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Biogenic amines and active peptides in extracts of the skin of thirty-two European amphibian species
Camp. Lfiochem. Physiol.
Vol. 94C, No. 2, pp. 455460, 1989 Pergamon Press plc
Printed in Great Britain
BIOGENIC AMINES AND ACTIVE PEPTIDES IN EXTRACTS OF THE SKIN OF THIRTY-TWO EUROPEAN AMPHIBIAN SPECIES M. ROSEGHINI,G. FALCONIERIERSPAMER,*C. SEVERINIand M. SIMMAcot Institute of Pharmacology III, Institute of Pharmacology and Pharmacognosy, Department of Biochemical Sciences and CNR Centre for Molecular Biology, and tuniversity “La Sapienza”, I-00185 Rome, Italy Telephone: 06/4457094 (Received 2 June 1989)
Abstract-l.
Extracts prepared from fresh or dried skins of 32 European amphibian species were submitted to chemical (colour reactions) and biological screening to determine the occurrence and contents of biogenic amines and peptides active on smooth muscle preparations and blood pressure. 2. Only indolealkylamines were detectable in the skins. They were represented by tryptamine, 5hydroxytryptamine, and its N-methylated, cyclized and sulphoconjugated derivatives. 3. The peptide families identified in the extracts were as follows: bombesins (bombesin and alytesin), bradykinins (bradykinin, bradykinin 1-8, bradykinin l-7), chemotactic peptides (RECP I, II and III), bombinins and TRH. Bombesins, bombinins and TRH (thyrotropin-releasing hormone) were isolated from skin extracts of discoglossid frogs; chemotactic peptides and again TRH from extracts of ranid frogs. 4. Further research will certainly lengthen the list of active peptides in the skin of European amphibians, as is the case with Australian, American and African amphibians.
MATERIALSAND METHODS
INTRODUCTION The occurrence and distribution of biogenic amines and active peptides in the skin of 100 amphibian species from Australia and Papua New Guinea, in more than 200 American and in 50 African species have been described in preceding papers (Roseghini et al., 1976, 1986, 1988; Erspamer et al., 1984; Erspamer et al., 1986). The purpose of this paper is to report our research on aromatic amines and active peptides occurring in skin extracts from 32 European amphibian species. Biogenic amines considered in our study were indole-, imidazole- and phenylalkylamines; active peptides were essentially those active on isolated and in situ smooth muscle preparations (intestine, gall bladder, uterus, urinary bladder) and systemic blood pressure. Thus, peptides included in the screening were chiefly those belonging to the bombesin, tachykinin, bradykinin and caerulein families. The presence of dermorphin, sauvagine and tryptophyllins was checked only in few selected species, i.e. in those rich in bombesins and bradykinins. Thyrotropin releasing hormone (TRH) and bombinins, identified in amphibian skin extracts by other research workers and chemotactic peptides (unpublished data by our group) are discussed to complete information. *Address for correspondence: G. Falconieri Erspamer, Istituto di Farrnaclogia e Farmacognosia, Universitl “La Sapienza”, Piazzale Aldo Moro 5, I-00185 Roma, Italy. CBPK) 9412-H
Amphibian material
The amphibian material examined in this study is listed in Table 1. Methanol extracts from fresh and dried skins were prepared in the customary way (Roseghini et al., 1976, 1986; Erspamer et al., 1984, 1986). Chromatography on paper and on alumina columns, colour reactions and bioassay systems
Methods used in the detection, separation and semiquantitative estimation of indole-, imidazole-, and phenylalkylamines were essentially the same as those reported in detail in a preceding paper (Roseghini et al., 1976). Similarly the purification procedures and bioassay systems employed in the study of peptides were the same as those previously described (Erspamer et al., 1984, 1986). The same limits of reliability of bioassay methods are also valid. Reagents and drugs
The following amines and peptides were available for comparison: in paper chromatography and bioassay: 5hydroxytryptamine creatinine sulphate, N-methyl-5hydroxytryptamine HCl, bufotenine picrate, bufotenidine picrate, bufoviridine, bufotenine 0-sulphate, caerulein, physalaemin, sauvagine and dermorphin (prepared or synthesized at the Farmitalia Carlo Erba Research Laboratories, Milan, Italy); tryptamine HCl, 5-hydroxyindoleacetic acid, bradykinin, [Des-Argq bradykinin, ranatensin (purchased from Sigma Chemical Co. St. Louis, M., U.S.A.). Natural bufothionine and dehydrobufotenine were prepared by ourselves from skin extracts of Bufo bufo and Eufo marinus. Similarly ala[Asp’] deltorphin was synthesized in our laboratory. Reagents and solvents used throughout the investigation were of analytical grade. 455
456
M. ROSZGHINI et af. PRIMARY AMINES
- CH, - CH,
5-HYDROXYTRYPTAMINE I!!!-HTI
TRYPTAMINE
N-METHYLATED DERIVATIVES
-CHz -TH2
CXJ
Ho’ ’
NH.CH3
’
-CH2-c;H3)2
0-J ct
-O’1
N Ii
BUFOTENINE
N-METHYL-5-HT
‘-CH2;~;YH3)3
BUFOTENIDINE
CYCLIZED DERIVATIVES
HZ
I
4%
H2
/C\
(CH,),-N
A\
CH, I
(CH,),-
eo \
N I
CH2 1
O,SH-O-
1’ IY
DEHYDROBUFGTENINE
CONJUGATED DERIVATIVES
HO-
03SH--O
BUFOTENINE 0-SULPHATE
BUFOVIRIDINE (BUFOTENINE 1-SULPHONlC AClOt
Fig. 1. The indolealkylamines in the skin of European amphibians.
RESULTS
on paper of the eluates and spraying of the chromatograms with suitable reagents makes it easy to separate and identify the different amines (Fig. 2).
The only category of aromatic biogenic amines occurring in the skin of the examined European amphibians was that of indolealkylamines, represented by their prototypes tryptamine and 5-hydroxytryptamine(SHT) and by N-methylated, cyclized and conjugated derivatives of S-HT (Fig. I). Amines in the Bufo skin. As shown in Table 1 as many as seven hydroxyindolealkylamines could be detected in the skin of the European toads, often in enormous amounts. A simple passage of the crude extracts through an alumina column, eluted with descending concentrations of ethanol, with subsequent chromatography
Amines in the skin of amphibians other than bufonids. Indolealkylamines occurred in the skin of
Biogenic antines
nearly all the examined species of Anura and in some species of Caudata (Table 2). The skin of the Salamandra species contained not only small amounts of 5-HT but also high concentrations of the rather unusual tryptamine; the Hydromantes skin, in turn, lacked tryptamine but contained 5-HT and its N-methylated derivatives bufotenine and bufotenidine. The figures in Table 2 refer to the total body contents; if calculated for the fresh skin they would have been approximately ten times greater.
457
Biogenic amines and peptides in amphibian skin Table 1. lndolealkylamines Species Source, date of capture, number of skins, average weight of a dry (D) or fresh skin (F) Bufo bufo bufo France, VIII. 1967, 7 skins (D,
5-HT
I .4 g)
Bufo bufo spinosus (a) Italy, IV. 1964, 7 skins F, 4.6 g) (b) Ibidem, IV. 1966, 15 skins (F, 8.2 g) (c) Ibidem, IV. 1967, 10 skins (F, 8.7g) Bufo calamita (a) Germany, X. 1954, 5 skins (F, 2.1 g) (b) Ibidem, VI. 1967, 3 skins (F, 3.4g) (c) Ibidem, X. 1967, 46 skins (D, 1.3 g)
N-Methylated derivatives M-S-HT BT BTD
Cyclized deriv. DH-BT
BTH
Conjugated deriv. BV BT-OS
10
0
175
215
700
1000
0
0
20 10 10
0 0 0
450 170
50 55
130 100
260 320
0 0
0 0
250
80
80
180
0
0
105 500 220
35 20 100
0 0 0
0 0 0
110 750 950
50 30 150
650 1000 800 2350 2000
30 80 70 850 450
0 0 0 0 0
0 0 0 0 0
480 500 400 2550 1900
0 ? ? ? ?
14
Bufo uiridis (a) Italy, IV. 1959, 150 skins (F, 3.1 g) (b) Ibidem, X. 1967, 116 skins (F, 1.8g) (c) Ibidem, V. 1970, 200 skins (F, 1.75 g) (d) Israel, VIII. 1967, 6 skins (D, 1.1 g) (e) Israel, V. 1970, 200 skins (F, 1.75 g)
@g/g tissue) in the skin of the four European toads
2:
15
40
10 15 20 65 40
25 1: 0 0
5-HT, 5-hydroxytryptamine; M-5-HT. N-methyl-S-HT. BT, bufotenine; BTD, bufotenidine; DH-BT, dehydrobufotenine; BV, bufoviridine; BT-OS, 0-sulphate of BT
Concerning the Anura, 5-HT was present, often in large amounts, in the skin of all examined discoglossid, ranid and hylid frogs; in the ranid frogs it was often accompanied by bufotenine and bufotenidine. Peptides
Representatives of five distinct peptide families have been so far identified in the skin of European amphibians: bombesins, bradykinins, chemotactic peptides (RECP, Rana esculenta chemotactic peptides), haemolytic peptides (bombinins) and TRH (Table 3). Their primary sequences are shown below:
BTH, bufothionine;
while they were apparently lacking, at least in active forms, in the skin of other frogs. The chemotactic peptides RECP were isolated from skin extracts of Rana esculenta; bombinin and its fragments from the skin of Bombina variegata; finally TRH from skin extracts of Rana ridibundu. DISCUSSION
1. Biogenic amines occurred, often in large amounts, in the skin of 22 out of the 32 examined European amphibians. They were represented by nine
I.
pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH~ pGlu-Gly-Arg-Leu-Gly-Thr-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH,
II.
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe Arg-Pro-Pro-Gly-Phe-Ser-Pro
Bombesin Alytesin
Bradykinin
Bradykinin Bradykinin
III. Phe-Leu-Pro-Ala-Ile-Ala-Gly-Ile-Leu-Ser-Gln-Leu-Phe-NH, Phe-Leu-Pro-Leu-Ile-Ala-Gly-Leu-Leu-Gly-Lys-Leu-Phe-NH* Phe-Leu-Pro-Leu-Leu-Ala-Gly-Leu-Ala-Ala-Asn-Phe-Leu-Pro(Lys, Ile)-Phe-NH,
l-8 l-7 RECP I RECP II ’ RECP III
IV. Gly-Ile-Gly-Ala-Leu-Ser-Ala-Lys-Gly-Ala-Leu-Leu-G~y-LeuLys-Gly-Leu-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asp-NH~ Bombinin Ser-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe [Ser’] Bombinin 18-25 Gly-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe [Gly’] Bombinin 18-25 Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asp-NH, Bombinin 19-27 V.
pGlu-His-Pro-NH,
TRH
Bombesins are represented by bombesin and alytesin, which occurred in high concentrations in the skin of the discoglossid frogs of the genera Bombina and Alytes, respectively, but were completely lacking in frogs of the Discoglossus genus. Bradykinins (authentic bradykinin l-9 and fragments l-8 and l-7) could be detected in extracts of the skin of several frogs (high concentrations were found especially in the skin of Rana temporaria),
different members, all belonging to the indolealkylamines and, except tryptamine, all deriving from 5-HT, by N-methylation, cychzation and 0- or Nsulphoconjugation of its molecule. It should be stressed that bufoviridine (bufotenine I-sulphonic acid) constituted the first example known in nature of a compound in which sulphuric acid was conjugated with the pyrolic nitrogen of the indole nucleus.
M. ROSEGHINI et al.
458
Table 2. Indolealkylamines (pg/g tissue) in the skin of European amphibians other than bufonids Species Source, date of capture, number of skins, average weight of a dry(D) or fresh (F) skin
T
5-HT
BT
BTD
0
2
6
2
0
0
0
0
0
0
0
0
3
5
0
0
55
IO
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
220
0
0
0 0 0
350 400 330
0 0 0
0 0 0
0
280
0
0 0
1100 1200
0 0
I. URODELA
PLETRODONTIDAE Hydromantes
italicus
Tuscania, Italy, XII. 1964, 8 total bodies (F, 2.8 g) SALAMANDRIDAE Euprocres platycephalus
Sardinia, Italy, IX. 1952, 3 total bodies (F, 2.5g) Pleurodeles
waltli
Spain, X. 1958, 3 total bodies (F, 0.19g) Salamandra
atra
North Italy, IX. 1963, 35 skins (F, 1.5 g) Salamandra
salamandra
North Italy, IX. 1963, 22 skins (F, 5.5 g) Triturus alpestris
Switzerland, V. 1967, 5 total bodies (F, 3 g) Triturus boscai
Spain, I. 1968, 4 total bodies (F, 1.1 g) Triturus carnifex
Italy, VII. 1972, 6 total bodies (F. 3.2 g) Triturw eristatus
Italy, VI. 1975, 5 total bodies (F, 3 g) Triturus marmoratus
Spain, V. 1967, 2 total bodies (F, 5.4g) Triturus vulgaris
North Italy, IV. 1964, 15 total bodies (F, 1.9 g) JJ. ANURA DISCOGLOSSJDAE Alytes cisternasi
Portugal, IV. 1974, 13 skins (D, 0.11 g) Alytes obstetricam (a) France, X. 1968, 30 skins (F, 0.58 g)
(b) Jbidem, XI. 1968, 71 skins (F, 0.48 g) (c) Jbidem, IX. 1969, 186 skins (F. 0.8 g) Alytes obstetricam
Portugal, V. 1974, Bombina
boscai
I I skins (D, 0.1 Ig)
bombina
(a) Germany, VI. 1968, 500 skins (F, 0.64 g) (b) Ibidem, VI. 1969, 233 skins (F, 0.48 g) Bombina variegata pachypus (a) South Italy, IV. 1953, 655 skins (F, 2.4g)
0 0
150 300
0 0
0 0 0
520 600 500
0 0 0
0 0
350 440
0 0
0
380
0
0
0
0
0
0
90
0
0
Yugoslavia, XI. 1963, 5 skins (D, 0.55 g)
0
JO0
2000
200
Ram (a) (b) (c) Raw
(F, 2.5 g) 2.8 g) (F, 2.6 g)
0 0 0
20 40 40
0 0 0
0 0 0
0.5 g)
0
0
0
0
0 0
15 7
120 60
0 0
0 0
10 20
15 100
15 15
(b) Jbidem, 1986-1988, 30 skins (F, 1.6g) Bombina uariegata variegata (a) France, VI. 1968, 10 skins (F, 1.3g) (b) Ibidem, VI. 1968, 50 skins (F, 1.3 g) (c) Jbidem, IX, 1969, 403 skins (F, 1.25 g) Discoglossus pictus (a) Sicily, III. 1950, 480 skins (F, 1.8 g)
(b) Jbidem, III. 1951, 102 skins (F, 1.6g) Discoglossus sardus Sardinia, Italy, V.1951, 210 skins (F, 1.7g) PELOBATIDAE P&bates cultripes Portugal, JV. 1971, 12 sins (D, 0.5 g) HYLIDAE Hyla arborea
North Italy, VIII. 1953, 50 skins (F, 0.46g) RANIDAE Rana dalmatina esculenta Parma, Italy, V. 1961, 800 skins Ibidem, V. 1962, 1100 skins (F, South Italy, 1986-1988, 82 skins graeca Tuscania, Italy, IV. 1967, 10 skins (F, Rana iberica
(a) Portugal, X. 1970, 3 skins (D, 0.23 g) (b) Ibidem, V. 1971, 30 skins (D, 0.1 g) Ram latastei (a) Cremona, Italy, IX. 1963, 24 skins (F, 1.3 g)
(b) Ferrara, Italy, IX. 1962, 5 skins (F, 1.9~) Ram ridibunda
Portugal, VII. 1971, 38 skins (D, 0.65 g)
0
25
0
0
Ram temporaria (a) South Germany, IX. 1963. 560 skins (F, 5.8 g) (b) North Italy, IX. 1962, 10 skins (F, 1.9 g)
0
120 _.
7
0 25
T, tryptamine; 5-HT. 5-hydroxytryptamine;
BT, bufotenine;
0 BTD, bufotenidine
20
100
459
Biogenic amines and peptides in amphibian skin
08
06
99
95, 95* 90, 902 80, 802 70, 702 60 ETHANOL
50
40
30 20 Hz0
ELUATE
Fig. 2. Schematic representation of elution profile from alumina columns (descending concentrations of ethanol) and Rf values of indolealkylamines occurring in skin extracts of European bufonids. Solvent: butanol-acetic acid-water; spraying reagents: NNCD and Ehrlich reagents. Black spots: BT, bufotenine; 5-HMT, S-hydroxy-r+methyl-tryptamine; BTD, bufotenidine; S-HT, 5-hydroxytryptamine; 5-HIAA, S-hydroxyindole-acetic acid; TRP, tryptophan. Hatched spots: cyclized (BTH, bufothionine; DH-BT, dehydrobufotenine) and sulpho-conjugated derivatives (B-VIR, bufoviridine; BT-0-SULPH, bufotenine 0-sulphate).
N-methylation occurred also in ranid frogs and even in Caudata. Of interest was the fact that the skin of the B. viridis specimens collected in Israel (B. viridis arabicus?) was considerably richer in amines than that of specimens collected in Europe (B. viridis viridis?) and that 0-sulphate of bufotenine was very abundant in B. calamita but not in B. viridis.
Both cyclization and conjugation produced a complete loss of activity, at least in the periphery, whereas N-methylation caused a progressive decay of 5-HT activity, with the appearance, however, of clear cut nicotinic effects in bufotenidine, its quaternary ammonium derivatives (unpublished observations). Cyclization and sulphoconjugation of hydroxyindolalkylamines seemed to be peculiar to toads;
Table 3. Active peptides in the skin of European discoglossid and ranid frogs @g/g tissue) Species
Bon&sins*
Alytes cistern&i Alytes obstetricans
Batch (a) Batch (b) Batch (c) Alytes obstetricans boscai Bombina bombina
Batch (a) Batch (b)
Bradvkinins*
Other Peutides
80
n.d.
900-1000 500-650 500-700 350
n.d. n.d. n.d. n.d.
200-300 300400
n.d. n.d.
250-350 500-650 500-700 300
n.d. n.d. n.d. n.d.
n.d. n.d. n.d.
n.d. n.d. n.d.
n.d.
30
Chemotactic peptides
n.d. n.d. n.d. n.d.
3tL35 n.d. n.d. 20-25
TRH
n.d. n.d.
200-250 100
Bombina uariegata uariegata
Batch (a) Batch (b) Batch (c) Bombina uariegata pachypus
Batch (b) Discoglossus pictus Discoglossus sardus Rana dalmatina Rana esculenta
Batch (c) Rana iberica
Batch (b) Rana Rana Rana Rana
graeca latastei ridibunda temporaria
Batch (a) Batch (b)
*Expressed in terms of bombesin and bradykinin, thyrotropin releasing hormone
respectively; n.d., not detectable (co.5 cg/g); TRH,
460
M. ROSEGHINI et al.
Representatives of five peptide familes have been so far isolated from the skin of European amphibians: bombesins, bradykinins, chemotactic peptides, haemolytic peptides and TRH. Bombesin and alytesin occurred in large amounts in the skin of discoglossid frogs of the genera Bombina and Alytes, respectively, but were lacking in the genus Discoglossus. They must be considered the prototypes of the bombesin family, now comprising as many as fourteen amphibian peptides and five avian and mammalian counterparts. The presence of ranatensin in the skin of European ranid frogs is possible but, if so, only in minute amounts (CO.5 pg/g). Authentic bradykinin was isolated from the skin of Rana temporaria (Anastasi, Erspamer and Bertaccini, 1965) and Rana esculenta, in the latter species together with its N-teXTnina octapeptide (bradykinin 1-8) and bradykinin l-7. The first fragment showed a sharply varying degree of activity in different bioassay systems (unpublished observations), the second was completely inactive. Active bradykinin forms were not detectable in skin extracts of R. dalmatina, R. graeca and R. latastei, whereas extracts from R. iberica and R. ridibunda showed a clear-cut bradykinin activity. On the basis of its elution profile from alumina and HPLC columns, this activity is attributable both to authentic bradykinin and bradykinin l-8. Chemotactic peptides, i.e. peptides displaying a strong chemotactic activity on macrophages, were first detected in skin extracts of Rana erythraea from the Philippines (Yasuhara et al., 1986) Now, Barra et al. (to be published) have isolated analogous peptides from the skin of Rana esculenta, indicating that this peptide family may have a widespread distribution in ranids and perhaps also in other amphibian families, where they may contribute to the immunological defence of the organism against microbial attack. The bombinins were isolated and sequence from skin extracts of Bombina variegata. The main property they possessed was a strong haemolytic activity (Csordas and Michl, 1969, 1970). Interestingly, skin extracts from Bombina variegata pachypus displayed also evident antimicrobial effects (Croce et al., 1973). Finally, TRH first isolated from skin extracts of Bombina orientalis (Yajima et al., 1975) and then of Rana pipiens (Jackson and Reichlin, 1977) has also been detected in the skin of Rana ridibunda (Ravazzola et al., I%)), again suggesting a widespread occurrence of the peptide in ranid and perhaps discoglossid frogs. Peptides belonging to other very important amphibian peptide families, such as tachykinins, caeruleins, sauvagine, tryptophyllins, dermorphins and deltorphins (Kreil et al., 1989; Erspamer et al.,
1989) were apparently European amphibians.
lacking in the skin of the
Acknowledgements-This work was supported by grants
from the Consiglio Nazionale delle Ricerche, Roma. REFERENCES Anastasi A., Erspamer V. and Bertaccini G. (1965) Occurrence of bradykinin in the skin of Rana temporaria.Comp. Biochem. Physiol. 14, 43-53. Croce G., Giglioli N. and Bolognani L. (1973) Antimicrobial activity in the skin secretions of Bombina uariegata pachypus. Toxicon 11, 99-100. Csordas A. and Michl H. (1969) Primary structure of two oligopeptides of the toxin of Bombina variegata L. Toxicon 7, 101-108. Erspamer V., Falconieri Erspamer G., Mazzanti G. and Endean R. (1984) Active peptides in the skin of one hundred amphibian species from Australia and Papua New Guinea. Comp. Biochem. Physiol. 77C, 88-108. Erspamer V., Falconieri Erspamer G. and Cei J. M. (1986) Active peptides in the skin of two hundred and thirty American amphibian species. Comp. Biochem. Physiol. UC,
125-137.
Erspamer V., Melchiorri P., Falconieri Erspamer G., Negri L., Corsi R., Severini C., Bossa D., Simmaco M. and Kreil G. (1989) Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for 6 opioid binding sites. Proc. Natl. Acad. Sci. USA 86, 5188-5192. Jackson I. and Reichlin S. (1977) Thyrotropin-releasing hormone: abundance in the skin of the frog, Ranapipiens. Science 198, 414415. Kiss G. and Michl H. (1962) Uber das Giftsekret der Gelbbauchunke. Toxicon 1, 33-39. Kreil G., Barra D., Simmaco M., Erspamer V., Falconieri Erspamer G., Negri L., Severini C., Corsi R. and Melchiorri P. (1989) Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for 6 opioid receptors, Eur. J. Pharmacol. 162, 123-128. Ravazzola M., Brown D., Leppaluoto J. and Orci L. (1979) Localization by immunofluorescence of thyrotropinreleasing hormone in the cutaneous glands of the frog, Rana ridibunda. Life Sci. 25, 1331-1335.
Roseghini M., Erspamer V. and Endean R. (1976) Indole-, imidazole- and phenyl-alkylamines in the skin of one hundred amphibian species from Australia and Papua New Guinea. Comp. Biochem. Physiol. 54C, 3143. Roseghini M., Erspamer V., Falconieri Erspamer G. and Cei J. M. (1986) Indole-. imidazole and uhenylalkylamines in‘the skin of one hundred and forty ‘American amphibian species other than bufonids. Comp. Biochem. Phvsiol. 85C. 139-147. Roseghini M.,* Falconieri Erspamer G. and Severini C. (1988) Biogenic amines and active peptides in the skin of fifty-two African amphibian species other than bufonids. Comp. Biochem. Physiol. 91C, 281-286.
Yajima H., Kitigawa K., Segawa T., Nakano M. and Kataoka K. (1975) Occurrence of Pvr-His-Pro-NH, in the frog skin. &em. kharm. Bull. (Tokyo) 23, 3301-3303. Yasuhara T., Nakajima T., Erspamer V., Falconieri Erspamer G., Tukamoto Y. and Mori M. (1986) Isolation and sequential analysis of peptides in Rana erythraea skin. In Peptide Chemistry 1985 (Ed. Y. Kiso), p. 363-368. Protein Research Foundation, Osaka 1986.
Vol. 94C, No. 2, pp. 455460, 1989 Pergamon Press plc
Printed in Great Britain
BIOGENIC AMINES AND ACTIVE PEPTIDES IN EXTRACTS OF THE SKIN OF THIRTY-TWO EUROPEAN AMPHIBIAN SPECIES M. ROSEGHINI,G. FALCONIERIERSPAMER,*C. SEVERINIand M. SIMMAcot Institute of Pharmacology III, Institute of Pharmacology and Pharmacognosy, Department of Biochemical Sciences and CNR Centre for Molecular Biology, and tuniversity “La Sapienza”, I-00185 Rome, Italy Telephone: 06/4457094 (Received 2 June 1989)
Abstract-l.
Extracts prepared from fresh or dried skins of 32 European amphibian species were submitted to chemical (colour reactions) and biological screening to determine the occurrence and contents of biogenic amines and peptides active on smooth muscle preparations and blood pressure. 2. Only indolealkylamines were detectable in the skins. They were represented by tryptamine, 5hydroxytryptamine, and its N-methylated, cyclized and sulphoconjugated derivatives. 3. The peptide families identified in the extracts were as follows: bombesins (bombesin and alytesin), bradykinins (bradykinin, bradykinin 1-8, bradykinin l-7), chemotactic peptides (RECP I, II and III), bombinins and TRH. Bombesins, bombinins and TRH (thyrotropin-releasing hormone) were isolated from skin extracts of discoglossid frogs; chemotactic peptides and again TRH from extracts of ranid frogs. 4. Further research will certainly lengthen the list of active peptides in the skin of European amphibians, as is the case with Australian, American and African amphibians.
MATERIALSAND METHODS
INTRODUCTION The occurrence and distribution of biogenic amines and active peptides in the skin of 100 amphibian species from Australia and Papua New Guinea, in more than 200 American and in 50 African species have been described in preceding papers (Roseghini et al., 1976, 1986, 1988; Erspamer et al., 1984; Erspamer et al., 1986). The purpose of this paper is to report our research on aromatic amines and active peptides occurring in skin extracts from 32 European amphibian species. Biogenic amines considered in our study were indole-, imidazole- and phenylalkylamines; active peptides were essentially those active on isolated and in situ smooth muscle preparations (intestine, gall bladder, uterus, urinary bladder) and systemic blood pressure. Thus, peptides included in the screening were chiefly those belonging to the bombesin, tachykinin, bradykinin and caerulein families. The presence of dermorphin, sauvagine and tryptophyllins was checked only in few selected species, i.e. in those rich in bombesins and bradykinins. Thyrotropin releasing hormone (TRH) and bombinins, identified in amphibian skin extracts by other research workers and chemotactic peptides (unpublished data by our group) are discussed to complete information. *Address for correspondence: G. Falconieri Erspamer, Istituto di Farrnaclogia e Farmacognosia, Universitl “La Sapienza”, Piazzale Aldo Moro 5, I-00185 Roma, Italy. CBPK) 9412-H
Amphibian material
The amphibian material examined in this study is listed in Table 1. Methanol extracts from fresh and dried skins were prepared in the customary way (Roseghini et al., 1976, 1986; Erspamer et al., 1984, 1986). Chromatography on paper and on alumina columns, colour reactions and bioassay systems
Methods used in the detection, separation and semiquantitative estimation of indole-, imidazole-, and phenylalkylamines were essentially the same as those reported in detail in a preceding paper (Roseghini et al., 1976). Similarly the purification procedures and bioassay systems employed in the study of peptides were the same as those previously described (Erspamer et al., 1984, 1986). The same limits of reliability of bioassay methods are also valid. Reagents and drugs
The following amines and peptides were available for comparison: in paper chromatography and bioassay: 5hydroxytryptamine creatinine sulphate, N-methyl-5hydroxytryptamine HCl, bufotenine picrate, bufotenidine picrate, bufoviridine, bufotenine 0-sulphate, caerulein, physalaemin, sauvagine and dermorphin (prepared or synthesized at the Farmitalia Carlo Erba Research Laboratories, Milan, Italy); tryptamine HCl, 5-hydroxyindoleacetic acid, bradykinin, [Des-Argq bradykinin, ranatensin (purchased from Sigma Chemical Co. St. Louis, M., U.S.A.). Natural bufothionine and dehydrobufotenine were prepared by ourselves from skin extracts of Bufo bufo and Eufo marinus. Similarly ala[Asp’] deltorphin was synthesized in our laboratory. Reagents and solvents used throughout the investigation were of analytical grade. 455
456
M. ROSZGHINI et af. PRIMARY AMINES
- CH, - CH,
5-HYDROXYTRYPTAMINE I!!!-HTI
TRYPTAMINE
N-METHYLATED DERIVATIVES
-CHz -TH2
CXJ
Ho’ ’
NH.CH3
’
-CH2-c;H3)2
0-J ct
-O’1
N Ii
BUFOTENINE
N-METHYL-5-HT
‘-CH2;~;YH3)3
BUFOTENIDINE
CYCLIZED DERIVATIVES
HZ
I
4%
H2
/C\
(CH,),-N
A\
CH, I
(CH,),-
eo \
N I
CH2 1
O,SH-O-
1’ IY
DEHYDROBUFGTENINE
CONJUGATED DERIVATIVES
HO-
03SH--O
BUFOTENINE 0-SULPHATE
BUFOVIRIDINE (BUFOTENINE 1-SULPHONlC AClOt
Fig. 1. The indolealkylamines in the skin of European amphibians.
RESULTS
on paper of the eluates and spraying of the chromatograms with suitable reagents makes it easy to separate and identify the different amines (Fig. 2).
The only category of aromatic biogenic amines occurring in the skin of the examined European amphibians was that of indolealkylamines, represented by their prototypes tryptamine and 5-hydroxytryptamine(SHT) and by N-methylated, cyclized and conjugated derivatives of S-HT (Fig. I). Amines in the Bufo skin. As shown in Table 1 as many as seven hydroxyindolealkylamines could be detected in the skin of the European toads, often in enormous amounts. A simple passage of the crude extracts through an alumina column, eluted with descending concentrations of ethanol, with subsequent chromatography
Amines in the skin of amphibians other than bufonids. Indolealkylamines occurred in the skin of
Biogenic antines
nearly all the examined species of Anura and in some species of Caudata (Table 2). The skin of the Salamandra species contained not only small amounts of 5-HT but also high concentrations of the rather unusual tryptamine; the Hydromantes skin, in turn, lacked tryptamine but contained 5-HT and its N-methylated derivatives bufotenine and bufotenidine. The figures in Table 2 refer to the total body contents; if calculated for the fresh skin they would have been approximately ten times greater.
457
Biogenic amines and peptides in amphibian skin Table 1. lndolealkylamines Species Source, date of capture, number of skins, average weight of a dry (D) or fresh skin (F) Bufo bufo bufo France, VIII. 1967, 7 skins (D,
5-HT
I .4 g)
Bufo bufo spinosus (a) Italy, IV. 1964, 7 skins F, 4.6 g) (b) Ibidem, IV. 1966, 15 skins (F, 8.2 g) (c) Ibidem, IV. 1967, 10 skins (F, 8.7g) Bufo calamita (a) Germany, X. 1954, 5 skins (F, 2.1 g) (b) Ibidem, VI. 1967, 3 skins (F, 3.4g) (c) Ibidem, X. 1967, 46 skins (D, 1.3 g)
N-Methylated derivatives M-S-HT BT BTD
Cyclized deriv. DH-BT
BTH
Conjugated deriv. BV BT-OS
10
0
175
215
700
1000
0
0
20 10 10
0 0 0
450 170
50 55
130 100
260 320
0 0
0 0
250
80
80
180
0
0
105 500 220
35 20 100
0 0 0
0 0 0
110 750 950
50 30 150
650 1000 800 2350 2000
30 80 70 850 450
0 0 0 0 0
0 0 0 0 0
480 500 400 2550 1900
0 ? ? ? ?
14
Bufo uiridis (a) Italy, IV. 1959, 150 skins (F, 3.1 g) (b) Ibidem, X. 1967, 116 skins (F, 1.8g) (c) Ibidem, V. 1970, 200 skins (F, 1.75 g) (d) Israel, VIII. 1967, 6 skins (D, 1.1 g) (e) Israel, V. 1970, 200 skins (F, 1.75 g)
@g/g tissue) in the skin of the four European toads
2:
15
40
10 15 20 65 40
25 1: 0 0
5-HT, 5-hydroxytryptamine; M-5-HT. N-methyl-S-HT. BT, bufotenine; BTD, bufotenidine; DH-BT, dehydrobufotenine; BV, bufoviridine; BT-OS, 0-sulphate of BT
Concerning the Anura, 5-HT was present, often in large amounts, in the skin of all examined discoglossid, ranid and hylid frogs; in the ranid frogs it was often accompanied by bufotenine and bufotenidine. Peptides
Representatives of five distinct peptide families have been so far identified in the skin of European amphibians: bombesins, bradykinins, chemotactic peptides (RECP, Rana esculenta chemotactic peptides), haemolytic peptides (bombinins) and TRH (Table 3). Their primary sequences are shown below:
BTH, bufothionine;
while they were apparently lacking, at least in active forms, in the skin of other frogs. The chemotactic peptides RECP were isolated from skin extracts of Rana esculenta; bombinin and its fragments from the skin of Bombina variegata; finally TRH from skin extracts of Rana ridibundu. DISCUSSION
1. Biogenic amines occurred, often in large amounts, in the skin of 22 out of the 32 examined European amphibians. They were represented by nine
I.
pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH~ pGlu-Gly-Arg-Leu-Gly-Thr-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH,
II.
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe Arg-Pro-Pro-Gly-Phe-Ser-Pro
Bombesin Alytesin
Bradykinin
Bradykinin Bradykinin
III. Phe-Leu-Pro-Ala-Ile-Ala-Gly-Ile-Leu-Ser-Gln-Leu-Phe-NH, Phe-Leu-Pro-Leu-Ile-Ala-Gly-Leu-Leu-Gly-Lys-Leu-Phe-NH* Phe-Leu-Pro-Leu-Leu-Ala-Gly-Leu-Ala-Ala-Asn-Phe-Leu-Pro(Lys, Ile)-Phe-NH,
l-8 l-7 RECP I RECP II ’ RECP III
IV. Gly-Ile-Gly-Ala-Leu-Ser-Ala-Lys-Gly-Ala-Leu-Leu-G~y-LeuLys-Gly-Leu-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asp-NH~ Bombinin Ser-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe [Ser’] Bombinin 18-25 Gly-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe [Gly’] Bombinin 18-25 Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asp-NH, Bombinin 19-27 V.
pGlu-His-Pro-NH,
TRH
Bombesins are represented by bombesin and alytesin, which occurred in high concentrations in the skin of the discoglossid frogs of the genera Bombina and Alytes, respectively, but were completely lacking in frogs of the Discoglossus genus. Bradykinins (authentic bradykinin l-9 and fragments l-8 and l-7) could be detected in extracts of the skin of several frogs (high concentrations were found especially in the skin of Rana temporaria),
different members, all belonging to the indolealkylamines and, except tryptamine, all deriving from 5-HT, by N-methylation, cychzation and 0- or Nsulphoconjugation of its molecule. It should be stressed that bufoviridine (bufotenine I-sulphonic acid) constituted the first example known in nature of a compound in which sulphuric acid was conjugated with the pyrolic nitrogen of the indole nucleus.
M. ROSEGHINI et al.
458
Table 2. Indolealkylamines (pg/g tissue) in the skin of European amphibians other than bufonids Species Source, date of capture, number of skins, average weight of a dry(D) or fresh (F) skin
T
5-HT
BT
BTD
0
2
6
2
0
0
0
0
0
0
0
0
3
5
0
0
55
IO
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
220
0
0
0 0 0
350 400 330
0 0 0
0 0 0
0
280
0
0 0
1100 1200
0 0
I. URODELA
PLETRODONTIDAE Hydromantes
italicus
Tuscania, Italy, XII. 1964, 8 total bodies (F, 2.8 g) SALAMANDRIDAE Euprocres platycephalus
Sardinia, Italy, IX. 1952, 3 total bodies (F, 2.5g) Pleurodeles
waltli
Spain, X. 1958, 3 total bodies (F, 0.19g) Salamandra
atra
North Italy, IX. 1963, 35 skins (F, 1.5 g) Salamandra
salamandra
North Italy, IX. 1963, 22 skins (F, 5.5 g) Triturus alpestris
Switzerland, V. 1967, 5 total bodies (F, 3 g) Triturus boscai
Spain, I. 1968, 4 total bodies (F, 1.1 g) Triturus carnifex
Italy, VII. 1972, 6 total bodies (F. 3.2 g) Triturw eristatus
Italy, VI. 1975, 5 total bodies (F, 3 g) Triturus marmoratus
Spain, V. 1967, 2 total bodies (F, 5.4g) Triturus vulgaris
North Italy, IV. 1964, 15 total bodies (F, 1.9 g) JJ. ANURA DISCOGLOSSJDAE Alytes cisternasi
Portugal, IV. 1974, 13 skins (D, 0.11 g) Alytes obstetricam (a) France, X. 1968, 30 skins (F, 0.58 g)
(b) Jbidem, XI. 1968, 71 skins (F, 0.48 g) (c) Jbidem, IX. 1969, 186 skins (F. 0.8 g) Alytes obstetricam
Portugal, V. 1974, Bombina
boscai
I I skins (D, 0.1 Ig)
bombina
(a) Germany, VI. 1968, 500 skins (F, 0.64 g) (b) Ibidem, VI. 1969, 233 skins (F, 0.48 g) Bombina variegata pachypus (a) South Italy, IV. 1953, 655 skins (F, 2.4g)
0 0
150 300
0 0
0 0 0
520 600 500
0 0 0
0 0
350 440
0 0
0
380
0
0
0
0
0
0
90
0
0
Yugoslavia, XI. 1963, 5 skins (D, 0.55 g)
0
JO0
2000
200
Ram (a) (b) (c) Raw
(F, 2.5 g) 2.8 g) (F, 2.6 g)
0 0 0
20 40 40
0 0 0
0 0 0
0.5 g)
0
0
0
0
0 0
15 7
120 60
0 0
0 0
10 20
15 100
15 15
(b) Jbidem, 1986-1988, 30 skins (F, 1.6g) Bombina uariegata variegata (a) France, VI. 1968, 10 skins (F, 1.3g) (b) Ibidem, VI. 1968, 50 skins (F, 1.3 g) (c) Jbidem, IX, 1969, 403 skins (F, 1.25 g) Discoglossus pictus (a) Sicily, III. 1950, 480 skins (F, 1.8 g)
(b) Jbidem, III. 1951, 102 skins (F, 1.6g) Discoglossus sardus Sardinia, Italy, V.1951, 210 skins (F, 1.7g) PELOBATIDAE P&bates cultripes Portugal, JV. 1971, 12 sins (D, 0.5 g) HYLIDAE Hyla arborea
North Italy, VIII. 1953, 50 skins (F, 0.46g) RANIDAE Rana dalmatina esculenta Parma, Italy, V. 1961, 800 skins Ibidem, V. 1962, 1100 skins (F, South Italy, 1986-1988, 82 skins graeca Tuscania, Italy, IV. 1967, 10 skins (F, Rana iberica
(a) Portugal, X. 1970, 3 skins (D, 0.23 g) (b) Ibidem, V. 1971, 30 skins (D, 0.1 g) Ram latastei (a) Cremona, Italy, IX. 1963, 24 skins (F, 1.3 g)
(b) Ferrara, Italy, IX. 1962, 5 skins (F, 1.9~) Ram ridibunda
Portugal, VII. 1971, 38 skins (D, 0.65 g)
0
25
0
0
Ram temporaria (a) South Germany, IX. 1963. 560 skins (F, 5.8 g) (b) North Italy, IX. 1962, 10 skins (F, 1.9 g)
0
120 _.
7
0 25
T, tryptamine; 5-HT. 5-hydroxytryptamine;
BT, bufotenine;
0 BTD, bufotenidine
20
100
459
Biogenic amines and peptides in amphibian skin
08
06
99
95, 95* 90, 902 80, 802 70, 702 60 ETHANOL
50
40
30 20 Hz0
ELUATE
Fig. 2. Schematic representation of elution profile from alumina columns (descending concentrations of ethanol) and Rf values of indolealkylamines occurring in skin extracts of European bufonids. Solvent: butanol-acetic acid-water; spraying reagents: NNCD and Ehrlich reagents. Black spots: BT, bufotenine; 5-HMT, S-hydroxy-r+methyl-tryptamine; BTD, bufotenidine; S-HT, 5-hydroxytryptamine; 5-HIAA, S-hydroxyindole-acetic acid; TRP, tryptophan. Hatched spots: cyclized (BTH, bufothionine; DH-BT, dehydrobufotenine) and sulpho-conjugated derivatives (B-VIR, bufoviridine; BT-0-SULPH, bufotenine 0-sulphate).
N-methylation occurred also in ranid frogs and even in Caudata. Of interest was the fact that the skin of the B. viridis specimens collected in Israel (B. viridis arabicus?) was considerably richer in amines than that of specimens collected in Europe (B. viridis viridis?) and that 0-sulphate of bufotenine was very abundant in B. calamita but not in B. viridis.
Both cyclization and conjugation produced a complete loss of activity, at least in the periphery, whereas N-methylation caused a progressive decay of 5-HT activity, with the appearance, however, of clear cut nicotinic effects in bufotenidine, its quaternary ammonium derivatives (unpublished observations). Cyclization and sulphoconjugation of hydroxyindolalkylamines seemed to be peculiar to toads;
Table 3. Active peptides in the skin of European discoglossid and ranid frogs @g/g tissue) Species
Bon&sins*
Alytes cistern&i Alytes obstetricans
Batch (a) Batch (b) Batch (c) Alytes obstetricans boscai Bombina bombina
Batch (a) Batch (b)
Bradvkinins*
Other Peutides
80
n.d.
900-1000 500-650 500-700 350
n.d. n.d. n.d. n.d.
200-300 300400
n.d. n.d.
250-350 500-650 500-700 300
n.d. n.d. n.d. n.d.
n.d. n.d. n.d.
n.d. n.d. n.d.
n.d.
30
Chemotactic peptides
n.d. n.d. n.d. n.d.
3tL35 n.d. n.d. 20-25
TRH
n.d. n.d.
200-250 100
Bombina uariegata uariegata
Batch (a) Batch (b) Batch (c) Bombina uariegata pachypus
Batch (b) Discoglossus pictus Discoglossus sardus Rana dalmatina Rana esculenta
Batch (c) Rana iberica
Batch (b) Rana Rana Rana Rana
graeca latastei ridibunda temporaria
Batch (a) Batch (b)
*Expressed in terms of bombesin and bradykinin, thyrotropin releasing hormone
respectively; n.d., not detectable (co.5 cg/g); TRH,
460
M. ROSEGHINI et al.
Representatives of five peptide familes have been so far isolated from the skin of European amphibians: bombesins, bradykinins, chemotactic peptides, haemolytic peptides and TRH. Bombesin and alytesin occurred in large amounts in the skin of discoglossid frogs of the genera Bombina and Alytes, respectively, but were lacking in the genus Discoglossus. They must be considered the prototypes of the bombesin family, now comprising as many as fourteen amphibian peptides and five avian and mammalian counterparts. The presence of ranatensin in the skin of European ranid frogs is possible but, if so, only in minute amounts (CO.5 pg/g). Authentic bradykinin was isolated from the skin of Rana temporaria (Anastasi, Erspamer and Bertaccini, 1965) and Rana esculenta, in the latter species together with its N-teXTnina octapeptide (bradykinin 1-8) and bradykinin l-7. The first fragment showed a sharply varying degree of activity in different bioassay systems (unpublished observations), the second was completely inactive. Active bradykinin forms were not detectable in skin extracts of R. dalmatina, R. graeca and R. latastei, whereas extracts from R. iberica and R. ridibunda showed a clear-cut bradykinin activity. On the basis of its elution profile from alumina and HPLC columns, this activity is attributable both to authentic bradykinin and bradykinin l-8. Chemotactic peptides, i.e. peptides displaying a strong chemotactic activity on macrophages, were first detected in skin extracts of Rana erythraea from the Philippines (Yasuhara et al., 1986) Now, Barra et al. (to be published) have isolated analogous peptides from the skin of Rana esculenta, indicating that this peptide family may have a widespread distribution in ranids and perhaps also in other amphibian families, where they may contribute to the immunological defence of the organism against microbial attack. The bombinins were isolated and sequence from skin extracts of Bombina variegata. The main property they possessed was a strong haemolytic activity (Csordas and Michl, 1969, 1970). Interestingly, skin extracts from Bombina variegata pachypus displayed also evident antimicrobial effects (Croce et al., 1973). Finally, TRH first isolated from skin extracts of Bombina orientalis (Yajima et al., 1975) and then of Rana pipiens (Jackson and Reichlin, 1977) has also been detected in the skin of Rana ridibunda (Ravazzola et al., I%)), again suggesting a widespread occurrence of the peptide in ranid and perhaps discoglossid frogs. Peptides belonging to other very important amphibian peptide families, such as tachykinins, caeruleins, sauvagine, tryptophyllins, dermorphins and deltorphins (Kreil et al., 1989; Erspamer et al.,
1989) were apparently European amphibians.
lacking in the skin of the
Acknowledgements-This work was supported by grants
from the Consiglio Nazionale delle Ricerche, Roma. REFERENCES Anastasi A., Erspamer V. and Bertaccini G. (1965) Occurrence of bradykinin in the skin of Rana temporaria.Comp. Biochem. Physiol. 14, 43-53. Croce G., Giglioli N. and Bolognani L. (1973) Antimicrobial activity in the skin secretions of Bombina uariegata pachypus. Toxicon 11, 99-100. Csordas A. and Michl H. (1969) Primary structure of two oligopeptides of the toxin of Bombina variegata L. Toxicon 7, 101-108. Erspamer V., Falconieri Erspamer G., Mazzanti G. and Endean R. (1984) Active peptides in the skin of one hundred amphibian species from Australia and Papua New Guinea. Comp. Biochem. Physiol. 77C, 88-108. Erspamer V., Falconieri Erspamer G. and Cei J. M. (1986) Active peptides in the skin of two hundred and thirty American amphibian species. Comp. Biochem. Physiol. UC,
125-137.
Erspamer V., Melchiorri P., Falconieri Erspamer G., Negri L., Corsi R., Severini C., Bossa D., Simmaco M. and Kreil G. (1989) Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for 6 opioid binding sites. Proc. Natl. Acad. Sci. USA 86, 5188-5192. Jackson I. and Reichlin S. (1977) Thyrotropin-releasing hormone: abundance in the skin of the frog, Ranapipiens. Science 198, 414415. Kiss G. and Michl H. (1962) Uber das Giftsekret der Gelbbauchunke. Toxicon 1, 33-39. Kreil G., Barra D., Simmaco M., Erspamer V., Falconieri Erspamer G., Negri L., Severini C., Corsi R. and Melchiorri P. (1989) Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for 6 opioid receptors, Eur. J. Pharmacol. 162, 123-128. Ravazzola M., Brown D., Leppaluoto J. and Orci L. (1979) Localization by immunofluorescence of thyrotropinreleasing hormone in the cutaneous glands of the frog, Rana ridibunda. Life Sci. 25, 1331-1335.
Roseghini M., Erspamer V. and Endean R. (1976) Indole-, imidazole- and phenyl-alkylamines in the skin of one hundred amphibian species from Australia and Papua New Guinea. Comp. Biochem. Physiol. 54C, 3143. Roseghini M., Erspamer V., Falconieri Erspamer G. and Cei J. M. (1986) Indole-. imidazole and uhenylalkylamines in‘the skin of one hundred and forty ‘American amphibian species other than bufonids. Comp. Biochem. Phvsiol. 85C. 139-147. Roseghini M.,* Falconieri Erspamer G. and Severini C. (1988) Biogenic amines and active peptides in the skin of fifty-two African amphibian species other than bufonids. Comp. Biochem. Physiol. 91C, 281-286.
Yajima H., Kitigawa K., Segawa T., Nakano M. and Kataoka K. (1975) Occurrence of Pvr-His-Pro-NH, in the frog skin. &em. kharm. Bull. (Tokyo) 23, 3301-3303. Yasuhara T., Nakajima T., Erspamer V., Falconieri Erspamer G., Tukamoto Y. and Mori M. (1986) Isolation and sequential analysis of peptides in Rana erythraea skin. In Peptide Chemistry 1985 (Ed. Y. Kiso), p. 363-368. Protein Research Foundation, Osaka 1986.