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Indole-, imidazole- and phenyl-alkylamines in the skin of one hundred amphibian species from Australia and Papua New Guinea
Camp.
Bmhm
Physd.,
IY76,
Vol. 54C. pp 31 to 43. Perqamon
Press. Prmted ,,I Grror Brimn
INDOLE-, IMIDAZOLEAND PHENYL-ALKYLAMINES IN THE SKIN OF ONE HUNDRED AMPHIBIAN SPECIES FROM AUSTRALIA AND PAPUA NEW GUINEA M. ROSEGHINI’,
V. ERSPAMER AND R. ENDEAN’
‘Institute of Medical Pharmacology and 2Department of Zoology, University (Received
I, University of Rome, I-00185 Rome, Italy; of Queensland, St. Lucia, Brisbane 4067, Australia 12 November
1975)
Abstract-l. Extracts prepared from the dried skins of approximately one hundred amphibian species from Australia and Papua New Guinea were subjected to chemical and biological screening in order to determine the nature and amounts of biogenic amines in their skins. 2. The most frequently and abundantly occurring amines were indolealkylamines, represented by their prototype 5-HT, by the entire series of N-methylated derivatives of 5-HT (N’-methyl-5-HT, N’,N’dimethyl-5-HT or bufotenine, bufotenidine) and by some new compounds: O-sulphate of bufotenine, O-sulphate of bufotenidine and the quaternary ammonium derivative of tryptamine. 3. Imidazolealkylamines were usually represented by histamine, N’-acetyl-histamine, N’-methylated N’,N’-dimethylhistamine) and cyclized histamines (spinaceamine, histamines (N’-methylhistamine, 6-methylspinaceamine) could be detected only in the skins of a limited number of species. 4. The above spectra of indole- and imidazolealkylamines are destined to broaden, because several other indole and imidazole derivatives in skin extracts have been identified and partly characterized by paper chromatography and electrophoresis. 5. The importance of amphibian skin as a rich source of biogenic amines, and their precursors and metabolites, is emphasized.
extracts were combined. When kept in dark bottles at 4°C the extracts could be stored for long periods of time (months and even years) without appreciable changes in their amine spectrum. Loss of weight during drying of the skins varied from one species to the next, generally it ranged between 70 and 75%
INTRODUCTION skin may be regarded as an enormous storehouse of biogenic amines and active polypeptides. Indeed, no other vertebrate or invertebrate tissue can compare with amphibian cutaneous tissue in regard to variety and concentration of these active compounds. During the past 10 years approx one hundred amphibian species were collected in different regions of Australia and Papua New Guinea. Methanol extracts of their dried skins were submitted to screening for detection of aromatic amines and polypeptides. This paper will present an account of the occurrence in the skins of species examined of indole-, imidazole- and phenyl-alkylamines as well as some of their possible metabolites and precursors. Although the chemical structures of some of these compounds are well known those of other compounds await elucidation. THE amphibian
METHODS Amphibian
AND
Chromatography
on alumina column
A very satisfactory separation of the different biogenic amines, their precursors and metabolites was often obtained by simple passage of crude skin extracts through an alkaline alumina column (Merck, Darmstadt). Chromatographic columns utilized were of different sizes according to the amount of material to be chromatographed. For amounts corresponding to 15-30 g of dry skin the columns were 3.3 cm wide and 50 cm high and the alumina weighed 140-17Og. Columns were double walled with a flow of tap water between the inner and the outer walls, to maintain constant temperature. The crude extract to be chromatographed was evaporated to dryness under reduced pressure, the residue washed with petroleum ether in order to remove fats and then taken up, by stirring in either 99 or in 95”/, ethanol. The liquid was passed through the column which was then eluted with descending concentrations of ethanol and ultimately with water. Fractions of 2&220 ml were collected.
MATERIALS
material
Paper chromatography
The amphibian material used in this study is listed in Table 1. Frogs were killed by a blow to the head, their skins removed immediately after killing, carefully spread out and dried in the shade. Soon after arrival in the laboratory the skins were cut into small pieces with scissors and immersed in a volume of 80% methanol equivalent to twenty times their weight. The liquid was decanted after a week and the skins again treated with a volume of 80% methanol fifteen to twenty times their weight. The two methanol
The ascending unidimensional technique on Whatman No. 1 paper was routinely employed. Chromatograms were run at 18°C for l&30 hr using the following solvent mixtures: n-butanol:acetic acid:water (40: 10:50), nbutanol: 35:! methylamine (80: 30), and l-pentanol:pyridine: water: 35% methylamine (40:40: 10: 1). For special purposes KC1 20% in water and methylethylketone:pyridine:water:35% methylamine (65: 15: 10:0.5) were also used. 31
M. ROSEGHINI, V. ERSPAMER AND R. ENDEAN
32 Table
1. Indolealkylamines
Species (Source, date of capture, number of skins, average weight of a skin)
in the skin of amphibians
from Australia
and Papua
New Guinea
S-HIAA 5-HTOL
5-HT
5-HMT
220
0
0
220 400
0 0
0 0
40, 10
500 400
0 0
0 0
30, 10 25, 5
180
0
0
20. 4
250
12
15
0
25
35
0
10
0
5
350
0
0
0
70
700
0
20
0
200
16
65
0
0
0
0
0
750
0
5-10
0
0
70
80-100
10
10
3500
90
15, 5
20
0
6000
200
150, 60
BT-S 600
0
7000
350
300
BT-S 500
100
0
25
0
30
20-25
0
60
0
3Wto
0
0
10
0
0
0
0
0
30
0
30
0
0
190
0
L. derlttrta near Brisbane, Q’ld., XII, 1965 5 I skins (0.74 g)
40-50
0
750
0
300
L. hicolor near Brisbane, Q’ld., XII. 1965 58 skins (0.028 g)
0
0
0
0
0
10
0
0
0
0
Litoria caerulea (1) near Brisbane, Q’ld., XII. 1964 179 skins (1.4g); (2) ibidem. 124 skins (1.66 g) 2 (3) ibidem. X-XI. 1965 380 skins (1.34 g) $ (4) ibidem, 302 skins (1.77 g) Y (5) ibidem, I-V. 1969 742 skins (I .64 g) (6) ibidem, I-III. 1970 587 skins (1.46 g) L. peroni (1) near Brisbane, Q’ld., XII. 1964 11 skins (0.24 g) (2) ibidem, XI-XII. 1969 9 skins (0.21 g)
(I) near Brisbane, Q’ld., XII. 1968 1 skin (0.28 g) (2) ibidem, XI-XII. 1969, 1I skins
BT
BTD
Other
indoles
I 50
(0.26 g) L. ,freyciwti near Brisbane, Q’ld., II. 1970 56 skins (0.05 g) L. pearsoniana (1) near Brisbane, Q’ld., II. 1964 40 skins (0.036 g) (2) ibidem, I. 1966 76 skins (0.039 FZ) (3) ibidem, ,1-X11.’ 1969 I17 skins (0.041 g) (4) ibidem, X. 1971 179 skins (0.04 g) (5) ibidem, 1~111. 1973 516 skins (0.045 g) L. grucilenttr near Brisbane, Q’ld., XII. 1965 69 skins (0.08 g) L. ruhellu (I) Darling Downs. Q’ld., IV. 1964 24 skins (0.045 g) (2) Chinchilla, Q’ld., XII. 1965 3 1 skins (0.076 g) L. latopalmutu (I) near Brisbane, Q’ld., I. 1965 X skins (0.087 g) (21 Chinchilla. O’ld.. V. 1968 ’ ‘19 skins (0.0&g)
L. nasufa (1) near Brisbane, Q’ld., XI. 1964 4 skins (0.17 g) (2) ibidem, XII. 1965, 4 skins (0.2 g)
3(f40
0
0
0
0
0
L. lesueuri near Brisbane, Q’ld., XII. 1965 38 skins (0.13 g)
150
0
5
0
20
L. qlandulosa Stanthorpe. Q’ld., XII. 1973 6 skins (0.17 g)
250
0
0
6000
100
I 30
BT-S 3&50 BT-S 150 BT-S 100-150
Biogenic
amines Table
Species (Source, date of capture, number of skins, average weight of a skin)
in amphibian
33
skin
l-continued
BT
BTD
5-HIAA .5-HTOL
5-HT
5-HMT
175
0
0
0
80
300 125
0 0
0 0
0 0
l(fl5 75
700
0
150
260 50
0 0
100 10
350
0
0
0
30
400
0
0
0
25
350
0
0
0
50
0
0
0
0
0
0
0
0
0
0
40
0
0
0
0
500
0
0
0
50
1750
0
0
0
400
330
0
0
0
80
2250
0
0
0
200
500
50
0
1850
50
400
0
0
1600
130
1600
0
0
1300
80
2200
0
0
550
0
0
25
80, 50
4500
0
0
55
320, 250
3000
0
0
60
80, 150
60
0
2300
280
70
0
0
870
270
0
60
0
0
0
0
750
80
3700
40
1700
200
5500
30, 30
600
40
5000
75. 30
Other
indoles
L. dayi
(1) Tinaroo Creek, North Q’ld., VIII. 1968, 9 skins (0.17 g) (2) ibidem, XI. 1968, 5 skins (0.1 g) (3) ibidem, I. 1969, 40 skins (0.1 g) .L. eucnemis
(1) Tinaroo Creek, North Q’ld., XII. 1968, 7 skins (0.19g) (2) ibidem, IV. 1969, 4 skins (0.19 g) (3) Madang, Papua New Guinea, II. 1970 32 skins (0.11 g) L. narmotis
(1) Mareeba, North Q’ld., XII. 1967 6 skins (0.13 g) (2) ibidem, VIII. 1968 39 skins (0.09 g) (3) Cairns, North Q’ld., XII. 1968 3 skins (0.2g) L. nigrofrenata
(1) Proserpine, North 10 skins (0.17g) (2) ibidem, iI1. 1969 6 skins (0.2 g)
Q’ld., XII. 1968
L. rothi
North Q’ld.. I. 1965 6 skins (0.19 g) L. infrafrena
ta
(1) Cairns, North Q’ld., I. 1965 3 skins (3.74 g) (2) Papua New Guinea, I. 1968 2 skins (1.8 g) (3) Madang, Papua New Guinea, II. 1970 7 skins (1.8 g) (4) ibidem, X. 1968, 15 skins (2.8 g) L. hooroolongensis
Armidale, N.S.W.. I. 1967 13 skins (0.074 g) L. ran~formis
N.S.W..
VI. 1972, 8 skins (0.5g)
I > 100
L. citropa
Sydney,
N.S.W., VI. 1974, 6 skins (0.25g)
L. aurea
(1) near Melbourne, Victoria, I-III. 178, 731 skins (0.48 g) (2) ibidem, XII. 1972-I. 1973 419 skins (0.62 g) (3) ibidem, II-III. 1973 230 skins (0.76 g) (4) ibidem, XI-XII. 1973 1018 skins (0.59 g)
3040
750, 120
L. ewingi
(1) Victoria, XII. 1968 15 skins (0.023 g) (2) ibidem, XII. 1969 12 skins (0.037 g) L. gilleni
Alice Springs, Central Australia, IV. 1967, 18 skins (1.2g) L. moorei
(1) near Perth, W.A., V. 1968 1 skin (0.37g) (2) Walpole, W.A., IV. 1973 29 skins (0.66 g) (3) Albany. W.A.. IV. 1973
TQA
3c-50
M. ROSEGHINI, V. ERSPAMER AND R. ENDEAN
34
Table Species (Source, date of capture, number of skins, average weight of a skin L. adeluidensis (1) near Perth, W.A., V. 1968 17 skins (0.05 g) (2) Walpole, W.A., IV. 1973 27 skins (0.12 g) L. cy&Jrh_Ynchus Albany, W.A., V. 1973, 12 skins (0.038g) L. darlingtoni (1) Goroka, Papua New Guinea, IV. 1971 24 skins (0.12 g) (2) Mt. Hagen, Papua New Guinea, IV. 1971 28 skins (0.12 g) (3) Arumanda, Papua New Guinea, III. 1972 161 skins (0.087 g) L. iris (1) Mt. Hagen, Papua New Guinea, IV. 1971 59 skins (0.035 g) (2) Arumanda, Papua New Guinea, III. 1972 73 skins (0.05 g)
l-continued
5-HIAA 5-HTOL
5-HT
5-HMT
150
75
0
300
50
600
140
100
I200
150
150
0
0
800
20
90
0
0
12
70
0
0
10
120
0
0
35
7
0
0
0
0
10
0
0
0
0
BT
BTD
L. angiana Goroka, Papua New Guinea, 49 skins (0.23 g)
III. 1972
30
0
0
65
10
L. micromenlhrana Madang, Papua New Guinea, 36 skins (0.14 g)
III. 1972
50
0
0
60
20
L. thesaurensis Goroka. Papua New Guinea, 75 skins (0.046g)
III. 1972
0
0
35
350
30
1
0
0
0
0
0
0
0
0
1
0
0
0
0
3
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
Linlnodynastes peroni (1) near Brisbane, Q’ld., VIII. 1969 28 skins (0.31 g) (2) Murray Bridge, S.A., IV. 1969 6 skins (0.4 g) L. or~lfltus near Brisbane.
2-3
I. 1965, 41 skins (0.12g)
L. ,jlrtcheri (1) Darling Downs, Q’ld.. III. 1966 9 skins (0.11 g) (2) Dalby, Q’ld., II. 1970 8 skins (0.2 g) L. dorscdis rerrarryinae (1) Rockhampton, North Q’ld., I. 1970 7 skins (1.38 g) (2) Darling Downs, Q’ld., XII. 1965 6 skins (0.65 g) (3) near Perth, W.A., V. 1970 12 skins (0.12 g)
5-7
L. dorsalis duwwrili (I) Castlemaine, Victoria, II. 1969 6 skins (0.31 g) (2) South Australia. III. 1969 3 skins (0.7 g) L. tasmaniensis (1) Mount Gambier, S.A., III-V. 1969 59 skins (0.061 g) (2) near Brisbane, Q’ld., XI. 1969 3 skins (0.18 g) L. salmini Rockhampton, North 5 skins (1.05 g)
Q’ld., IX. 1969
Adrlotus hrevis Killarney, Q’ld., XII. 1964 39 skins (0.12 g)
l-2
Other
indoles
I 50 I 12(f180
III
Biogenic
amines Table
Species (Source, date of capture, number of skins, average weight of a skin Lechriodus j!etcheri Mt. Tamborine, Q’ld., I. 1965 33 skins (0.12 g)
in amphibian
35
skin
l-continued
BT
BTD
5-HIAA 5-HTOL
5-HT
5-HMT
l&12
0
0
0
0
Other
K~urranus loveridgei near Brisbane, Q’ld., V. 1965 18 skins (0.065 g)
0
0
0
0
0
Mixophvrs fasciolatus near Brisbane. Q’ld., XII. 1964 17 skins (0.63 g)
0
0
0
0
0
M. schetiilli Speerwah, North 2 skins (1.05g)
0
0
0
0
0
0
0
0
0
0
75
0
0
0
5
200
0
0
0
30
100
0
30
5
10
60
0
40
20
I 100
5
50
0
100
0
I 150
20
20
0
0
25
I 50
280
180
0
0
60
I 120
700
0
0
0
40
10
0
0
0
0
7
80
0
40
5
80
0
0
0
110
0
0
0
0
0
0
30
0
0
0
20
0
0
0
40
0
0
5
100
0
0
10
50
0
0
0
0
0
Q’ld., I. 1970
Notaden hrnnetti Condobolin. N.S.W., II-III. 28 skins (0.96 g)
1973
Cyclorana australis (1) Dalby, Q’ld., III. 1966 2 skins (1.24 g) (2) Darling Downs, Q’ld., III. 1971 15 skins (1.5 g) C. alboguttatus (I) Dalby. Q’ld., I. 1965 6 skins (0.38 g) (2) Darling Downs, Q’ld., XII. 1965 14 skins (0.36 g) (3) Condobolin, N.S.W., I-II. 1973 18 skins (0.46 g) C. cultripes (1) Darling Downs, Q’ld., III. 1966 I2 skins (0.175 g) (2) Condobolin, N.S.W., II. 1973 34 skins (0. IX g) C. breripes Condobolin. N.S.W., II. 1973 22 skins (0.2 g)
2G25
C. platprephalus (1) South Q’ld., XI 1969 14 skins (0.15g) (2) Condobolin, N.S.W., II. 1973 20 skins (0.35 g) Uprroleia rugosa South Q’ld. and N.S.W., 108 I skins (0.055 g)
196991973
U. marmorata near Brisbane, Q’ld., 196991971 147 skins (0.061 g) U. laerigata near Brisbane, Q’ld.. II. 1970 8 skins (0.09 g) Taudact)alus acutirostris (1) Tinaroo Creek, North Q’ld., III. 1968. 12 skins (0.025 g) (2) Kuranda, North Q’ld., II. 1970 56 skins (0.024 g) T. diurrzus (I) Mt. Glorious, Q’ld., XII. 1968 19 skins (0.047 g) (2) ibidem, II. 1969 39 skins (0.043 g) (3) ibidem, III. 1969 53 skins (0.053 g) Sphenophrynr plwialis Kuranda, North Q’ld., X. 1968 28 skins (0.02 g)
3-t
2-3
0
indoles
I 50
I 100-150
III 10
36
M. ROSEGHINI, V. ERSPAMER AND Table Species (Source, date of capture, number of skins, average weight of a skin
Crinia
R. ENDEAN
l&continued
5-HIAA 5-HTOL
5-HT
5-HMT
1000
0
0
0
20-30
380
0
0
0
20
170
0
0
0
1200
0
0
0
300
0
0
0
0
0
0
0
0
70
0
0
0
0
80
0
0
0
0
3t340
0
0
0
0
440
0
0
0
30
0
0
0
3&35
130
0
0
0
10
25530
0
0
0
0
40
0
0
0
0
25-30
0
0
0
0
500-600
0
0
0
20
2200
0
0
0
40
360
0
0
0
40-50
300
0
0
0
20
90-120
0
0
0
0
90-110
0
0
0
0
0
0
0
0
0
0
0
40
BT
BTD
Other
indoles
pseudoinsiynijera
(1) near Perth, W.A., V. 1968 32 skins (0.024 g) (2) Albany, W.A., IV. 1973 176 skins (0.028 g) Cr. ,suhinsignijereru Albany, W.A., III. 1973 124 skins (0.021 g) Cr. insignifera near Perth, W.A., IX. 1971 17 skins (0.02 g) Cr. rosen Albany, W.A., IV. 1973 38 skins (0.037 g)
lo-15
70
3(f35
Cr. leai
Walpole, W. A., IV. 1973 300 skins (0.034 g) Cr. glauerti (1) near Perth, W.A., V. 1970 43 skins (0.007 g) (2) Albany, W.A., IV. 1973 168 skins (0.012 g) Cr. georgiana near Perth, W.A., IX. 1971 16 skins (0.056 g) Cr. signijera
South Australia, 196991970 52 skins (0.027 g) Cr. riparia Adelaide, S.A., IX. 1971 67 skins (0.053 g) Cr. parinsignifera Castlemaine, Victoria, 27 skins (0.022 g)
II. 1969
45G-500
Cr. tinnula
(1) Beerwah, South Q’ld., III. 1970 95 skins (0.013 g) (2) ibidem. IV. 1970, 48 skins (0.01 g) Metacrinia
nichollsi
Albany, W.A., III-IV. 88 skins (0.023 g) Pseudophryne
1973
giintheri
(1) Wagin, W.A., V. 1968 44 skins (0.071 g) (2) near Perth. Wi., V. 1970 76 skins (0.083 g) (3) Walpole, W.A., IV. 1973 105 skins
III 50-60
Ps. coriacea
(1) Bald Mountain, Q’ld., I. 1972 90 skins (0.083 g) (2) ibidem, XI. 1972, 76 skins (0.085 g)
III 8-10
Ps. hihroni
(1) Armidale, N.S.W., IV. 1966 34 skins (0.045 g) (2) Adelaide, S.A., V. 1970 23 skins (0.056g) (3) Moggill, Q’ld., V. 1972 27 skins (0.064 g) Ps. corroboree New South Wales,
12 skins (0.06 g)
III. 1967
40
80-100
III 15
Biogenic
amines Table
Species (Source, date of capture, number of skins, average weight of a skin) Ps. svmimarmorata South Australia, IV. 1969 20 skins (0.057 g)
5-HT
in amphibian
skin
37
l-continued
5-HMT
BT
BTD
5-HIAA 5-HTOL
700
0
50
0
0
0
N. pelohatoides near Perth. W.A., V. 197O/IV. 1973 45 skins (0.14 g)
0
0
0
N. centralis Walpole, W.A., IV. 1973 25 skins (0.24 g)
0
0
0
30
0
15
120
0
10
5-20
0
35
0
Neohutruchus pictus (1) near Adelaide, S.A., III. 1969 5 skins (0.47g) (2) Condobolin, N.S.W., V. 1972 20 skins (0.2 g)
He2eioporus eyrei (1) near Perth, W.A., V. 1970 68 skins (0.32 g) (2) Albany, W.A., IV. 1973 16 skins (0.5 g) H. psammophilus (1) near Perth, W.A., V. 1970 37 skins (0.3 g) (2) Albany, W.A., IV. 1973 38 skins (0.18 g) H. albopunctatus (1) Wagin, W.A., V. 1968 1 skin: glandular areas (0.3 g) remaining skin (0.85-g) (2) Walpole. W.A.. IV. 1973 ‘36 skins (1.2g) H. inornatus near Perth, W.A., V. 1970 2 skins (0.55 g) Myobatrachus gouldii Walpole, W.A., IV. 1973 36 skins (0.28 g) Nictimystes tympanocryptis (1) Speerwah, North Q’ld., I. 1970 22 skins (0.06 g) (2) ibidem, II-III. 1970 47 skins (0.055 g) (3) Millaa Millaa Falls, North Q’ld., V. 1970, 105 skins (0.06g) (4) ibidem, XI. 1970 152 skins (0.058 g) N. oestigea (I) North Q’ld., I. 1970 26 skins (0.08 g) (2) Speerwah, North Q’ld., II-III. 1970 18 skins (0.06 g) (3) Palmerston, North Q’ld., XI. 1970 55 skins (0.06g)
10
600 10 250
0 0 0
0 0
0 0
0
0
45-50
0
0
0
2-3
50
0
0
0
3-4
0
0
8800
0
BTD-S
300
0
0
9500
0
BTD-S
300
0
0
8400
0
BTD-S
225
0
0
5100
0
BTD-S
250
0
0
550
0
0
115
0
0
20 0 10
65
III. 1972
1200
400
0
4300
N. kubori Goroka, Papua New Guinea, 71 skins (0.11 g)
III. 1972
0
0
85
250
20
0
0
0
0
0
III. 1971
indoles
610
N. disrupta Goroka, Papua New Guinea, 3 1 skins (0.46 g)
Asterophrys rufescens Lae, Papua New Guinea, 2 skins (0.085 g)
Other
60, 20
II 150
38
M. ROSEGHINI, V. ERSPAMER AND Table Species (Source, date of capture, number of skins, average weight of a skin
R. ENDEAN
l--continued
S-HT
5-HMT
BT
BTD
S-HIAA 5-HTOL
A. stictogaster Goroka, Papua New Guinea, 23 skins (1.9 g)
III. 1972
0
0
0
0
0
Xenobatrachus rostratus Goroka, Papua New Guinea, 9 skins (0.08 g)
III. 1973
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
70
0
0
0
0
0
0
0
0
0
20
0
0
0
0
40
0
0
0
15-20
120
0
0
0
10
40
0
0
0
20
70
0
0
0
50
125
0
0
0
80
25
0
0
0
10
4
0
0
70
0
0
Platymantis popuensis Lae, Papua New Guinea, 16 skins (0.12 g)
III. 1971
PI. corrugatus Madang. Papua New Guinea, 20 skins (0.084 g)
III. 1972
Cophixalus auriegatus Goroka, Papua New Guinea, 186 skins (0.012 g)
III. 1972
C. cryptotympanum Wau, Papua New Guinea, 51 skins (0.04 g)
III. 1971
C. ornatus North Q’ld., VIII. 1968/I. 122 skins (0.024 g)
1970
Rana Papua (1) Wau, Papua New Guinea, III. 1971 4 skins (0.8 g) (2) Madang, Papua New Guinea, III. 1972 9 skins (0.55 g) R. kreffti Wau, Papua New Guinea, 3 1 skins (0.32 g)
III. 1971
R. grisea Goroka, Papua New Guinea, 3 1 skins (0.52 g)
III. 1972
R. arfhki (1) Madang, Papua New Guinea, I. 1970 2 skins (5.6 g) (2) ibidem, III. 1972, 12 skins (2.03g) R. daemeli (1) Papua New Guinea, I. 1968, 29 skins (0.35 g) (2) Speerwah, North Q’ld., I. 1970 5 skins (0.3 g) (3) Markham Valley, Papua New III. 1971 8 skins (0.53 g)
3-1
0
Other
indoles
0
Guinea,
5-HT, 5-hydroxytryptamine; 5-HMT, 5-hydroxyindoleacetic acid; 5-HTOL, phate; TQA, tryptamine quaternary pounds of supposedly indole nature skin. Values for unknown indoles are
0
10
5-hydroxy-N-methyltryptamine; BT, bufotenine, BTD, bufotenidine; 5-HIAA, 5-hydroxytryptophol; BT-S, bufotenine 0-sulphate; BTD-S, bufotenidine O-sulammonium derivative; I-III, unknown indole compounds. For additional comsee text. Indolealkylamine contents are expressed in pg free base per g dried only approximate, and expressed in pg 5-HT/g.
The spraying reagents used for the detection and identification of the different compounds were as follows: (a) an aqueous solution of diazotized sulphanilic acid (Pauly reagent) followed by 3-5% aqueous sodium carbonate; (b) an aqueous solution of diazotized p-nitroaniline followed by sodium carbonate; (c) a 0.1-0.3% solution of HeinriCh and Schuler NNCD reagent (2-chloro-4_nitrobenzenediazoniumnaphthalene-2-sulphonate) in 0.1 N HCl; (d) a 0.05-0.1% alcoholic solution of dichloroquinone chlorimide (Gibbs reagent) followed by sodium carbonate; (f) a l-2:/, alcoholic solution of p-dimethylaminobenzaldehyde, followed by exposure of the chromatograms to HCl
vapours in a glass cabinet; (g) a 1% alcoholic solution of Folin reagent for amino acids (1,2-naphthoquinone-4sulphonic sodium salt) followed by sodium carbonate; (h) a saturated aqueous solution of Reinecke salt, followed by a gentle washing of the paper sheets under running tap water until the background colour was completely removed; (i) Dragendorff reagent; and finally (j) a 0.2% solution of barium chloride in 70% ethanol followed by 0.05% sodium rhodizonate in 50% ethanol (reagent for sulphates, which appear as yellow spots on a reddish background). Imidazole derivatives are characterized by the positivity
Biogenic
amines
in amphibian
ammonium bases, the frog rectus abdominis muscle. Methysergide (0.2 @g/ml) was used as a 5-HT antagonist and mepyramine maleate (0.1 pg/ml) as a histamine antagonist.
of reactions (a), (b) and eventually (g); phenolic derivatives by the positivity of the same reactions and occasionally of reaction (d); 5-hydroxy-indole compounds by the positivity of all reactions from (a) to (g) inclusive. With tryptamines lacking the phenolic hydroxy group or having this group occupied by conjugation or alkylation, only reactions (c), (f) and (g) are positive. The positivity of reactions (h) and (i) is indicative of the presence of a quaternary ammonium group. Semi-quantitative estimations of the individual compounds on paper-chromatograms were carried out chiefly by visual comparison of spots produced by different amounts of crude skin extracts or ethanol eluates with spots produced by known amounts of the corresponding pure synthetic compounds. If a particular synthetic compound was not available, a closely related synthetic compound was used.
Reagents
A high voltage electrophoresis apparatus from L. Hormuth, Inh. W. E. Vetter (Heidelberg) was used for the electrophoretic experiments. Electropherograms were run at pH 1.2 (glacial acetic acid:85% formic acid:distilled water, 15:25: 110) and pH 5.8 (99% pyridine:glacial acetic acid: distilled water, 15.5: 1.8: 135). Acid hydrolysis Treatment in a boiling water bath, for 3&90 min, with pure acetic acid, which does not attack the indole nucleus appreciably, was routinely used (a) to hydrolyse sulphoconjugated indolealkylamines and (b) to check the effect of acid treatment on unknown compounds.
RESULTS lndolealkylamines Analytical matographic
The qualitative and quantitative bioassay of amines was carried out on the following smooth muscle preparations: for 5-HT and related amines, the oestrous rat uterus; for histamines, the guinea-pig ileum; for quaternary
a9
IN
THE
and related
data, obtained semiquantitative
compounds mainly from paper chroestimations and, if
necessary, supplemented by bioassay, are summarized in Fig. 1 and Table 1. Table 1 not only shows the amine content of skins from different species but also the content in different batches of the same species. For Litoria caerulea the amine content of skins from
Bioassay
I
and drugs
The following synthetic compounds were available for comparison: tryptamine HCl, N,N-dimethyltryptamine 5-hydroxytryptamine creatinine sulphate methiodide*, (5-HT), bufotenine base*, bufotenine 0-sulphate*, bufotenidine*, bufotenidine 0-sulphate*, 5-hydroxyindole acetic acid (5-HIIA), 5-hydroxytryptophol, 5-hydroxy-r-tryptophan, L-tryptophan, histamine 2 HCI, N-methyl-histamine. 2 HCI, N,N-dimethylhistamine 2 HCI, spinaceamine.2 HC1. + HZO, 6-methylspinaceamine.2HC1, N-acetylhistamine, N-acetyl-L-histidine, L-histidine, imidazole acetic acid, p-tyramine HCl, m-tyramine HCl, octopamine tartrate, leptodactyline picrate, L-tyrosine, L-noradrenaline tartrate, N-methyl-5-hydroxytryptamine, which was not available as a synthetic compound, was expressed in terms of 5-HT. Compounds marked with * were synthetised at the Farmitalia Research Laboratories, Milan. by Dr. A. Temperilli. The NNCD reagent was purchased from Hopkin & Williams Ltd., Chadwell Heath, Essex, England. Reagents and solvents used throughout the investigation were of analytical grade.
Paper electrophoresis
RI
39
skin
1
INOOLE COMPOUNDS
1
SKIN
AMPHiBIANSi
OF AUSTRALIAN
08
a7
a6
05
04
a3
a2
0.1
ETHANOL ELUATE Fig. 1. Extracts of the skin of amphibians from Australia and Papua New Guinea. Paper chromatograms run in butanol:acetic acid:water showing Rf values of the different indole compounds eluted from alumina columns by descending concentrations of ethanol. 5-HTOL, 5-hydroxytryptophol; TQA, quaternary ammonium derivative of tryptamine; BT, bufotenine; BTD, bufotenidine; BTD-S, O-sulphate of bufotenidine; BT-S, 0-sulphate of bufotenine; 5-HMT, 5-hydroxy-N’-methyltryptamine; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid; TP, tryptophan; IIIIIIII, indole compounds IIIIIIII, see text.
M.
40
ROSEGHINI,V. ERSPAMERAND R. ENDEAN
male and female specimens respectively is shown. To obtain some idea of the amine content of fresh skin the tabulated data should be divided by 3.554.0. Numerous experiments that will be reported on in detail elsewhere have demonstrated that drying of the skin does not cause qualitative changes in the spectrum of biogenic amines present, nor does it result in important quantitative changes. The identification of the new or rare indole derivatives bufotenine O-sulphate, bufotenidine O-sulphate and tryptamine quaternary ammonium base, has been discussed in another paper (Roseghini, Endean & Temperilli, 1976).
: m
’
’ CH*-T ‘+tN(CH,),
“‘o,s.o~~CHz~t~~~“~,~
H” Tryptomine
trimethylommonium
Bufotenidine
0-sulphote
Tabulated data do not cover the full spectrum of indole derivatives occurring in the skin of Australian amphibians. To complete this spectrum it is necessary to describe briefly some hitherto unidentified compounds that are peculiar to a limited number of species or even to a single species. Some of them are certainly of indole nature (indoles I-III) but for others their indole nature is only presumed. Indole I. This compound is present in extracts of several Litoria and Cyclorana species (see last column of Table 1). It shows the following characteristics: elution from an alumina column by 9&80x ethanol; on paper chromatograms, Rf 0.5 in butanol:acetic acid and 0.594.66 in butanol:methylamine; yellow to dirty orange colour with NNCD, brown-red with the Pauly reagent, dirty blue-violet with the Gibbs reagent, and grey-blue with p-dimethylaminobenzaldehyde. Electrical mobility: E1,z = 0.88 5-HT, E5.s = 0.65 5-HT. Iadole II. This compound is present in extracts of Nictimystes disrupta. It is similar to indole I in its colour reactions. However, it is eluted from alumina columns, together with bufotenidine, by 95-90% ethanol, and its Rf values are 0.52 in butanol:acetic acid, and 0.86 in butanol:methylamine. Other minor spots similar to those produced by mdoles I and II may be occasionally seen on chromatograms of eluates of Cyclorana. Indole 111. This compound is present in Pseudophryne, Uperoleia and probably in several Litoriu species. Elution from alumina by 80% ethanol, immediately before 5-HT; RfO.47Xl.5 in butanol:acetic acid; livid red colour with NNCD. Litoriu moorei. Ethanol eluates 70, and 702. Chromatographic spots with Rf values of 0.76 in butanol: acetic acid and 0.2 in butanol:methylamine; plasmayellow colour with NNCD and slight blue, turning to intense blue, with p-dimethylaminobenzaldehyde. Pauly and Gibbs reactions were apparently negative. Litoriu lesueuri, L. dentutu and L. rubella. Crude extracts. Chromatographic spot showing Rf O&%0.9 in butanol:acetic acid; yellow or orange colour with NNCD, yellow-brownish with the Pauly reagent and violet with p-dimethylaminobenzaldehyde. Cyclorunu platycephulus. Ethanol eluate 99. Chromatographic spot with Rf value of 0.55 in butanol: acetic acid and 0.83 in butanol:methylamine; intense
peach red colour with NNCD, grey-blue with the Gibbs reagent, faint brown-red with the Pauly reagent, doubtful reaction with p-dimethylaminobenzaldehyde; E,,, = 0.82 5-HT, E,,, = 0.73 5-HT. Adelotus hreuis. Crude extracts. Two chromatographic spots presenting Rf values of 0.9 and 0.72, respectively, in butanol:acetic acid; lemon-yellow colour with NNCD, light orange-brown with the Pauly reagent and light greenish with the Gibbs reagent; the p-dimethylaminobenzaldehyde reaction was negative. Pseudophryne coriaceu. Ethanol eluates 951-952-90. Two large chromatographic spots. Spot (1): Rf 0.7 in butanol:acetic acid and Rf 0.9 in butanol:methylamine; intense orange-yellow colour with NNCD, slight lemon-yellow with the Pauly reagent, bluish or greenish with the Gibbs reagent, light yellow-brown turning to green with pdimethylaminobenzaldehyde; Ei,* = 0.84 5-HT, E5.s = 0.9 5-HT. Spot (2): Rf0.63 in butanol:acetic acid and Rf 0.9 in butanol:methylamine; intense orange colour with NNCD, slight brown with the Pauly reagent, very slight greenish with the Gibbs reagent; E,.* = 1.16 5-HT. Es,s = 1.2 5-HT. Ethanol eluates 50 and 40. One large chromatographic spot : IZf 0.760.8 in butanol : acetic acid and Rf 0.67 in butanol:methylamine; orange colour with NNCD, faint yellowish with the Pauly reagent, faint yellow-greenish with the Gibbs reagent, and lilac with p-dimethylaminobenzaldehyde; E1,2 = 0.84 5-HT, E 5,8 = 0.24 5-HT. Pseudophryneglntheri. Ethanol eluates 702-60,-602. Large chromatographic spot: Rf 0.63 in butanol:acetic acid; orange-yellow turning to greenish-yellow with NNCD, red-brown with the Pauly reagent, grey-blue with the Gibbs reagent, and sky blue with p-dimethylaminobenzaldehyde. Uperoleia rugosa and U. marmoruta. Ethanol eluates (95,)-952-90. Two chromatographic spots. Spot (1): Rf 0.87 in butanol:acetic acid and 0.75 in butanol:methylamine; yellow colour with NNCD and the Pauly reagent, brownish-yellow turning to slight brownish-violet with p-dimethylaminobenzaldehyde; E1,2 - 0.484.56 5-HT, E5,s = 0.25 5-HT. Spot (2): Rf 0.74 in butanol:acetic acid and 0.32 in butanol:methylamine; violet colour with NNCD, slight brownrose with the Pauly reagent, green-blue with p-dimethylaminobenzaldehyde; E1.2 = 0.4 5-HT, E5,s = 0.1 5-HT. Ethanol eluates 50, and 502. One chromatographic spot: Rf0.85 in butanol:acetic acid and 0.25 in butanol: methylamine; yellow colour with NNCD. Myobatrachus youldii. Ethanol eluate 60. Two very large chromatographic spots. Spot (1): Rf 0.68 in butanol:acetic acid and 0.75 in butanol:methylamine; plasma-yellow turning to intense brown-yellow with NNCD, light yellow-brown with the Pauly reagent, light grey turning to violetgrey with the Gibbs reagent, yellowish turning to greenish and then to livid red with p-dimethylaminobenzaldehyde; E1.2 = 0.05 5-HT, E5.a = 0.15 5-HT. Upon hydrolysis for 2 hr at 100°C with glacial acetic acid Rf values changed to 0.75 in butanol:acetic acid and to 0.87FO.92 in butanol:methylamine; NNCD gave a rose red colour, Pauly reagent an in-
Biogenic amines in amphibian skin tense wine-red colour, p-dimethylaminobenzaldehyde a slowly developing greenish colour. Spot (2): Rf 0.53 in butanol: acetic acid and 0.6 in butanol: methylamine; light rose colour with NNCD and the Pauly reagent, light bluish with the Gibbs reagent. Upon hydrolysis with acetic acid, Rf values changed to 0.75 in butanol:acetic acid and to 0.83-0.85 in butanol:methylamine. Colour reactions were nearly the same as for spot (1) after hydrolysis. Ethanol eluates 8&(70). One large chromatographic spot : Rf 0.63 in butanol : acetic acid and 0.77 in butanol:methylamine; dirty yellow colour with NNCD, light yellow turning to canary-yellow and later to brownish or dirty green with p-dimethylaminobenzaldehyde, no reaction with the Pauly and the Gibbs reagents; El,* = 0.12 5-HT, E5,s = 0.36 5-HT. Upon treatment with acetic acid, Rf values changed to 0.7M.73 in butanol:acetic acid and to 0.93 in butanol:methylamine. NNCD gave a light brownish spot surrounded by a rose-red boundary. Visual comparison of the sizes of spots produced on paper chromatograms by the above unidentified compounds with the sizes of spots produced by known amounts of 5-HT demonstrated that these compounds occur in the skin in conspicuous amounts, ranging from 5&100 up to lOOOpg/g dry skin. Imidazolealkylamines
and related compounds
Like indolealkylamines, imidazole compounds are well represented in the amphibian skin. However, they are not as common as indolealkylamines. Apart from histidine, an obligatory constituent of all amphibian tissue extracts, and N’-acetylhistidine, present in 5-20 pg/g amounts in all skin extracts, histamine was the imidazole derivative which occurred most frequently and abundantly. The species in which it was present are listed below; the letters in parentheses refer to the different batches (see Table 1). The concentrations of histamine and related amines, in pg per g dry tissue, are expressed in terms of free bases. Litoria L. L. L. L. L. L. L. L. L. L.
aurea
chloris glandulosa dayi eucnemis nannotis infiafienata booroolongensis raniformis citropa
aui-ea
L. gilleni L. moorei L. adelaidensis Taudactylus acutirostris 7: diurnus Limnodynastes dorsalis terrareginae Kyarranus loueridgei Asterophrys rufescens
140(a), 240(b), 300(c), 330(d), 240(e), 150(f) 85590(b) 500 10(a), 75(b), 25(a) Wa), 35(b), 67(c) 65(a), 22(b), 24(c)
170(a), 320(b), 150(c), 390(d) 90(a) 350(a) 600-700
45&500(a), 230(d)
50-60(b), 440(c),
25 80(a), 600(b), 380(c) 25(b) 20 25(a), 6&65(b), 32(c)
3-5(a) 8-12 18-20
41
HN~CH2-iL C” 3
N’- Acetylhistamine
h2
Spinaceamine N’, N’-Dimethylhistomine
Wherever histamine is present in large amounts it appears to be accompanied by N’-acetylhistamine. However, because acetylhistamine is present in low concentration it may be traced and estimated only on chromatograms of eluates from alumina columns. A few quantitative data are as follows: Litoria caerulea L. aurea L. moorei
5(a), 3-5(d), 335(e) 15(a), 18-20(b), 25(c), 5(d) 12-20(b), 30(c)
N’-methylation and cyclization processes of histamine were uncommon. However, N’-methylated and cyclized histamines could be clearly demonstrated in three species: Nictimystes disrupta: N’-methylhistamine, 100-150 pg/g; N’N-dimethylhistamine, 5 pg/g; spinaceamine, 7 pg/g; 6-methylspinaceamine, 30-35 pg/g. Litoria glandulosa: N’-methylhistamine, 15 pg/g. Litoria moorei: spinaceamine, 30 pg/g. Other imidazole derivatives of unknown structure are occasionally found in skin extracts. They will be briefly described in the order in which they emerge from an alumina column. Imidazole I. Elution from alumina by 80-70x ethanol; Rf 0.15-0.23 in butanol: acetic acid and 0.09 in butanol:methylamine; gold-yellow or orange-yellow colour with the Pauly reagent, light dirty violet with the Gibbs reagent and pink orange with the Dragendorff reagent. Imidazole I was detectable in skin extracts of the following species: Crinia pseudoinsignifera, C. tinnula, C. georgiana (3&40 pg/g), C. riparia, C. leai (10 &g), Cyclorana australis, Litoria pearsoniana, Uperoleia rugosa, U. marmorata. Values in parentheses are only
approximate as they are based on comparison with histidine spots. Imidazole II. Present only in skin extracts of Crinia leai and C. rosea. Elution from alumina by 50% ethanol; Rf 0.3330.37 in butanol:acetic acid and 0.07 in butanol:methylamine; pink colour with the Pauly reagent. Imidazole II was scarcely distinguishable from N’-acetylhistidine, its spot showing up on paper chromatograms either slightly above or slightly below but always in contact with that of N’-acetylhistidine. Expressed in terms of this amino acid the content of imidazole II in Crinia leai was 25G3OOpg/g and in C. rosea it was 5Opg/g. Imidazole III. Present in skin extract of C. pseudinsignifera and C. subinsignifera. Elution from alumina by 40% ethanol; Rf 0.880.85 in butanol:acetic acid; pink colour with the Pauly reagent. Expressed in terms of imidazole acetic acid the content of imidazole III for C. pseudoinsignifera was 10&150 pg/g and for C. subinsignifera it was 2540 pg/g.
42
M. ROSEGHINI, V. ERSPAMER AND R. ENDEAN
3
40
30
20
Hz0
ETHANOL E_LUATE
Fig. 2. Extracts of the skin of amphibians from Australia and Papua New Guinea. Paper chromatograms run in butanol:acetic acid:water showing Rf values of the different imidazole compounds eluted from alumina columns by descending concentrations of ethanol. AcH, N’-acetylhistamine; DMH, N’,N’dimethylhistamine; MSP, 6-methylspinaceamine; MH, N’-methylhistamine; SP, spinaceamine; H, histamine; AcHD, N’acetylhistidine; HD, histidine; ILII-III, imidazole spots I-II-III, see text. A schematic representation of the most important known and unknown imidazole derivatives found in Australian amphibians is given in Fig. 2. Still other spots of probable imidazole nature may be visualized on paper chromatograms by the Pauly reagent. Because they are usually small they have not been investigated further.
Catecholamines have never been detected in our skin extracts. Although it is possible that the apparent absence of these compounds stems from their alteration during drying of the skins and preparation of the extracts it is more likely that they are really lacking. Indeed, catecholamines, or their metabolites ‘could hardly have escaped detection during the chemical and pharmacological screening procedures used. Likewise leptodactyline, the quaternary ammonium base of m-tyramine that occurs sometimes in very large amounts in the skins of South American leptodactylid frogs was never found in Australian amphibians Occasionally the Pauly and Gibbs reactions showed up on chromatograms small spots that were probably of a phenolic nature. However, none of these spots could be identified using the pure phenylalkylamines available. Attempts to isolate compounds responsible for the positivity of chromatographic reactions were not made because their low concentration in the skin indicated that insufficient material would be available for a detailed analysis. DISCUSSION
The data presented in this paper were obtained from a study of the skins of approximately one
hundred amphibian species belonging to a representative selection of the amphibian species known to occur in Australia and Papua New Guinea. Thus the spectrum of aromatic biogenic amines and related compounds found in these skins may be regarded as reasonably complete. In the indolealkylamine series, 5-HT appeared to be a normal constituent of the skin in most of the species examined. The concentration of the amine varied from a few pg up to ZOOO,ng/g dry skin. Large differences in 5-HT content could be seen in different batches of skins from the same species. This was true also for all other amines detected. 5-Hydroxytryptophan, the necessary precursor amino acid of 5-HT, was not detected in the skins. In some species the cutaneous tissue appeared to lack N’-methyltransferase activity, but in other species this activity was intense, as shown by the presence of the whole series of N’-methylated derivatives of 5-HT: N’-methyl-5-HT, bufotenine and bufotenidine. The concentration of the latter quaternary ammonium base may attain values as high as 9500 pg/g (Nictimystes disrupta). Tryptamine, which has never been detected as such in the materials examined, occurred in Litoria moorei as its quaternary ammonium derivative (Roseghini, Endean & Temperilli, 1976). Skin extracts often contained appreciable amounts, up to 15Opg/g, of 5-hydroxyindoleacetic acid and of 5-hydroxytryptophol, both originating from the oxidative deamination of 5-HT, N’-methyl-5-HT and, to a lesser degree, bufotenine. It is probable that most of the above metabolites are the result of post-mortal deamination processes occurring during drying of the skin. However, the possibility cannot be excluded that minor aliquots may occur in the living tissue. Examples of O-conjugation with sulphuric acid were relatively rare. However, the skin of L. pear-
Biogenic amines in amphibian skin soniana contained large amounts of the O-sulphate of bufotenine, and that of Nictimystes disrupta large amounts of the 0-sulphate of bufotenidine (Roseghini, Endean & Temperilli, 1976). Cyclized 5-hydroxyindoles, such as dehydrobufotenine and bufothionine were completely lacking. In the imidazolealkylamine series the compound occurring most frequently in the skin was histamine, in concentrations ranging from a few pg up to SOOpg/g. All histamine derivatives, known to occur in amphibian skin (Erspamer, Vitali & Roseghini, 1964; Erspamer, Roseghini & Cei, 1964) were represented in Australian amphibians: N’-acetylhistamine was relatively common, whereas N’-methylated histamines (N’-methylhistamine and N’,N’-dimethylhistamine) and histamines cyclized to give origin to tetrahydroimidazopyridine compounds (spinaceamine and 6-methylspinaceamine) occurred less frequently. The constellation of amines herein described presupposes, of course, a constellation of enzymes catalysing first the formation of the primary amines from their precursor amino acids (L-tryptophan-5-hydroxylase, aromatic acid decarboxylase(s)), then the transformation of the primary amines into their different derivatives (N’-acetyltransferase, N’-methyltransferase(s), 0-sulphoconjugase, cyclizing enzymes).
43
Some of the indole and imidazole compounds occurring in the amphibian skin have been identified with known substances; several other compounds still await isolation and elucidation of their structure. We are convinced that the study of these unknown comrepresenting novel biological molecules, pounds, would be rewarding. Among other things, new findings in the amphibian skin might well pave the way for new findings in mammals and man. This paper will be supplemented by another dealing with active polypeptides in the same batches of skins. Acknowledgement-This investigation was supported by grants from the Consiglio Nazionale delle Ricerche, Rome. REFERENCES
ERSPAMER V., ROSEGHINI M. & CEI J. M. (1964) Indole-,
imidazole- and phenylalkylamines in the skin of thirteen Leptodactylus s&xi&. B&hem. Pharmac. 13, 1083-1093. ERSPAMERV.. VITALI T. & ROSEGHINI M. (1964) The identification of’new histamine derivatives in the skin of Leptodactylus. Archs. Biochem. Biophys. 105, 62&629. ROSEGHINI M., ENDEAN R. & TEMPERILLI A. (1976) New and uncommon indole- and imidazole-alkylamines in the skin of amphibians from Australia and from Papua New Guinea. Z. Naturforschg. (In press).
Bmhm
Physd.,
IY76,
Vol. 54C. pp 31 to 43. Perqamon
Press. Prmted ,,I Grror Brimn
INDOLE-, IMIDAZOLEAND PHENYL-ALKYLAMINES IN THE SKIN OF ONE HUNDRED AMPHIBIAN SPECIES FROM AUSTRALIA AND PAPUA NEW GUINEA M. ROSEGHINI’,
V. ERSPAMER AND R. ENDEAN’
‘Institute of Medical Pharmacology and 2Department of Zoology, University (Received
I, University of Rome, I-00185 Rome, Italy; of Queensland, St. Lucia, Brisbane 4067, Australia 12 November
1975)
Abstract-l. Extracts prepared from the dried skins of approximately one hundred amphibian species from Australia and Papua New Guinea were subjected to chemical and biological screening in order to determine the nature and amounts of biogenic amines in their skins. 2. The most frequently and abundantly occurring amines were indolealkylamines, represented by their prototype 5-HT, by the entire series of N-methylated derivatives of 5-HT (N’-methyl-5-HT, N’,N’dimethyl-5-HT or bufotenine, bufotenidine) and by some new compounds: O-sulphate of bufotenine, O-sulphate of bufotenidine and the quaternary ammonium derivative of tryptamine. 3. Imidazolealkylamines were usually represented by histamine, N’-acetyl-histamine, N’-methylated N’,N’-dimethylhistamine) and cyclized histamines (spinaceamine, histamines (N’-methylhistamine, 6-methylspinaceamine) could be detected only in the skins of a limited number of species. 4. The above spectra of indole- and imidazolealkylamines are destined to broaden, because several other indole and imidazole derivatives in skin extracts have been identified and partly characterized by paper chromatography and electrophoresis. 5. The importance of amphibian skin as a rich source of biogenic amines, and their precursors and metabolites, is emphasized.
extracts were combined. When kept in dark bottles at 4°C the extracts could be stored for long periods of time (months and even years) without appreciable changes in their amine spectrum. Loss of weight during drying of the skins varied from one species to the next, generally it ranged between 70 and 75%
INTRODUCTION skin may be regarded as an enormous storehouse of biogenic amines and active polypeptides. Indeed, no other vertebrate or invertebrate tissue can compare with amphibian cutaneous tissue in regard to variety and concentration of these active compounds. During the past 10 years approx one hundred amphibian species were collected in different regions of Australia and Papua New Guinea. Methanol extracts of their dried skins were submitted to screening for detection of aromatic amines and polypeptides. This paper will present an account of the occurrence in the skins of species examined of indole-, imidazole- and phenyl-alkylamines as well as some of their possible metabolites and precursors. Although the chemical structures of some of these compounds are well known those of other compounds await elucidation. THE amphibian
METHODS Amphibian
AND
Chromatography
on alumina column
A very satisfactory separation of the different biogenic amines, their precursors and metabolites was often obtained by simple passage of crude skin extracts through an alkaline alumina column (Merck, Darmstadt). Chromatographic columns utilized were of different sizes according to the amount of material to be chromatographed. For amounts corresponding to 15-30 g of dry skin the columns were 3.3 cm wide and 50 cm high and the alumina weighed 140-17Og. Columns were double walled with a flow of tap water between the inner and the outer walls, to maintain constant temperature. The crude extract to be chromatographed was evaporated to dryness under reduced pressure, the residue washed with petroleum ether in order to remove fats and then taken up, by stirring in either 99 or in 95”/, ethanol. The liquid was passed through the column which was then eluted with descending concentrations of ethanol and ultimately with water. Fractions of 2&220 ml were collected.
MATERIALS
material
Paper chromatography
The amphibian material used in this study is listed in Table 1. Frogs were killed by a blow to the head, their skins removed immediately after killing, carefully spread out and dried in the shade. Soon after arrival in the laboratory the skins were cut into small pieces with scissors and immersed in a volume of 80% methanol equivalent to twenty times their weight. The liquid was decanted after a week and the skins again treated with a volume of 80% methanol fifteen to twenty times their weight. The two methanol
The ascending unidimensional technique on Whatman No. 1 paper was routinely employed. Chromatograms were run at 18°C for l&30 hr using the following solvent mixtures: n-butanol:acetic acid:water (40: 10:50), nbutanol: 35:! methylamine (80: 30), and l-pentanol:pyridine: water: 35% methylamine (40:40: 10: 1). For special purposes KC1 20% in water and methylethylketone:pyridine:water:35% methylamine (65: 15: 10:0.5) were also used. 31
M. ROSEGHINI, V. ERSPAMER AND R. ENDEAN
32 Table
1. Indolealkylamines
Species (Source, date of capture, number of skins, average weight of a skin)
in the skin of amphibians
from Australia
and Papua
New Guinea
S-HIAA 5-HTOL
5-HT
5-HMT
220
0
0
220 400
0 0
0 0
40, 10
500 400
0 0
0 0
30, 10 25, 5
180
0
0
20. 4
250
12
15
0
25
35
0
10
0
5
350
0
0
0
70
700
0
20
0
200
16
65
0
0
0
0
0
750
0
5-10
0
0
70
80-100
10
10
3500
90
15, 5
20
0
6000
200
150, 60
BT-S 600
0
7000
350
300
BT-S 500
100
0
25
0
30
20-25
0
60
0
3Wto
0
0
10
0
0
0
0
0
30
0
30
0
0
190
0
L. derlttrta near Brisbane, Q’ld., XII, 1965 5 I skins (0.74 g)
40-50
0
750
0
300
L. hicolor near Brisbane, Q’ld., XII. 1965 58 skins (0.028 g)
0
0
0
0
0
10
0
0
0
0
Litoria caerulea (1) near Brisbane, Q’ld., XII. 1964 179 skins (1.4g); (2) ibidem. 124 skins (1.66 g) 2 (3) ibidem. X-XI. 1965 380 skins (1.34 g) $ (4) ibidem, 302 skins (1.77 g) Y (5) ibidem, I-V. 1969 742 skins (I .64 g) (6) ibidem, I-III. 1970 587 skins (1.46 g) L. peroni (1) near Brisbane, Q’ld., XII. 1964 11 skins (0.24 g) (2) ibidem, XI-XII. 1969 9 skins (0.21 g)
(I) near Brisbane, Q’ld., XII. 1968 1 skin (0.28 g) (2) ibidem, XI-XII. 1969, 1I skins
BT
BTD
Other
indoles
I 50
(0.26 g) L. ,freyciwti near Brisbane, Q’ld., II. 1970 56 skins (0.05 g) L. pearsoniana (1) near Brisbane, Q’ld., II. 1964 40 skins (0.036 g) (2) ibidem, I. 1966 76 skins (0.039 FZ) (3) ibidem, ,1-X11.’ 1969 I17 skins (0.041 g) (4) ibidem, X. 1971 179 skins (0.04 g) (5) ibidem, 1~111. 1973 516 skins (0.045 g) L. grucilenttr near Brisbane, Q’ld., XII. 1965 69 skins (0.08 g) L. ruhellu (I) Darling Downs. Q’ld., IV. 1964 24 skins (0.045 g) (2) Chinchilla, Q’ld., XII. 1965 3 1 skins (0.076 g) L. latopalmutu (I) near Brisbane, Q’ld., I. 1965 X skins (0.087 g) (21 Chinchilla. O’ld.. V. 1968 ’ ‘19 skins (0.0&g)
L. nasufa (1) near Brisbane, Q’ld., XI. 1964 4 skins (0.17 g) (2) ibidem, XII. 1965, 4 skins (0.2 g)
3(f40
0
0
0
0
0
L. lesueuri near Brisbane, Q’ld., XII. 1965 38 skins (0.13 g)
150
0
5
0
20
L. qlandulosa Stanthorpe. Q’ld., XII. 1973 6 skins (0.17 g)
250
0
0
6000
100
I 30
BT-S 3&50 BT-S 150 BT-S 100-150
Biogenic
amines Table
Species (Source, date of capture, number of skins, average weight of a skin)
in amphibian
33
skin
l-continued
BT
BTD
5-HIAA .5-HTOL
5-HT
5-HMT
175
0
0
0
80
300 125
0 0
0 0
0 0
l(fl5 75
700
0
150
260 50
0 0
100 10
350
0
0
0
30
400
0
0
0
25
350
0
0
0
50
0
0
0
0
0
0
0
0
0
0
40
0
0
0
0
500
0
0
0
50
1750
0
0
0
400
330
0
0
0
80
2250
0
0
0
200
500
50
0
1850
50
400
0
0
1600
130
1600
0
0
1300
80
2200
0
0
550
0
0
25
80, 50
4500
0
0
55
320, 250
3000
0
0
60
80, 150
60
0
2300
280
70
0
0
870
270
0
60
0
0
0
0
750
80
3700
40
1700
200
5500
30, 30
600
40
5000
75. 30
Other
indoles
L. dayi
(1) Tinaroo Creek, North Q’ld., VIII. 1968, 9 skins (0.17 g) (2) ibidem, XI. 1968, 5 skins (0.1 g) (3) ibidem, I. 1969, 40 skins (0.1 g) .L. eucnemis
(1) Tinaroo Creek, North Q’ld., XII. 1968, 7 skins (0.19g) (2) ibidem, IV. 1969, 4 skins (0.19 g) (3) Madang, Papua New Guinea, II. 1970 32 skins (0.11 g) L. narmotis
(1) Mareeba, North Q’ld., XII. 1967 6 skins (0.13 g) (2) ibidem, VIII. 1968 39 skins (0.09 g) (3) Cairns, North Q’ld., XII. 1968 3 skins (0.2g) L. nigrofrenata
(1) Proserpine, North 10 skins (0.17g) (2) ibidem, iI1. 1969 6 skins (0.2 g)
Q’ld., XII. 1968
L. rothi
North Q’ld.. I. 1965 6 skins (0.19 g) L. infrafrena
ta
(1) Cairns, North Q’ld., I. 1965 3 skins (3.74 g) (2) Papua New Guinea, I. 1968 2 skins (1.8 g) (3) Madang, Papua New Guinea, II. 1970 7 skins (1.8 g) (4) ibidem, X. 1968, 15 skins (2.8 g) L. hooroolongensis
Armidale, N.S.W.. I. 1967 13 skins (0.074 g) L. ran~formis
N.S.W..
VI. 1972, 8 skins (0.5g)
I > 100
L. citropa
Sydney,
N.S.W., VI. 1974, 6 skins (0.25g)
L. aurea
(1) near Melbourne, Victoria, I-III. 178, 731 skins (0.48 g) (2) ibidem, XII. 1972-I. 1973 419 skins (0.62 g) (3) ibidem, II-III. 1973 230 skins (0.76 g) (4) ibidem, XI-XII. 1973 1018 skins (0.59 g)
3040
750, 120
L. ewingi
(1) Victoria, XII. 1968 15 skins (0.023 g) (2) ibidem, XII. 1969 12 skins (0.037 g) L. gilleni
Alice Springs, Central Australia, IV. 1967, 18 skins (1.2g) L. moorei
(1) near Perth, W.A., V. 1968 1 skin (0.37g) (2) Walpole, W.A., IV. 1973 29 skins (0.66 g) (3) Albany. W.A.. IV. 1973
TQA
3c-50
M. ROSEGHINI, V. ERSPAMER AND R. ENDEAN
34
Table Species (Source, date of capture, number of skins, average weight of a skin L. adeluidensis (1) near Perth, W.A., V. 1968 17 skins (0.05 g) (2) Walpole, W.A., IV. 1973 27 skins (0.12 g) L. cy&Jrh_Ynchus Albany, W.A., V. 1973, 12 skins (0.038g) L. darlingtoni (1) Goroka, Papua New Guinea, IV. 1971 24 skins (0.12 g) (2) Mt. Hagen, Papua New Guinea, IV. 1971 28 skins (0.12 g) (3) Arumanda, Papua New Guinea, III. 1972 161 skins (0.087 g) L. iris (1) Mt. Hagen, Papua New Guinea, IV. 1971 59 skins (0.035 g) (2) Arumanda, Papua New Guinea, III. 1972 73 skins (0.05 g)
l-continued
5-HIAA 5-HTOL
5-HT
5-HMT
150
75
0
300
50
600
140
100
I200
150
150
0
0
800
20
90
0
0
12
70
0
0
10
120
0
0
35
7
0
0
0
0
10
0
0
0
0
BT
BTD
L. angiana Goroka, Papua New Guinea, 49 skins (0.23 g)
III. 1972
30
0
0
65
10
L. micromenlhrana Madang, Papua New Guinea, 36 skins (0.14 g)
III. 1972
50
0
0
60
20
L. thesaurensis Goroka. Papua New Guinea, 75 skins (0.046g)
III. 1972
0
0
35
350
30
1
0
0
0
0
0
0
0
0
1
0
0
0
0
3
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
Linlnodynastes peroni (1) near Brisbane, Q’ld., VIII. 1969 28 skins (0.31 g) (2) Murray Bridge, S.A., IV. 1969 6 skins (0.4 g) L. or~lfltus near Brisbane.
2-3
I. 1965, 41 skins (0.12g)
L. ,jlrtcheri (1) Darling Downs, Q’ld.. III. 1966 9 skins (0.11 g) (2) Dalby, Q’ld., II. 1970 8 skins (0.2 g) L. dorscdis rerrarryinae (1) Rockhampton, North Q’ld., I. 1970 7 skins (1.38 g) (2) Darling Downs, Q’ld., XII. 1965 6 skins (0.65 g) (3) near Perth, W.A., V. 1970 12 skins (0.12 g)
5-7
L. dorsalis duwwrili (I) Castlemaine, Victoria, II. 1969 6 skins (0.31 g) (2) South Australia. III. 1969 3 skins (0.7 g) L. tasmaniensis (1) Mount Gambier, S.A., III-V. 1969 59 skins (0.061 g) (2) near Brisbane, Q’ld., XI. 1969 3 skins (0.18 g) L. salmini Rockhampton, North 5 skins (1.05 g)
Q’ld., IX. 1969
Adrlotus hrevis Killarney, Q’ld., XII. 1964 39 skins (0.12 g)
l-2
Other
indoles
I 50 I 12(f180
III
Biogenic
amines Table
Species (Source, date of capture, number of skins, average weight of a skin Lechriodus j!etcheri Mt. Tamborine, Q’ld., I. 1965 33 skins (0.12 g)
in amphibian
35
skin
l-continued
BT
BTD
5-HIAA 5-HTOL
5-HT
5-HMT
l&12
0
0
0
0
Other
K~urranus loveridgei near Brisbane, Q’ld., V. 1965 18 skins (0.065 g)
0
0
0
0
0
Mixophvrs fasciolatus near Brisbane. Q’ld., XII. 1964 17 skins (0.63 g)
0
0
0
0
0
M. schetiilli Speerwah, North 2 skins (1.05g)
0
0
0
0
0
0
0
0
0
0
75
0
0
0
5
200
0
0
0
30
100
0
30
5
10
60
0
40
20
I 100
5
50
0
100
0
I 150
20
20
0
0
25
I 50
280
180
0
0
60
I 120
700
0
0
0
40
10
0
0
0
0
7
80
0
40
5
80
0
0
0
110
0
0
0
0
0
0
30
0
0
0
20
0
0
0
40
0
0
5
100
0
0
10
50
0
0
0
0
0
Q’ld., I. 1970
Notaden hrnnetti Condobolin. N.S.W., II-III. 28 skins (0.96 g)
1973
Cyclorana australis (1) Dalby, Q’ld., III. 1966 2 skins (1.24 g) (2) Darling Downs, Q’ld., III. 1971 15 skins (1.5 g) C. alboguttatus (I) Dalby. Q’ld., I. 1965 6 skins (0.38 g) (2) Darling Downs, Q’ld., XII. 1965 14 skins (0.36 g) (3) Condobolin, N.S.W., I-II. 1973 18 skins (0.46 g) C. cultripes (1) Darling Downs, Q’ld., III. 1966 I2 skins (0.175 g) (2) Condobolin, N.S.W., II. 1973 34 skins (0. IX g) C. breripes Condobolin. N.S.W., II. 1973 22 skins (0.2 g)
2G25
C. platprephalus (1) South Q’ld., XI 1969 14 skins (0.15g) (2) Condobolin, N.S.W., II. 1973 20 skins (0.35 g) Uprroleia rugosa South Q’ld. and N.S.W., 108 I skins (0.055 g)
196991973
U. marmorata near Brisbane, Q’ld., 196991971 147 skins (0.061 g) U. laerigata near Brisbane, Q’ld.. II. 1970 8 skins (0.09 g) Taudact)alus acutirostris (1) Tinaroo Creek, North Q’ld., III. 1968. 12 skins (0.025 g) (2) Kuranda, North Q’ld., II. 1970 56 skins (0.024 g) T. diurrzus (I) Mt. Glorious, Q’ld., XII. 1968 19 skins (0.047 g) (2) ibidem, II. 1969 39 skins (0.043 g) (3) ibidem, III. 1969 53 skins (0.053 g) Sphenophrynr plwialis Kuranda, North Q’ld., X. 1968 28 skins (0.02 g)
3-t
2-3
0
indoles
I 50
I 100-150
III 10
36
M. ROSEGHINI, V. ERSPAMER AND Table Species (Source, date of capture, number of skins, average weight of a skin
Crinia
R. ENDEAN
l&continued
5-HIAA 5-HTOL
5-HT
5-HMT
1000
0
0
0
20-30
380
0
0
0
20
170
0
0
0
1200
0
0
0
300
0
0
0
0
0
0
0
0
70
0
0
0
0
80
0
0
0
0
3t340
0
0
0
0
440
0
0
0
30
0
0
0
3&35
130
0
0
0
10
25530
0
0
0
0
40
0
0
0
0
25-30
0
0
0
0
500-600
0
0
0
20
2200
0
0
0
40
360
0
0
0
40-50
300
0
0
0
20
90-120
0
0
0
0
90-110
0
0
0
0
0
0
0
0
0
0
0
40
BT
BTD
Other
indoles
pseudoinsiynijera
(1) near Perth, W.A., V. 1968 32 skins (0.024 g) (2) Albany, W.A., IV. 1973 176 skins (0.028 g) Cr. ,suhinsignijereru Albany, W.A., III. 1973 124 skins (0.021 g) Cr. insignifera near Perth, W.A., IX. 1971 17 skins (0.02 g) Cr. rosen Albany, W.A., IV. 1973 38 skins (0.037 g)
lo-15
70
3(f35
Cr. leai
Walpole, W. A., IV. 1973 300 skins (0.034 g) Cr. glauerti (1) near Perth, W.A., V. 1970 43 skins (0.007 g) (2) Albany, W.A., IV. 1973 168 skins (0.012 g) Cr. georgiana near Perth, W.A., IX. 1971 16 skins (0.056 g) Cr. signijera
South Australia, 196991970 52 skins (0.027 g) Cr. riparia Adelaide, S.A., IX. 1971 67 skins (0.053 g) Cr. parinsignifera Castlemaine, Victoria, 27 skins (0.022 g)
II. 1969
45G-500
Cr. tinnula
(1) Beerwah, South Q’ld., III. 1970 95 skins (0.013 g) (2) ibidem. IV. 1970, 48 skins (0.01 g) Metacrinia
nichollsi
Albany, W.A., III-IV. 88 skins (0.023 g) Pseudophryne
1973
giintheri
(1) Wagin, W.A., V. 1968 44 skins (0.071 g) (2) near Perth. Wi., V. 1970 76 skins (0.083 g) (3) Walpole, W.A., IV. 1973 105 skins
III 50-60
Ps. coriacea
(1) Bald Mountain, Q’ld., I. 1972 90 skins (0.083 g) (2) ibidem, XI. 1972, 76 skins (0.085 g)
III 8-10
Ps. hihroni
(1) Armidale, N.S.W., IV. 1966 34 skins (0.045 g) (2) Adelaide, S.A., V. 1970 23 skins (0.056g) (3) Moggill, Q’ld., V. 1972 27 skins (0.064 g) Ps. corroboree New South Wales,
12 skins (0.06 g)
III. 1967
40
80-100
III 15
Biogenic
amines Table
Species (Source, date of capture, number of skins, average weight of a skin) Ps. svmimarmorata South Australia, IV. 1969 20 skins (0.057 g)
5-HT
in amphibian
skin
37
l-continued
5-HMT
BT
BTD
5-HIAA 5-HTOL
700
0
50
0
0
0
N. pelohatoides near Perth. W.A., V. 197O/IV. 1973 45 skins (0.14 g)
0
0
0
N. centralis Walpole, W.A., IV. 1973 25 skins (0.24 g)
0
0
0
30
0
15
120
0
10
5-20
0
35
0
Neohutruchus pictus (1) near Adelaide, S.A., III. 1969 5 skins (0.47g) (2) Condobolin, N.S.W., V. 1972 20 skins (0.2 g)
He2eioporus eyrei (1) near Perth, W.A., V. 1970 68 skins (0.32 g) (2) Albany, W.A., IV. 1973 16 skins (0.5 g) H. psammophilus (1) near Perth, W.A., V. 1970 37 skins (0.3 g) (2) Albany, W.A., IV. 1973 38 skins (0.18 g) H. albopunctatus (1) Wagin, W.A., V. 1968 1 skin: glandular areas (0.3 g) remaining skin (0.85-g) (2) Walpole. W.A.. IV. 1973 ‘36 skins (1.2g) H. inornatus near Perth, W.A., V. 1970 2 skins (0.55 g) Myobatrachus gouldii Walpole, W.A., IV. 1973 36 skins (0.28 g) Nictimystes tympanocryptis (1) Speerwah, North Q’ld., I. 1970 22 skins (0.06 g) (2) ibidem, II-III. 1970 47 skins (0.055 g) (3) Millaa Millaa Falls, North Q’ld., V. 1970, 105 skins (0.06g) (4) ibidem, XI. 1970 152 skins (0.058 g) N. oestigea (I) North Q’ld., I. 1970 26 skins (0.08 g) (2) Speerwah, North Q’ld., II-III. 1970 18 skins (0.06 g) (3) Palmerston, North Q’ld., XI. 1970 55 skins (0.06g)
10
600 10 250
0 0 0
0 0
0 0
0
0
45-50
0
0
0
2-3
50
0
0
0
3-4
0
0
8800
0
BTD-S
300
0
0
9500
0
BTD-S
300
0
0
8400
0
BTD-S
225
0
0
5100
0
BTD-S
250
0
0
550
0
0
115
0
0
20 0 10
65
III. 1972
1200
400
0
4300
N. kubori Goroka, Papua New Guinea, 71 skins (0.11 g)
III. 1972
0
0
85
250
20
0
0
0
0
0
III. 1971
indoles
610
N. disrupta Goroka, Papua New Guinea, 3 1 skins (0.46 g)
Asterophrys rufescens Lae, Papua New Guinea, 2 skins (0.085 g)
Other
60, 20
II 150
38
M. ROSEGHINI, V. ERSPAMER AND Table Species (Source, date of capture, number of skins, average weight of a skin
R. ENDEAN
l--continued
S-HT
5-HMT
BT
BTD
S-HIAA 5-HTOL
A. stictogaster Goroka, Papua New Guinea, 23 skins (1.9 g)
III. 1972
0
0
0
0
0
Xenobatrachus rostratus Goroka, Papua New Guinea, 9 skins (0.08 g)
III. 1973
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
70
0
0
0
0
0
0
0
0
0
20
0
0
0
0
40
0
0
0
15-20
120
0
0
0
10
40
0
0
0
20
70
0
0
0
50
125
0
0
0
80
25
0
0
0
10
4
0
0
70
0
0
Platymantis popuensis Lae, Papua New Guinea, 16 skins (0.12 g)
III. 1971
PI. corrugatus Madang. Papua New Guinea, 20 skins (0.084 g)
III. 1972
Cophixalus auriegatus Goroka, Papua New Guinea, 186 skins (0.012 g)
III. 1972
C. cryptotympanum Wau, Papua New Guinea, 51 skins (0.04 g)
III. 1971
C. ornatus North Q’ld., VIII. 1968/I. 122 skins (0.024 g)
1970
Rana Papua (1) Wau, Papua New Guinea, III. 1971 4 skins (0.8 g) (2) Madang, Papua New Guinea, III. 1972 9 skins (0.55 g) R. kreffti Wau, Papua New Guinea, 3 1 skins (0.32 g)
III. 1971
R. grisea Goroka, Papua New Guinea, 3 1 skins (0.52 g)
III. 1972
R. arfhki (1) Madang, Papua New Guinea, I. 1970 2 skins (5.6 g) (2) ibidem, III. 1972, 12 skins (2.03g) R. daemeli (1) Papua New Guinea, I. 1968, 29 skins (0.35 g) (2) Speerwah, North Q’ld., I. 1970 5 skins (0.3 g) (3) Markham Valley, Papua New III. 1971 8 skins (0.53 g)
3-1
0
Other
indoles
0
Guinea,
5-HT, 5-hydroxytryptamine; 5-HMT, 5-hydroxyindoleacetic acid; 5-HTOL, phate; TQA, tryptamine quaternary pounds of supposedly indole nature skin. Values for unknown indoles are
0
10
5-hydroxy-N-methyltryptamine; BT, bufotenine, BTD, bufotenidine; 5-HIAA, 5-hydroxytryptophol; BT-S, bufotenine 0-sulphate; BTD-S, bufotenidine O-sulammonium derivative; I-III, unknown indole compounds. For additional comsee text. Indolealkylamine contents are expressed in pg free base per g dried only approximate, and expressed in pg 5-HT/g.
The spraying reagents used for the detection and identification of the different compounds were as follows: (a) an aqueous solution of diazotized sulphanilic acid (Pauly reagent) followed by 3-5% aqueous sodium carbonate; (b) an aqueous solution of diazotized p-nitroaniline followed by sodium carbonate; (c) a 0.1-0.3% solution of HeinriCh and Schuler NNCD reagent (2-chloro-4_nitrobenzenediazoniumnaphthalene-2-sulphonate) in 0.1 N HCl; (d) a 0.05-0.1% alcoholic solution of dichloroquinone chlorimide (Gibbs reagent) followed by sodium carbonate; (f) a l-2:/, alcoholic solution of p-dimethylaminobenzaldehyde, followed by exposure of the chromatograms to HCl
vapours in a glass cabinet; (g) a 1% alcoholic solution of Folin reagent for amino acids (1,2-naphthoquinone-4sulphonic sodium salt) followed by sodium carbonate; (h) a saturated aqueous solution of Reinecke salt, followed by a gentle washing of the paper sheets under running tap water until the background colour was completely removed; (i) Dragendorff reagent; and finally (j) a 0.2% solution of barium chloride in 70% ethanol followed by 0.05% sodium rhodizonate in 50% ethanol (reagent for sulphates, which appear as yellow spots on a reddish background). Imidazole derivatives are characterized by the positivity
Biogenic
amines
in amphibian
ammonium bases, the frog rectus abdominis muscle. Methysergide (0.2 @g/ml) was used as a 5-HT antagonist and mepyramine maleate (0.1 pg/ml) as a histamine antagonist.
of reactions (a), (b) and eventually (g); phenolic derivatives by the positivity of the same reactions and occasionally of reaction (d); 5-hydroxy-indole compounds by the positivity of all reactions from (a) to (g) inclusive. With tryptamines lacking the phenolic hydroxy group or having this group occupied by conjugation or alkylation, only reactions (c), (f) and (g) are positive. The positivity of reactions (h) and (i) is indicative of the presence of a quaternary ammonium group. Semi-quantitative estimations of the individual compounds on paper-chromatograms were carried out chiefly by visual comparison of spots produced by different amounts of crude skin extracts or ethanol eluates with spots produced by known amounts of the corresponding pure synthetic compounds. If a particular synthetic compound was not available, a closely related synthetic compound was used.
Reagents
A high voltage electrophoresis apparatus from L. Hormuth, Inh. W. E. Vetter (Heidelberg) was used for the electrophoretic experiments. Electropherograms were run at pH 1.2 (glacial acetic acid:85% formic acid:distilled water, 15:25: 110) and pH 5.8 (99% pyridine:glacial acetic acid: distilled water, 15.5: 1.8: 135). Acid hydrolysis Treatment in a boiling water bath, for 3&90 min, with pure acetic acid, which does not attack the indole nucleus appreciably, was routinely used (a) to hydrolyse sulphoconjugated indolealkylamines and (b) to check the effect of acid treatment on unknown compounds.
RESULTS lndolealkylamines Analytical matographic
The qualitative and quantitative bioassay of amines was carried out on the following smooth muscle preparations: for 5-HT and related amines, the oestrous rat uterus; for histamines, the guinea-pig ileum; for quaternary
a9
IN
THE
and related
data, obtained semiquantitative
compounds mainly from paper chroestimations and, if
necessary, supplemented by bioassay, are summarized in Fig. 1 and Table 1. Table 1 not only shows the amine content of skins from different species but also the content in different batches of the same species. For Litoria caerulea the amine content of skins from
Bioassay
I
and drugs
The following synthetic compounds were available for comparison: tryptamine HCl, N,N-dimethyltryptamine 5-hydroxytryptamine creatinine sulphate methiodide*, (5-HT), bufotenine base*, bufotenine 0-sulphate*, bufotenidine*, bufotenidine 0-sulphate*, 5-hydroxyindole acetic acid (5-HIIA), 5-hydroxytryptophol, 5-hydroxy-r-tryptophan, L-tryptophan, histamine 2 HCI, N-methyl-histamine. 2 HCI, N,N-dimethylhistamine 2 HCI, spinaceamine.2 HC1. + HZO, 6-methylspinaceamine.2HC1, N-acetylhistamine, N-acetyl-L-histidine, L-histidine, imidazole acetic acid, p-tyramine HCl, m-tyramine HCl, octopamine tartrate, leptodactyline picrate, L-tyrosine, L-noradrenaline tartrate, N-methyl-5-hydroxytryptamine, which was not available as a synthetic compound, was expressed in terms of 5-HT. Compounds marked with * were synthetised at the Farmitalia Research Laboratories, Milan. by Dr. A. Temperilli. The NNCD reagent was purchased from Hopkin & Williams Ltd., Chadwell Heath, Essex, England. Reagents and solvents used throughout the investigation were of analytical grade.
Paper electrophoresis
RI
39
skin
1
INOOLE COMPOUNDS
1
SKIN
AMPHiBIANSi
OF AUSTRALIAN
08
a7
a6
05
04
a3
a2
0.1
ETHANOL ELUATE Fig. 1. Extracts of the skin of amphibians from Australia and Papua New Guinea. Paper chromatograms run in butanol:acetic acid:water showing Rf values of the different indole compounds eluted from alumina columns by descending concentrations of ethanol. 5-HTOL, 5-hydroxytryptophol; TQA, quaternary ammonium derivative of tryptamine; BT, bufotenine; BTD, bufotenidine; BTD-S, O-sulphate of bufotenidine; BT-S, 0-sulphate of bufotenine; 5-HMT, 5-hydroxy-N’-methyltryptamine; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid; TP, tryptophan; IIIIIIII, indole compounds IIIIIIII, see text.
M.
40
ROSEGHINI,V. ERSPAMERAND R. ENDEAN
male and female specimens respectively is shown. To obtain some idea of the amine content of fresh skin the tabulated data should be divided by 3.554.0. Numerous experiments that will be reported on in detail elsewhere have demonstrated that drying of the skin does not cause qualitative changes in the spectrum of biogenic amines present, nor does it result in important quantitative changes. The identification of the new or rare indole derivatives bufotenine O-sulphate, bufotenidine O-sulphate and tryptamine quaternary ammonium base, has been discussed in another paper (Roseghini, Endean & Temperilli, 1976).
: m
’
’ CH*-T ‘+tN(CH,),
“‘o,s.o~~CHz~t~~~“~,~
H” Tryptomine
trimethylommonium
Bufotenidine
0-sulphote
Tabulated data do not cover the full spectrum of indole derivatives occurring in the skin of Australian amphibians. To complete this spectrum it is necessary to describe briefly some hitherto unidentified compounds that are peculiar to a limited number of species or even to a single species. Some of them are certainly of indole nature (indoles I-III) but for others their indole nature is only presumed. Indole I. This compound is present in extracts of several Litoria and Cyclorana species (see last column of Table 1). It shows the following characteristics: elution from an alumina column by 9&80x ethanol; on paper chromatograms, Rf 0.5 in butanol:acetic acid and 0.594.66 in butanol:methylamine; yellow to dirty orange colour with NNCD, brown-red with the Pauly reagent, dirty blue-violet with the Gibbs reagent, and grey-blue with p-dimethylaminobenzaldehyde. Electrical mobility: E1,z = 0.88 5-HT, E5.s = 0.65 5-HT. Iadole II. This compound is present in extracts of Nictimystes disrupta. It is similar to indole I in its colour reactions. However, it is eluted from alumina columns, together with bufotenidine, by 95-90% ethanol, and its Rf values are 0.52 in butanol:acetic acid, and 0.86 in butanol:methylamine. Other minor spots similar to those produced by mdoles I and II may be occasionally seen on chromatograms of eluates of Cyclorana. Indole 111. This compound is present in Pseudophryne, Uperoleia and probably in several Litoriu species. Elution from alumina by 80% ethanol, immediately before 5-HT; RfO.47Xl.5 in butanol:acetic acid; livid red colour with NNCD. Litoriu moorei. Ethanol eluates 70, and 702. Chromatographic spots with Rf values of 0.76 in butanol: acetic acid and 0.2 in butanol:methylamine; plasmayellow colour with NNCD and slight blue, turning to intense blue, with p-dimethylaminobenzaldehyde. Pauly and Gibbs reactions were apparently negative. Litoriu lesueuri, L. dentutu and L. rubella. Crude extracts. Chromatographic spot showing Rf O&%0.9 in butanol:acetic acid; yellow or orange colour with NNCD, yellow-brownish with the Pauly reagent and violet with p-dimethylaminobenzaldehyde. Cyclorunu platycephulus. Ethanol eluate 99. Chromatographic spot with Rf value of 0.55 in butanol: acetic acid and 0.83 in butanol:methylamine; intense
peach red colour with NNCD, grey-blue with the Gibbs reagent, faint brown-red with the Pauly reagent, doubtful reaction with p-dimethylaminobenzaldehyde; E,,, = 0.82 5-HT, E,,, = 0.73 5-HT. Adelotus hreuis. Crude extracts. Two chromatographic spots presenting Rf values of 0.9 and 0.72, respectively, in butanol:acetic acid; lemon-yellow colour with NNCD, light orange-brown with the Pauly reagent and light greenish with the Gibbs reagent; the p-dimethylaminobenzaldehyde reaction was negative. Pseudophryne coriaceu. Ethanol eluates 951-952-90. Two large chromatographic spots. Spot (1): Rf 0.7 in butanol:acetic acid and Rf 0.9 in butanol:methylamine; intense orange-yellow colour with NNCD, slight lemon-yellow with the Pauly reagent, bluish or greenish with the Gibbs reagent, light yellow-brown turning to green with pdimethylaminobenzaldehyde; Ei,* = 0.84 5-HT, E5.s = 0.9 5-HT. Spot (2): Rf0.63 in butanol:acetic acid and Rf 0.9 in butanol:methylamine; intense orange colour with NNCD, slight brown with the Pauly reagent, very slight greenish with the Gibbs reagent; E,.* = 1.16 5-HT. Es,s = 1.2 5-HT. Ethanol eluates 50 and 40. One large chromatographic spot : IZf 0.760.8 in butanol : acetic acid and Rf 0.67 in butanol:methylamine; orange colour with NNCD, faint yellowish with the Pauly reagent, faint yellow-greenish with the Gibbs reagent, and lilac with p-dimethylaminobenzaldehyde; E1,2 = 0.84 5-HT, E 5,8 = 0.24 5-HT. Pseudophryneglntheri. Ethanol eluates 702-60,-602. Large chromatographic spot: Rf 0.63 in butanol:acetic acid; orange-yellow turning to greenish-yellow with NNCD, red-brown with the Pauly reagent, grey-blue with the Gibbs reagent, and sky blue with p-dimethylaminobenzaldehyde. Uperoleia rugosa and U. marmoruta. Ethanol eluates (95,)-952-90. Two chromatographic spots. Spot (1): Rf 0.87 in butanol:acetic acid and 0.75 in butanol:methylamine; yellow colour with NNCD and the Pauly reagent, brownish-yellow turning to slight brownish-violet with p-dimethylaminobenzaldehyde; E1,2 - 0.484.56 5-HT, E5,s = 0.25 5-HT. Spot (2): Rf 0.74 in butanol:acetic acid and 0.32 in butanol:methylamine; violet colour with NNCD, slight brownrose with the Pauly reagent, green-blue with p-dimethylaminobenzaldehyde; E1.2 = 0.4 5-HT, E5,s = 0.1 5-HT. Ethanol eluates 50, and 502. One chromatographic spot: Rf0.85 in butanol:acetic acid and 0.25 in butanol: methylamine; yellow colour with NNCD. Myobatrachus youldii. Ethanol eluate 60. Two very large chromatographic spots. Spot (1): Rf 0.68 in butanol:acetic acid and 0.75 in butanol:methylamine; plasma-yellow turning to intense brown-yellow with NNCD, light yellow-brown with the Pauly reagent, light grey turning to violetgrey with the Gibbs reagent, yellowish turning to greenish and then to livid red with p-dimethylaminobenzaldehyde; E1.2 = 0.05 5-HT, E5.a = 0.15 5-HT. Upon hydrolysis for 2 hr at 100°C with glacial acetic acid Rf values changed to 0.75 in butanol:acetic acid and to 0.87FO.92 in butanol:methylamine; NNCD gave a rose red colour, Pauly reagent an in-
Biogenic amines in amphibian skin tense wine-red colour, p-dimethylaminobenzaldehyde a slowly developing greenish colour. Spot (2): Rf 0.53 in butanol: acetic acid and 0.6 in butanol: methylamine; light rose colour with NNCD and the Pauly reagent, light bluish with the Gibbs reagent. Upon hydrolysis with acetic acid, Rf values changed to 0.75 in butanol:acetic acid and to 0.83-0.85 in butanol:methylamine. Colour reactions were nearly the same as for spot (1) after hydrolysis. Ethanol eluates 8&(70). One large chromatographic spot : Rf 0.63 in butanol : acetic acid and 0.77 in butanol:methylamine; dirty yellow colour with NNCD, light yellow turning to canary-yellow and later to brownish or dirty green with p-dimethylaminobenzaldehyde, no reaction with the Pauly and the Gibbs reagents; El,* = 0.12 5-HT, E5,s = 0.36 5-HT. Upon treatment with acetic acid, Rf values changed to 0.7M.73 in butanol:acetic acid and to 0.93 in butanol:methylamine. NNCD gave a light brownish spot surrounded by a rose-red boundary. Visual comparison of the sizes of spots produced on paper chromatograms by the above unidentified compounds with the sizes of spots produced by known amounts of 5-HT demonstrated that these compounds occur in the skin in conspicuous amounts, ranging from 5&100 up to lOOOpg/g dry skin. Imidazolealkylamines
and related compounds
Like indolealkylamines, imidazole compounds are well represented in the amphibian skin. However, they are not as common as indolealkylamines. Apart from histidine, an obligatory constituent of all amphibian tissue extracts, and N’-acetylhistidine, present in 5-20 pg/g amounts in all skin extracts, histamine was the imidazole derivative which occurred most frequently and abundantly. The species in which it was present are listed below; the letters in parentheses refer to the different batches (see Table 1). The concentrations of histamine and related amines, in pg per g dry tissue, are expressed in terms of free bases. Litoria L. L. L. L. L. L. L. L. L. L.
aurea
chloris glandulosa dayi eucnemis nannotis infiafienata booroolongensis raniformis citropa
aui-ea
L. gilleni L. moorei L. adelaidensis Taudactylus acutirostris 7: diurnus Limnodynastes dorsalis terrareginae Kyarranus loueridgei Asterophrys rufescens
140(a), 240(b), 300(c), 330(d), 240(e), 150(f) 85590(b) 500 10(a), 75(b), 25(a) Wa), 35(b), 67(c) 65(a), 22(b), 24(c)
170(a), 320(b), 150(c), 390(d) 90(a) 350(a) 600-700
45&500(a), 230(d)
50-60(b), 440(c),
25 80(a), 600(b), 380(c) 25(b) 20 25(a), 6&65(b), 32(c)
3-5(a) 8-12 18-20
41
HN~CH2-iL C” 3
N’- Acetylhistamine
h2
Spinaceamine N’, N’-Dimethylhistomine
Wherever histamine is present in large amounts it appears to be accompanied by N’-acetylhistamine. However, because acetylhistamine is present in low concentration it may be traced and estimated only on chromatograms of eluates from alumina columns. A few quantitative data are as follows: Litoria caerulea L. aurea L. moorei
5(a), 3-5(d), 335(e) 15(a), 18-20(b), 25(c), 5(d) 12-20(b), 30(c)
N’-methylation and cyclization processes of histamine were uncommon. However, N’-methylated and cyclized histamines could be clearly demonstrated in three species: Nictimystes disrupta: N’-methylhistamine, 100-150 pg/g; N’N-dimethylhistamine, 5 pg/g; spinaceamine, 7 pg/g; 6-methylspinaceamine, 30-35 pg/g. Litoria glandulosa: N’-methylhistamine, 15 pg/g. Litoria moorei: spinaceamine, 30 pg/g. Other imidazole derivatives of unknown structure are occasionally found in skin extracts. They will be briefly described in the order in which they emerge from an alumina column. Imidazole I. Elution from alumina by 80-70x ethanol; Rf 0.15-0.23 in butanol: acetic acid and 0.09 in butanol:methylamine; gold-yellow or orange-yellow colour with the Pauly reagent, light dirty violet with the Gibbs reagent and pink orange with the Dragendorff reagent. Imidazole I was detectable in skin extracts of the following species: Crinia pseudoinsignifera, C. tinnula, C. georgiana (3&40 pg/g), C. riparia, C. leai (10 &g), Cyclorana australis, Litoria pearsoniana, Uperoleia rugosa, U. marmorata. Values in parentheses are only
approximate as they are based on comparison with histidine spots. Imidazole II. Present only in skin extracts of Crinia leai and C. rosea. Elution from alumina by 50% ethanol; Rf 0.3330.37 in butanol:acetic acid and 0.07 in butanol:methylamine; pink colour with the Pauly reagent. Imidazole II was scarcely distinguishable from N’-acetylhistidine, its spot showing up on paper chromatograms either slightly above or slightly below but always in contact with that of N’-acetylhistidine. Expressed in terms of this amino acid the content of imidazole II in Crinia leai was 25G3OOpg/g and in C. rosea it was 5Opg/g. Imidazole III. Present in skin extract of C. pseudinsignifera and C. subinsignifera. Elution from alumina by 40% ethanol; Rf 0.880.85 in butanol:acetic acid; pink colour with the Pauly reagent. Expressed in terms of imidazole acetic acid the content of imidazole III for C. pseudoinsignifera was 10&150 pg/g and for C. subinsignifera it was 2540 pg/g.
42
M. ROSEGHINI, V. ERSPAMER AND R. ENDEAN
3
40
30
20
Hz0
ETHANOL E_LUATE
Fig. 2. Extracts of the skin of amphibians from Australia and Papua New Guinea. Paper chromatograms run in butanol:acetic acid:water showing Rf values of the different imidazole compounds eluted from alumina columns by descending concentrations of ethanol. AcH, N’-acetylhistamine; DMH, N’,N’dimethylhistamine; MSP, 6-methylspinaceamine; MH, N’-methylhistamine; SP, spinaceamine; H, histamine; AcHD, N’acetylhistidine; HD, histidine; ILII-III, imidazole spots I-II-III, see text. A schematic representation of the most important known and unknown imidazole derivatives found in Australian amphibians is given in Fig. 2. Still other spots of probable imidazole nature may be visualized on paper chromatograms by the Pauly reagent. Because they are usually small they have not been investigated further.
Catecholamines have never been detected in our skin extracts. Although it is possible that the apparent absence of these compounds stems from their alteration during drying of the skins and preparation of the extracts it is more likely that they are really lacking. Indeed, catecholamines, or their metabolites ‘could hardly have escaped detection during the chemical and pharmacological screening procedures used. Likewise leptodactyline, the quaternary ammonium base of m-tyramine that occurs sometimes in very large amounts in the skins of South American leptodactylid frogs was never found in Australian amphibians Occasionally the Pauly and Gibbs reactions showed up on chromatograms small spots that were probably of a phenolic nature. However, none of these spots could be identified using the pure phenylalkylamines available. Attempts to isolate compounds responsible for the positivity of chromatographic reactions were not made because their low concentration in the skin indicated that insufficient material would be available for a detailed analysis. DISCUSSION
The data presented in this paper were obtained from a study of the skins of approximately one
hundred amphibian species belonging to a representative selection of the amphibian species known to occur in Australia and Papua New Guinea. Thus the spectrum of aromatic biogenic amines and related compounds found in these skins may be regarded as reasonably complete. In the indolealkylamine series, 5-HT appeared to be a normal constituent of the skin in most of the species examined. The concentration of the amine varied from a few pg up to ZOOO,ng/g dry skin. Large differences in 5-HT content could be seen in different batches of skins from the same species. This was true also for all other amines detected. 5-Hydroxytryptophan, the necessary precursor amino acid of 5-HT, was not detected in the skins. In some species the cutaneous tissue appeared to lack N’-methyltransferase activity, but in other species this activity was intense, as shown by the presence of the whole series of N’-methylated derivatives of 5-HT: N’-methyl-5-HT, bufotenine and bufotenidine. The concentration of the latter quaternary ammonium base may attain values as high as 9500 pg/g (Nictimystes disrupta). Tryptamine, which has never been detected as such in the materials examined, occurred in Litoria moorei as its quaternary ammonium derivative (Roseghini, Endean & Temperilli, 1976). Skin extracts often contained appreciable amounts, up to 15Opg/g, of 5-hydroxyindoleacetic acid and of 5-hydroxytryptophol, both originating from the oxidative deamination of 5-HT, N’-methyl-5-HT and, to a lesser degree, bufotenine. It is probable that most of the above metabolites are the result of post-mortal deamination processes occurring during drying of the skin. However, the possibility cannot be excluded that minor aliquots may occur in the living tissue. Examples of O-conjugation with sulphuric acid were relatively rare. However, the skin of L. pear-
Biogenic amines in amphibian skin soniana contained large amounts of the O-sulphate of bufotenine, and that of Nictimystes disrupta large amounts of the 0-sulphate of bufotenidine (Roseghini, Endean & Temperilli, 1976). Cyclized 5-hydroxyindoles, such as dehydrobufotenine and bufothionine were completely lacking. In the imidazolealkylamine series the compound occurring most frequently in the skin was histamine, in concentrations ranging from a few pg up to SOOpg/g. All histamine derivatives, known to occur in amphibian skin (Erspamer, Vitali & Roseghini, 1964; Erspamer, Roseghini & Cei, 1964) were represented in Australian amphibians: N’-acetylhistamine was relatively common, whereas N’-methylated histamines (N’-methylhistamine and N’,N’-dimethylhistamine) and histamines cyclized to give origin to tetrahydroimidazopyridine compounds (spinaceamine and 6-methylspinaceamine) occurred less frequently. The constellation of amines herein described presupposes, of course, a constellation of enzymes catalysing first the formation of the primary amines from their precursor amino acids (L-tryptophan-5-hydroxylase, aromatic acid decarboxylase(s)), then the transformation of the primary amines into their different derivatives (N’-acetyltransferase, N’-methyltransferase(s), 0-sulphoconjugase, cyclizing enzymes).
43
Some of the indole and imidazole compounds occurring in the amphibian skin have been identified with known substances; several other compounds still await isolation and elucidation of their structure. We are convinced that the study of these unknown comrepresenting novel biological molecules, pounds, would be rewarding. Among other things, new findings in the amphibian skin might well pave the way for new findings in mammals and man. This paper will be supplemented by another dealing with active polypeptides in the same batches of skins. Acknowledgement-This investigation was supported by grants from the Consiglio Nazionale delle Ricerche, Rome. REFERENCES
ERSPAMER V., ROSEGHINI M. & CEI J. M. (1964) Indole-,
imidazole- and phenylalkylamines in the skin of thirteen Leptodactylus s&xi&. B&hem. Pharmac. 13, 1083-1093. ERSPAMERV.. VITALI T. & ROSEGHINI M. (1964) The identification of’new histamine derivatives in the skin of Leptodactylus. Archs. Biochem. Biophys. 105, 62&629. ROSEGHINI M., ENDEAN R. & TEMPERILLI A. (1976) New and uncommon indole- and imidazole-alkylamines in the skin of amphibians from Australia and from Papua New Guinea. Z. Naturforschg. (In press).