The distribution of various amines and polypeptides of the skin in 90 species of five frog families show general agreement with classifications based on other criteria. In particular, the skin biochemistry generally supports the subgeneric divisions of Leptodactylus based on secondary sex characters; the isolated position of Odontophrynus among the Cerato- phryidae indicated by serological evidence is supported; Phyllomedusa differs strongly from other hylid genera in its possession of large amounts of polypeptides; and the presence of bufotenine in Melanophryniscus confirms the assignment of the genus to the Bufonidae. There is great variation within and between species of Bufo in amounts of amines of the skin.Material digitalizado en SEDICI gracias a la colaboración del Dr. Jorge Williams (FCNM-UNLP).Facultad de Ciencias Naturales y Muse

Cei, José Miguel Alfredo María

Erspamer, V.

English

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Made in United States of AmericaReprinted from Copeia, 1966, No. 1, March 22 pp. 74-78ÇQIECCION HERPETOLOGYYBIBLIOTECADr José Migu&l CeiBIBLIOTECAJORGE D. WILLIAMSBiochemical Taxonomy of South American Amphibians by Means of Skin Amines and PolypeptidesJose M. Cei and Vittorio Erspamer8IBLI0TECAJCRGE D. WIL LIAMSBiochemical Taxonomy of South American Amphibians by Means of Skin Amines and PolypeptidesJose M. Cei and Vittorio ErspamerThe distribution of various amines and polypeptides of the skin in 90 species of five frog families show general agreement with classifications based on other criteria. In particular, the skin biochemistry generally sup­ports the subgeneric divisions of Leptodactylus based on secondary sex characters; the isolated position of Odontophrynus among the Cerato- phryidae indicated by serological evidence is supported; Phyllomedusa dif­fers strongly from other hylid genera in its possession of large amounts of polypeptides; and the presence of bufotenine in Melanophryniscus confirms the assignment of the genus to the Bufonidae. There is great variation within and between species of Bufo in amounts of amines of the skin.IntroductionATTENTION has been focused in the past decade on comparative biochemistry as capable of offering keys to the solution of problems of evolution and classification as well as elucidating problems of general bio­chemistry. Wittliff, for example, used amines and other skin secretions of toads to dis­tinguish species and species hybrids (Wittliff, 1962, 1964). For the past four years we have been carrying out a systematic study of amines and polypeptides present in skins of am­phibians of South and Central America. Re­sults of this survey appear to have important taxonomic implications as well as being of biochemical and pharmacological interest (identification of at least five new phar­macologically active polypeptides and about 10 new naturally occurring amines).The different anuran groups represent exceptional material for biochemical com­parisons because of the large number of species included. Data include analyses of indole-, phenyl-, and imidazolealkylamines; polypeptides studied are principally those which have physiological effects upon smooth muscle preparations. Sex differences or sea­sonal variation were not evidenced at the present stage of our comparative researches.Materials and MethodsAnimals examined included 90 species dis­tributed among five families of neotropical anurans: Leptodactylidae, Ceratophryidae, Hylidae, Bufonidae, and Ranidae.Collection and extraction of the skins.—The skins, removed from the animals immediately after sacrifice, were either extracted in loco with three to four parts of methanol (w/v) or spread out and dried in the shade. Dried skins were extracted in the laboratory with eight to 10 times their weight of 70% ace­tone and 80% methanol. Methanol was nec­essary to extract active polypeptides; generally acetone was best for the extraction of the alkylamines since it dissolves less inert ma­terial. However, the best procedure for ex­tracting specific active compounds had to be established in each case by experiment. Skins were soaked for a week in the solvent, the extract was decanted, and the skins were re­extracted for another week with the same solvent. Extracts were pooled, filtered, and stored in a refrigerator in dark bottles. Such crude extracts do not lose activity if stored for months or even years.Chromatographic analysis.—After evaporating the solvent and defatting with petroleum CEI AND ERSPAMER—FROG SKIN SUBSTANCES 75ether, extracts were chromatographed on Whatman No. 1 paper, using an ascending technique. Solvents employed were n-butanol: acetic acid : water (40 : 10 : 50) and n-bu­tanol : 30% methylamine (80 : 30). Chromato­grams were developed with the following reagents: diazotized sulphanilic acid (Pauly reagent), diazotized /»-nitroaniline, Heinrich and Schuler’s NNCD reagent (2-chloro-4- nitro-l-diazobenzene-a-naphthalene sulphuric acid), dichloroquinone-chlorimide reagent (Gibbs’ reagent), /»-dimethylaminobenzalde- hyde reagent, Folin reagent for amino acids (1, 2-naphthoquinone-4-sulphonic sodium salt), and ninhydrin reagent. Crude estimates of the concentration of constituents in ex­tracts were made by visual comparison of the intensity of spots on developed chro­matograms with spots produced by known amounts of corresponding synthetic com­pounds. For quantitative estimations, frac­tions were eluted from chromatograms into test tubes. Fractions and standards were treated with appropriate reagents and color intensities compared in a spectrophotometer. Bioassay.—Biological activity of all the ex­tracts was assayed on the following prepara­tions: rabbit large intestine, rat large intes­tine, rat estrous uterus, guinea pig ileum, frog rectus abdominis muscle. Effects on blood pressure and respiration were assayed using dogs. Diuretic effects were measured on the rat. Earlier reports (Erspamer, 1959a, 1959b; Anastasi and Erspamer, 1963) cited details of procedures employed in purifica­tion, isolation, and identification of biologi­cally active substances discovered during this survey.Standards.—The following synthetic com­pounds were available for chromatographic and bioassay standards: 5-hydroxytryptamine creatinine sulfate (5-HT), N-methyl-5-HT creatinine sulfate, bufotenine base, dehydro­bufotenine base, 5-hydroxyindoleacetic acid, tryptamine hydrochloride, indole acetic acid, tryptophan, 5-hydroxytryptophan, jb-tyra- mine, m-tyramine, /j-tyrosine, m-tyrosine, o- tyrosine, candicine iodide, leptodactyline pic­rate, histamine dihydrochloride, N-methylhis- tamine dihydrochloride, N,N-dimethylhista- mine dihydrochloride, spinaceamine dihydro­chloride, 6-methylspinaceamine dihydrochlo­ride, histidine, bradykinin (Sandoz), eleidosin (Farmitalia), hypertensin (CIBA) and vaso­pressin (Sandoz) .Results and DiscussionLeptodactylidae.—Skin extracts of primitive Leptodactylidae either lack or contain only scanty amounts of biologically active polypep­tides and amines. Genera examined include Elosia (Elosiinae), Calyptocephalella (Ca- lyptocephalellinae), Eleutherodactylus (Eleu- therodactylinae), Telmatobius and Batra- chophrynus (Telmatobiinae), and Thoropa, Cycloramphus, and Eupsophus. Maximum values of leptodactyline and 5-HT occurred in Calyptocephalella gayi and Thoropa mili- aris (10-12 p,g/g of dry skin).The Leptodactylinae include species which virtually lack and others which contain high concentrations of active polypeptides and amines. The genus Pleurodema, for exam­ple, contains little leptodactyline (2-25 p,g/g of dry skin), occasionally 5-HT (200-250 pg/g in the coxal glands of Pleurodema bu- fonina), but lacks biologically active poly­peptides. Physalaemus, while virtually lack­ing amines, contains large amounts of physa- laemin, an eleidosin-like polypeptide, which potently lowers blood pressure and stimu­lates extravascular smooth muscle (Erspamer, Bertaccini, and Cei, 1962).Skin of true Leptodactylus lacks poly­peptides but contains a formidable array of amines (Table 1). The spectrum of amines present in different species is in fair accord­ance with the scheme of subgeneric division suggested by Miranda-Ribeiro (1926) and Lutz (1930). The Cavicola species group (i.e., Leptodactylus prognathus, L. bufonius, L. sibilatrix) lacks amines; the Platimantis group (L. podicipinus, L. melanonotus) contains large amounts of leptodactyline and indole- alkylamines. Using amine content of their skins, the Pachypus species group may be dif­ferentiated into two groups in full accord­ance with morphological characteristics such as the stout forearm and sex characters. One group, represented by L. ocellatus, L. chaquensis, and L. bolwianus, is rich in phenylalkylamines (up to 9,000 pg leptodacty­line base per g of dry tissue), but lacks indolealkylamines and imidazolealkylamines. The second group, represented by L. penta­dactylus and L. laticeps, both characterized by peculiar horny pads on the chest of the males, is poor in phenylalkylamines but rich in indolealkylamines and, what is more un­usual, in imidazolealkylamines. These in­clude, not only widely distributed histamine, but also the less common and poorly known76 COPEIA, 1966, NO. 1T5815<L>bJD si.c8cdÏ0>^5ccdOH E 1Òo©©oo©o©oo? Ï4< coo©©©oo©oo©o © co©©o©©ÌOco r>©oro oroorI o oro©©© oro or s o© oooooo©ooio coTt<io *7i—I I—< © r-H©©oo 00 orI o © orCO I or©©©©© © 0000I © 00©o©oo© GOI1O ÌOo©©©©©00 Tt<©©©©©© ©orCEI AND ERSPAMER—FROG SKIN SUBSTANCES 77Table 2. Content of Indolealkylamines (Mg of free bases/g dry skin) in South American Bufo.1Bufo species 5-HTN-Methyl-5-HTBufote­nineBufoteni- dineDehydro- bufotenineBufo- thionineB. marinusm. marinusSurinamParamaribo I) 100 130 4 0 3,000 350-400II) 135 100 n.d. 0 6,000 15-30Costa Rica 100 50 n.d. 0 2,200 260m. poeppigiBolivia( Coroico )Perù150-180 120-170 n.d. n.d. 1,100 230(Tingo Maria) 150-170 80-120 n.d. n.d. 270 230B. ictericusBrasil (Rio)Argentina10-20 10-20 n.d. 0 4,500 250(Misiones) 50-70 40-60 15 0 3,300 375B. paracnemisArgentina (Formosa) 70 80 4,300 180 180 290(Tucuman) 25 0 2,800 220 120 110(Corrientes) 10-20 0 5,000 650 250 300(Laguna Ibera) 35 0 4,500 400 270 300( Misiones) 100 200 2,500 250 1,500 360B. arenarumArgentina(Tucuman) 60-600 60-400 1,100-2,600 40-60 550-700 250-450(Cordoba) 40-120 30-100 800-1,600 50 230-600 70-170(Mendoza) 35-75 0 1,200-2,100 65-130 350-600 220-450B. spimi losuss. spinulosusPerù, Cuzco n.d. n.d. 220-250 30 180 830s. aruncoCibile, Santiago n.d. n.d. 15-20 230 360s. trifoliusPerù n.d. n.d. 500-600 100 n.d. n.d.lì. gramilosusg. majoì'Argentina-FormosaChaco-60 20-25 140 0 n.d. 300Resistencia-S2 250 130 200 0 550 780g. fernandezaeArgentinaChaco-Resistencia-S- 150 500 150 0 2,400 1,150Buenos Aires 60 330 150 0 1,200 9001 n.d. = not detectable.2 S = sympatric populations.78 COPEIA, 1966, NO. 1N-methylhistamine and N,N-dimethylhista- mine, and the hitherto unknown spinacea- mine and 6-methylspinaceamine. Further, a clear-cut biochemical distinction can be made between three subspecies of L. pentadactylus (Table 1).Ceratophryidae.—Skins of this peculiar neo­tropical stock of anurans are very poor in indolealkylamines, with the exception of the genus Odontophrynus (O. americanus, O. oc- cidentalis: 5-HT, 310 to 2,600 ^g/g of dry skin). Serologically this genus lies far away from other studied representatives of this ancient anuran branch (Cei, 1965).Hylidae.—Among the Hylidae, the genera Hyla, Sphoenorhynchus, Osteocephalus, Trachycephalus, Corythomantis, probably Triprion, and Gastrotheca, lack or are poor in amines and polypeptides. However, species of the highly specialized genus Phyllomedusa (Phyllomedusa sauvagii, P. hypochondrialis, P. annae, P. rohdei, P. burmeisteri, P. calli- dryas, P. dacnicolor) contain large amounts of powerful bradykinin-like and physalaemin- like polypeptides. To date, at least five active polypeptides have been identified and it seems possible that each species of Phyl­lomedusa has a characteristic polypeptide spectrum. A strikingly specialized position of Phyllomedusa as an independent hylid branch is suggested by this material (cf. Cei, 1963).Bufonidae.—Skins of Bufo lack biologically active polypeptides, but contain phenylalkyl­amines and large quantities of a variety of indolealkylamines (Table 2). Marked differ­ences in the spectrum of indolealkylamines occur among different species, and occasion­ally even between populations from different geographic areas. Melanophryniscus moreirae skin contains a new hydroxyphenylalkyla­mine, as well as considerable bufotenine (more than 2,000 ¿¿g/g of dry skin). The presence of bufotenine confirms the relation­ship of Melanophryniscus with other bufonid genera as suggested, on a morphological basis, by Griffiths (1959) and other herpetol­ogists.Ranidae — The few species of Central and South American Ranidae examined were similar to European and Japanese Ranidae in that their skins also contained indolealkyl­amines (5-HT) and bradykinin-like poly­peptides.ConclusionsIt appears as if the amines and poly­peptides present in amphibian skin offer valu­able biochemical evidence for determining the positions of suprageneric taxa, and also for the solution of controversial problems concerning interspecific and subspecific rela­tionships. As yet it is not possible to state the adaptive and biological significance of these pharmacologically active compounds.We wish to extend sincere thanks to Dr. H. C. Dessauer (Louisiana State University, New Orleans) for his courtesy and assistance in rewriting the manuscript. This investiga­tion was supported by grants from the Con­siglio Nazionale delle Ricerche, Roma, and the Rockefeller Foundation, New York.Literature CitedAnastasi, A. and V. Erspamer. 1963. The isola­tion and amino acid sequence of eleidosin, the active endocapeptide of the posterior salivary glands of Eledone. Arch. Biochem. Biophys. 101:56-65.Cei, J. M. 1963. Some precipitin tests and pre­liminary remarks on the systematic relation­ships of four South American families of frogs. Serol. Mus. Bull. 30:4-6.-------- . 1965. The relationships of some cera- tophryid and leptodactylid genera as indicated by precipitin tests. Herpetologica 20:217-224. Erspamer, V. 1959a. Isolation of leptodactyline (m-hydroxyphenylethyltrimethylammonium) from extracts of Leptodactylus skin. Arch. Biochem. Biophys. 82:431-438.-------- . 1959b. Isolation of bufoviridine from the skin of Bufo viridis and its identification as di- hydrobufothionine. Biochem. Pharm. 2:270- 275.--------, G. Bertaccini, and J. M. Cei. 1962. Occurrence of an eledoisin-like polypeptide (Physalaemin) in skin extracts of Physalaemus fuscumaculatus. Experientia 18:562-563.Griffiths, I. 1959. The phylogeny of Sminthil- lus limbatus and the status of the Brachycephal- idae (Amphibia, Salientia) . Proc. Zool. Soc. London 132:457-487.Lutz, A. 1930. Segunda memoria sobre especics brasileiras do genero Leptodactylus incluindo outras aliadas. Mem. Inst. O. Cruz 23:1-20.Miranda-Ribeiro, A. 1926. Notas para servir ao estudo dos Gymnobatrachios brasileiros. Arch. Mus. Nac. Rio Janeiro 27:1-227.Wittliff, J. L. 1962. Parotoid gland secretions in two species groups of toads (Genus Bufo) . Evolution 16:143-153.-------- . 1964. Venom constituents of Bufo fow- leri, Bufo valliceps and their natural hybrid analyzed by electrophoresis and chromatog­raphy. In Leone, C. A. (ed.) Proc. Intern. Conf, on Taxonomic Biochem., Physiol, and Serol. Ronald Press, New York.Institute of Biology, University of Cuyo, Mendoza, Argentina, and Institute of Pharmacology, University of Parma, Parma, Italy.

Biochemical Taxonomy of South American Amphibians by Means of Skin Amines and Polypeptides

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Biochemical Taxonomy of South American Amphibians by Means of Skin Amines and Polypeptides

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