250Crossref PubMed Scopus (8) Google Scholar). However, according to 8Duvaux-Miret O. Capron A. Adv. Neuroimmunol. 1991; 1: 41-57Abstract Full Text PDF Scopus (12) Google Scholar, the extremely high homology between the genes of S. mansoni and vertebrates may be the result of a transfer of genetic material from the host toward the parasite, probably by a viral mechanism. The other possibility is a high evolutionary conservation through selective pressure, which has allowed S. mansoni to synthesize molecules highly similar to the host endogenous signals; this could be used to avoid the host defense (8Duvaux-Miret O. Capron A. Adv. Neuroimmunol. 1991; 1: 41-57Abstract Full Text PDF Scopus (12) Google Scholar). Recently, a peptide belonging to the POMC vertebrate family has been isolated in T. tessulatum brains. This peptide is related to the Vertebrate γ-MSH and presented ~80% sequence homology with vertebrates γ1-MSH (32Salzet M. Wattez C. Bulet P. Malecha J. FEBS Lett. 1994; 348: 102-106Crossref PubMed Scopus (17) Google Scholar). However, in the leech T. tessulatum, the γ-MSH-like peptide seems be localized in a multipeptidic precursor different from POMC (31Salzet M. Wattez C. Baert J.L. Malecha J. Brain Res. 1993; 631 (b): 247-255Crossref PubMed Scopus (32) Google Scholar, 30Salzet M. Bulet P. Wattez C. Verger-Bocquet M. Malecha J. J. Biol. Chem. 1995; 270 (b): 1575-1582Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Other peptides present in this multiple hormones precursor have recently been isolated in leeches, i.e. AI and AII (21Laurent V. Bulet P. Salzet M. Neurosci. Lett. 1995; 190: 175-179Crossref PubMed Scopus (32) Google Scholar; 31Salzet M. Wattez C. Baert J.L. Malecha J. Brain Res. 1993; 631 (b): 247-255Crossref PubMed Scopus (32) Google Scholar, 30Salzet M. Bulet P. Wattez C. Verger-Bocquet M. Malecha J. J. Biol. Chem. 1995; 270 (b): 1575-1582Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore, these results tend to favor the absence in leeches of a true POMC precursor. If we consider that POMC is absent in annelids, the hypothesis of a prodynorphin-like precursor can be suggested. Furthermore as suggested by 40Stefano G.B. Leung M.K. Zhao X. Scharrer B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 626-630Crossref PubMed Scopus (156) Google Scholar, detection by immunocytochemistry of the prodynorphin end-products makes it difficult to imagine that proenkephalin is the only source of Leu-enkephalin in neural tissue. It could also derive from a prodynorphin. Although epitopes of the different prodynorphin end-products are immunologically recognized in nervous system of different invertebrates, their chemical nature is unknown. However, in the leech T. tessulatum, α-NE and the dynorphin-like peptides are expressed in different neurons (44Verger-Bocquet M. Malecha J. Tramu G. Cell Tissue Res. 1987; 250 (b): 63-71Crossref PubMed Scopus (11) Google Scholar, 45Verger-Bocquet M. Malecha J. Wattez C. Tramu G. Comp. Endocrinol. 1988; 7: 21-27Google Scholar). Only the characterization of the α-NE precursor would demonstrate precisely if a prodynorphin-like gene existed in annelids. Our discovery of the α-NE confirms the very ancient stature of opioids in metazoans and the hypothesis emitted by Stefano et al. (1989) that the highly regulated vertebrate immune system probably had its origin in the invertebrates. These molecules would be used since the beginning of the evolution to start a type of integrated reply in order to maintained the body homeostasis (14Franchesi C. Ottaviani E. J. Immunol. Res. 1992; 4: 53-55Google Scholar) and notably in neuroimmunity reactions (39Stefano G.B. Cell. Mol. Neurobiol. 1992; 12: 357-366Crossref PubMed Scopus (45) Google Scholar), as well as documented neuroregulatory actions (20Kream R.M. Zukin R.S. Stefano G.B. J. Biol. Chem. 1980; 255: 9218-9224Abstract Full Text PDF PubMed Google Scholar; 37Stefano G.B. Cell. Mol. Neurobiol. 1982; 2: 167-178Crossref PubMed Scopus (85) Google Scholar). We are indebted to Dr. J. Hoffmann (Institut de Biologie Moléculaire et Cellulaire, UPR 9022 CNRS, Strasbourg, France) for facilities provided to us for the peptide sequencing. We also thank Dr. A. Van Dorsselaer (Laboratoire de spectrométrie de masse bioorganique, UA 31 CNRS, Strasbourg, France) for the mass spectrometry determination." @default.
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