I'cl,~,dc~. Vol. 6. Suppl. 3. pp. 7-12. 1985. ' AnkhoInternationalInc. Printedin the U.S.A. 0196-978185 S3 t~,) + .

00

Phyllomedusa Skin: A Huge Factory

and Store-House of
a Variety of Active Peptides
V. E R S P A M E R , I P. MELCHIORRI, G. F A L C O N I E R I E R S P A M E R ,
P. C. M O N T E C U C C H I A N D R. DE C A S T I G L I O N E

Institutes o f Medical Pharmacology ill and o f Pharmacology and Pharmacognosy

University o f Rome " L a Sapienza,'" 00100 Rome, Italy
and Farmitalia Carlo Erba S.p.A.. Cheotical Research and Developntent
via dei Gracchi 35, 20146 Milan, ltah'

ERSPAMER. V.. P. MELCHIORRi. G. FALCONIER! ERSPAMER. P. C. MONTECUCCHI AND R. DE CASTIG-

LIONE. Phyllomedusa sMn: A huge .f~wtoO' aml stm'e-house ,~?"a variety of active peptide,~. PEPTIDES 6: Suppl. 3,
7-12. 1985.~The skin of the neotropical hylid frogs belonging to the subfamily Phyllomedusinae is a formidable factory
and store-house of a variety of active peptides belonging to seven distinct families: the caeruleins (represented by phyl-
Iocaeruleinl. the bradykinins (phyllokinin). the tachykinins (phyllomedusin). the bombesins (phyllolitorin. [Leu~]phyl-
Iolitorin. rohdei-litorin), sauvagine, the dermorphins (dermorphin, [Hyp~]dermorphin) and finally the tryptophyllins (a set
of 8-11 members). Another linear peptide and three diketopiperazines should be added to the list. The biochemical and
pharmacological positions of the Phyllomedttsa peptides within their families is briefly discussed, dwelling upon some
recent and controversial data.

Phylh,m'du.~, skin Phyllocaerulein Phyllomedusin Phyllokinin Phyllolitorins Rohdei-litorin

Sauvagine Dermorphins Tryptophyllins Diketopiperazines

T H E subfamily Phyllomedusinae of the Hylidae amphibian
family is represented by a number of Neotropical species
distributed in South and Central America from northern
Argentina to Mexico. The toxonomical collocation and the
peculiar characteristics of these frogs will be illustrated in
more detail elsewhere in this symposium [5,17].
Here we wish to lay emphasis on the fact that phyl-
lomedusid hylids are of exceptional interest in the study of
amphibian skin peptides, because their cutaneous tissue ap-
pears to be an inexhaustible mine of these molecules. No
other amphibian skin can compete with that of the Phyl-
h,medusae, which has already yielded as many as 23 pep-
tides, belonging to at least seven peptide families.
This report summarizes the status of research in this par-
ticular field. The different peptide families will be discussed
in succession, dwelling upon some fresh data.
CAERULEINS
Authentic caerulein is present in the skin of several South
American species of Leptodacty/us, but not in the Phyl-
h,medusa skin, which contains the nonapeptide phyllocaeru-
lein differing from caerulein by the absence of aspartic acid
at position 3 and the substitution ofglutamine with glutamic
acid at position 2.
pGlu-GIn-Asp-Tyr(SO~H)-Thr-Gly-Trp-Met-
Asp-Phe-NH._, Caerulein
pGlu-Glu Tyr(SO:~H)-Thr-Gly-Trp-Met-
Asp-Phe-NH~ Phyllocaerulein
In its spectrum of biological activity phyllocaerulein was
virtually indistinguishable from caerulein and CCK-8, the
C-terminal octapeptide of cholecystokinin.
However. quantitatively, phyllocaerulein was slightly
more potent (by 10 to 50%) than caerulein on dog and rat
gastric acid secretion, dog pancreatic secretion, guinea-pig
gall bladder motility, and dog blood pressure [7].
TACHYKININS
The only tachykinin so far isolated from Phyllomedusa
skin (Phyl. bicolorl is the decapeptide phyllomedusin, the
shortest member of this peptide family.

~Requests for reprints should be addressed to Prof. Vittorio Erspamer, Institute of Medical Pharmacology !11, University of Rome "La
Sapienza.'" Cittb, universitaria, 00100 Rome. Italy.
 8 ERSPAMER ET AL.

Comparing the six above structural formulae some con-

pGlu-Pro-Ser-Lys-Asp-Ala-Phe-lle-Gly-
Leu-Met-NHe Eledoisin
pGlu Asn-Pro-Asn-Arg-Phe-Ile-Gly-
Leu-Met-NH~ Phyllomedusin
pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-
Leu-Met-NH~ Physalaemin
It may be seen that phyllomedusin shares with eledoisin the
Ile residue at position 4 from the C-terminus and with
physalaemin the tripeptide Pro-Asn-Arg(Lys) at the centre of
the molecule. Substitution of the Ile residue for the Tyr resi-
due implies a two base change in the mRNA code.
In keeping with its intermediate primary structure, phyl-
lomedusin also occupies an intermediate position, in its spec-
trum of biological activity, between the eledoisin/kassinin
and the physalaemin/substance P tachykinin subfamilies [9].
It would be interesting to establish the relative affinities of
phyllomedusin for the tachykinin receptor subtypes P and K.
BRADYKININS
The only bradykinin found in Phvllomedusa skin was the
endecapeptide phyllokinin, which is nothing but bradykinin
prolonged at its C-terminus by the dipeptide Ile-Tyr(SO:~H).
Trypsin digestion produced cleavage of this dipeptide with
the formation of bradykinin.
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Bradykinin
Arg-Pro-Pro-Giy-Phe-Ser-Pro-Phe-Arg-Ile- Phyllokinin
Tyr(SO:~H)
On a molar basis phyllokinin was approximately three
times more potent than bradykinin on the dog blood pres-
sure, but only 30-40% as potent on the guinea-pig ileum and
the rat uterus, and 6.5-8% as potent on the rat duodenum.
Removing the O-sulphate from the molecule of phyllokinin
produced a considerable reduction of potency [7].
BOMBESINS
Bombesin peptides of the bombesin/alytesin subfamily
(possessing the C-terminal heptapeptide Trp-Ala-VaI-Gly-
His-Leu-Met-NH2) are apparently lacking in the Phyl-
h.nedusa skin. This. however, contains at least three
litorin-like nonapeptides, the primary structure of which is
shown below, together with that of litorin, the C-terminal
nonapeptide of ranatensin C and the mammalian neuromedin B
[1, 7, 8. 10. 18, 31].
siderations can be made:
(a) The two phyllolitorins isolated from the skin of Phyl-
Iomedusa sauvagei may be clearly distinguished from the
other 16 bombesin-like peptides so far described in amphi-
bian skin and mammalian tissues in having the His residue at
position 3 from the C terminus replaced by a Set residue.
This substitution is not a simple single base change in mRNA
code, but implies a more complex amino acid replacement,
involving a two base change. Parallel bioassay and receptor
studies with specific antisera will decide whether we have to
deal with a new bombesin subfamily or not. It will be simi-
larly of interest to know whether counterparts of the phyl-
lolitorins exist in mammalian tissues.
(b) All the three litorins occurring in Phvlh.ncdusa skin
possess, like neuromedin B, but unlike litorin and
ranatensin-C, a Leu residue at position 8 from the
C-terminus. Substitution of the Leu residue for the more
usual Gin residue (amphibian bombesins) or His residue
(mammalian bombesins) involves a single base change in the
mRNA code.
(c) Rohdei-litorin shares with neuromedin B the entire
C-terminal octapeptide and presents, again like neuromedin
B and ranatensin-C, a Thr residue substituted for the usual
Val residue at position 5 from the C-terminus. This substitu-
tion involves a two base change in mRNA code.
The spectrum of activity of the Phyllomedusa litorins is
similar to that of litorin, xvith striking quantitative differ-
ences, however, in the different preparations, as shown in
Table 1.
Experiments are in progress to determine, with the aid of
synthetic litorin analogues, the influence of the different
amino acid substitutions on the spectrum of biological activ-
ity.
The content of phyllolitorin [LeuS]phyllolitorin in fresh
Phyll. sauvagei skin is of the order of 6--7/.Lg/g. and similar is
that of rohdei-litorin in fresh Phyll. rohdei skin.
The phyllolitorins seem to be present, in considerable
amounts also in the skin of Phy'll. burmeisteri (12-16 p.g/g)
and Phyll. hypochondrialis {10-12 /xg/g). but only in trace
amounts (<0.5 p.g/g) in the skin of Phyll. bit'oh." and
Pachymethtsa dacnicoh.'. It is possible, on the basis of par-
allel bioassay, that still another litorin occurs in the skin of
Phyll. trinitatus.
SAUVAGINE
This peptide, isolated from the skin of Phyllomedusa
sauvagei, is a linear peptide with the following forty amino
acid residues [19,20]:

pGlu-Gln-Trp-Ala-VaI-Gly-His-
Phe-Met-NH~
pGlu-Leu-Trp-Ala-Val-Gly-Ser-
Phe-Met-NH2
pGlu-Leu-Trp-Ala-VaI-Gly-Ser-
Leu-Met-NH~
pGlu-Leu-Trp-Ala-Thr-Gly-His-
Phe-Met-NH~
Asn-Gln-Trp-Ala-Thr-Gly-His-
Phe-Met-NH~
Gly-Asn-Leu-Trp-Ala-Thr-Gly-His-
Phe-Met-NH~
Pyr-Gly-Pro-Pro-lle-Ser-lte-Asp-Leu-Ser-Leu-Glu-Leu-Leu-Arg-
Litorin Lys-Met-lle-Glu-lle-Glu-Lys-Gln-Glu-Lys-Glu-Lys-GIn-Gln-Ala-
Ala-Asn-Asn-Arg-Leu-Leu-Leu-Asp-Thr-lle-NH~ Sauvagine
Phyllolitorin
[Leu"] phyllolitorin Sauvagine presents a close similarity in its primary struc-
ture to urotensin I, a peptide from bony fish urophysis, and
Rohdei-litorin the corticotropin-releasing factor (CRF). later isolated from
ovine hypothalamus. Both are linear peptides with 41 amino
C-terminal nonapeptide acid residues. In catostomus-urotensin 1. 21 amino acids
of ranatensin-C occur in an equivalent region to sauvagine, and 12 additional
Neuromedin B amino acids may represent single base changes: in ovine
CRF. figures are 20 and 12. respectivel~ [10].
During the purification of sauvagine a minor component
PEPTIDES IN PH)LLO.IlEI)USA SKIN
TABLE I
THE RELATIVE POTENCY. ON A MOLAR BASIS. OF PHYLLOLITORIN. LEU"-PHYLLOLiTORINAND
ROHDEI LITORIN EXPRESSED AS A PERCENTAGE OF THAT OF LITORIN (TAKEN AS 100)ON FIVE
ISOLATED AND IN SITU SMOOTH MUSCLE PREPARATIONS
Preparation Phyllolitorin
Rat uterus 3-15
Guinea-pig colon 1.5-3
Rat urinary bladder in situ 110-120
Guinea-pig gall bladder in situ 2-4
Chick intestine 60
was isolated having the same biological spectrum as
sauvagine, a similar molecular weight and an identical amino
acid composition. However. this second form of sauvagine
ISAU 11) sho~ed a different electrophoretic motility, with a
more acidic behaviour, suggesting the presence of an addi-
tional carbox)l group, probably arising from deamidation of
the Gin residue at position 23. SAU I1 could be easily sepa-
rated fi-om the main form by reversed phase HPLC (Rusconi
and Montecucchi, personal communication).
Sauvagine possesses a peculiar spectrum of biological ac-
tivity, similar to that of urotensin i and CRF. which may be
summarized as follows [8,10]:
(1) Potent. long-lasting hypotensive action in dogs and
rats, due to striking vasodilatation, affecting in dogs mainly
the mesenteric vascular bed and in rats the musculo-
cutaneous vessels.
(2} Potent antidiuretic effect in hydrated rats, which is not
merel) due to hypotension, but probably also to a direct
action of the peptide on renal tubules.
3) A number of behavioural effects, generally appreci-
able following intracerebroventricular injection in rats: in-
crease in motor response and emotionality, increase in
grooming, decrease in rearing, disruptive effects on feeding,
with reduction of food intake [2. I1 ], and other effects, which
will be discussed elsewhere in this symposium.
(4} A hyperglycaemic effect in the rat by ICV injection,
with increase in plasma concentration of catecholamines.
Activation of the sympathetic nervous system could also be
seen in the dog. even by peripheral administration.
(5) Finall). but most importantly, complex effects on the
anterior pituitary. Release of ACTH and /3-endorphin was
potently stimulated by subcutaneous sauvagine, whereas the
release of PRL, GH and TSH was inhibited, As a conse-
quence of ACTH release, the adrenal cortex was activated,
and as a con,equence of inhibition of TSH release, uptake of
'2:'I by the th.x roid gland was markedly reduced. The effects
of sauvagine on the pituitary have been extensively studied
in the rat. and verified in the dog, monkey and man. The
inhibitory effect of sauvagine on GH release has also been
confirmed for CRF [24], whereas inhibition of TRF and PR
release still lacks confirmation. In this regard even synthetic
s~uvagine gaxe results partially at variance with those of the
natural peptide. We are now eagerly engaged in an attempt to
elucidate these discrepancies.
In all experiments in which sauvagine was assayed in
parallel with urotensin l and CRF, the frog peptide proved to
be as potent as urotensin I, except in fish, and generally
more potent, even by several times, than CRF.
Using a sauvagine antiserum raised in rabbits with natural
9
Leu~-phyllolitorin Rohdeilitorin
4-18 140-170
1.5-3 20-25
50-80 220-240
2-4 6-9
20-25 250-350
sauvagine, sauvagine immunostaining has been observed in
the following tissues: fish urophysis and spinal cord. frog
medulla oblongata, midbrain, encephalic sensitive ganglia
and retina. The exact localization of sauvagine/CRFqike
peptides in the retina is controversial. Sauvagine is believed
to be contained in the non neuronal Mfiller glia cells of the
avascular frog retina [6]: CRF in the neuronal amacrine cells
of the avascular bird retina [ 15] and the vascular rat retina [29].
DERMORPHINS
Dermorphins are two heptapeptide amides isolated from
the skin of Phyllomedu.~a sam'agei. Phvll. rohdei and Phyll.
burmeisteri [2 I]:
Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 Dermorphin
Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser-NH~ [Hyp~qdermorphin
Methanol extracts of Phvll. sauvagei and Phyll. rohdei
skin also contain the corresponding deamidated peptides,
but it is questionable whether they pre-exist in the living
tissue or are artifacts arising during storage of the extracts,
or purification procedures.
It may be seen that the structure of the dermorphins pre-
sents the unique, intriguing characteristic of having a D-amino
acid residue in the molecule, which is critical for biological
activity. Moreover, the amino acid sequence of the dermor-
phins differs sharply from that of the enkephalins and of all
other endogenous opioid peptides, which invariably carry, at
their N-terminal, the enkephalin pentapeptide Tyr-Gly-Gly-
Phe-Met(Leu).
Dermorphin displayed a very potent central and periph-
eral opioid activity [8,10]. No natural opioid peptide could
compete with dermorphin in its effects on the CNS. The
antinociceptive effect of dermorphin, given in rats, mice and
rabbits, by intracerebroventricular injection, was 100 to 1000
times that of morphine, more than 10 times that of
/3-endorphin, and more than 10,000 times that of the
enkephalins. Development of tolerance to dermorphin in rats
was rapid, similar to that of morphine, occurring over a
maximum period of 48 hr. Naloxone produced the usual
precipitation of withdrawal symptoms. However, dermor-
phin seemed 10 times less addictive than morphine [3].
Other central actions of dermorphin, particularly evident
upon ICV injection, were catatonia, behavioural and EEG
changes; inhibition of gastric emptying, intestinal propulsion
and gastric acid secretion; stimulation of prolactin release in
 10
mammals and birds. In rats the peptide showed also an ex-
tremely potent antidipsogenic effect (threshold dose I-5
ng/rat) [ 16].
Concordant results from several laboratories have
demonstrated that dermorphin has a very great affinity for
/.t-receptors (even surpassing that of morphine), very mod-
erate, questionable affinity for 8 and K receptors, and no
affinity for ~ receptors.
Inactivation of dermorphin by mammalian tissues (liver,
kidney, intestine, brain) was rapid, as was excretion of un-
modified peptide through the bile. Approximately 8OZ of
dermorphin that reached the liver was cleared in a single
passage. The main degradation product in the rat liver and
kidney was the N-terminal tripeptide, devoid of any opioid
activity, in the brain the N-terminal tetrapeptide, which still
retained about 10% of the analgesic potency of the parent
peptide [23].
More than 200 dermorphin analogues have been so far
synthesized in Italy, Poland and Japan, and submitted to
parallel bioassay. Results will be discussed, in part,
elsewhere in this symposium. On the whole, no analogue
surpassed dermorphin in its analgesic potency by 1CV injec-
tion, while apparently attaining a similar or even greater po-
tency by peripheral administration. This seems to be the case
of [D-Arg2]dermorphin and some o f its N-terminal tetrapep-
tide analogues [28], and of [D-MetO2]dermorphin-4 [27].
And now a final question of great importance. Does der-
morphin have counterparts, as nearly all other amphibian
skin peptides, in vertebrate extracutaneous tissues, espe-
cially gut and brain, and in invertebrates? In spite of some
positive results, the question remains unanswered.
Buffa et al. [4] have obtained a clear cut immunostaining
of nerve cells and fibres in nucleus arcuatus and other cere-
bral structures, using a highly specific anti dermorphin
Tryptophyllin pGlu-Pro-Trp-Met-NH._,
tetrapeptides
(TPH 4) pGlu-Pro-Trp-Val-NH2
Phe-Pro-Trp-Leu-NH2
Tryptophyllin Phe- Pro-Pro-Trp-Met-NH2
pentapeptides
(TPH 5) Phe- Pro-Pro-Trp-VaI-NH2
Phe- Pro-Pro-Trp-Leu-NH~
Tryptophyllin
heptapeptide VaI-Pro-Pro-Leu-Gly-Trp-Met-OH
(TPH 7)
Tryptophyllin
decatriapeptide pGlu-Glu-Lys-Pro-Trp-Pro-Pro-Pro-lle-Tyr-Pro-Met-OH
(TPH 13)
Some of the above tryptophyllins or other similar
tryptophan-containing peptides seem to occur, in greater or
lesser amounts, in all examined Phyllomedusa species. It is
not possible to decide whether the deamidated peptides pre-
exist in the living skin. or are artifacts arising during drying
and extraction of the skin or successive purification proce-
dures.
ERSPAMER ETAL.
serum raised in o u r laboratory. Reaction was particularly
evident after colchicine pretreatment.
On the other hand, quite recently Tsou et al. [30] suc-
ceeded in demonstrating the occurrence of a dermorphin-like
peptide in the guinea-pig and rat stomach (98 and 33 ng/g
tissue, respectively), but not in the CNS.
A very sensitive anti dermorphin serum was used, capa-
ble of working at a dilution of I: 120,000 and positive results
were achieved only after HCI extraction of the stomach, but
not after the usual methanol extraction. Because of this
characteristic and the molecular weight of the peptide (2000
versus 805), the Chinese investigators conclude that the
peptide is not an authentic dermorphin. This conclusion
seems correct and. reasonably, we ought to search for
dermorphin-like peptides in mammalian tissues, possibly of
different molecular size Icf. the gastrins, the CCKs, the
bombesins etc.), rather than for dermorphin itself.
At any rate, should the occurrence of dermorphin-like
peptides in mammalian tissues be proved unequivocally, as
we firmly believe it will be, we have the impression that the
frog peptide would not be accepted with excessive
enthusiasm in the superfamily of mammalian opioid pep-
tides, as it would impose a re-examination of several con-
clusions and theories.
TRYPTOPHYLLINS
This name has been suggested to indicate a non
homogeneous family of tryptophan-containing tetra-, penta-.
hepta- and decatria-peptides isolated from methanol extracts
of Phyllomedusa rohdei skin [12,22]. They generally have in
common a Trp residue at position 2 from the C-terminus and
one or two Pro residues, at positions 2 (and 31 from the
N-terminus.
pGlu-Pro-Trp-Met-OH
p-Glu-Ala-Trp-Met-OH
Phe-Pro-Pro-Trp-OH
Phe- Pro-Pro-Trp-Leu-OH
Tryptophyllins represent the first example of frog pep-
tides identified, in our laboratory, not by bioassay, but sim-
ply by colour reactions on paperchromatograms.
The activity of the tryptophyllins remains to be estab-
lished. Bioassay on a number of isolated and in situ smooth
muscle preparations and on systemic blood pressure, gave
negative results. However. in preliminary experiments.
PEPTIDES IN PHYLLOMEDUSA SKIN 11

TABLE 2
PEPTIDE CONTENTS IN THE SKIN OF ELEVEN PHYLLOMEDUSID FROGS (IN ~ag/g FRESH SKIN)

Phyllocaerulein Phyllokinin Phyllomedusin Sauvagine Dermorphins

Phyll..~auvat, ei 200-770 75-220 1.5-5 100-130 50-60

Phyll. rohdei < I 70-130 15-35 <5 60-80
Phyll. hurmeisteri 230-275 140-220 14-50 55-120 50-60
Phyll. hypochondrialis < 1 20-30 4-6 3-5 2
Phyll. bicolor 520-650 300-480 550-1400 50-210
Phyll. edentula 180 130 10 40
Phyll. palliata < I 80 70 30
Phyll. trinitatus 190 150 40
Pachyoledtt,sa dacnicolor 25 25 310 25
Agalychnis helenae 20-80 10 65-190 2-3
Agalychnis annae 75-125 I-2 15-50 <5

~hich await confirmation, the penta-tryptophyllins showed
-ome effect on gastric emptying in the rat (by intracerebro-
entricular injection) and, surprisingly, on protein synthesis
in rat liver, following subcutaneous injection. The same
promoting activity on protein synthesis was displayed also
b.~ TPH 7 peptides [14].
Recently an antiserum against [Mets]TPH 5 was raised in
the rabbit in our laboratory. With this antiserum a population
of cells was immunostained in the rat, rabbit and frog
anterior pituitary [25].
The tryptophyllins will be discussed in more detail
else~ here in this symposium. Here we wish to point out that
they do not seem to be peculiar to Phylhm~edusa skin. In
fact. tryptophan-containing peptides showing, on
paperchromatograms, colour reactions and Rf values very
similar to those of the tryptophyllins, occur in methanol ex-
tracts of the skin of other hylid frogs of Central and South
America. belonging to the genera Smilisca (S. sila. S. sor-
dida. S. cyam~stica. S. phaeota) and Triprion (Tr. spatulatus
,cticulatus). A study of these materials would probably be
re,yarding.
MISCELLANEOUS PEPTIDES
One additional linear peptide and three diketopiperazines
~ere identified in methanol extracts of Phyllomedusa skin
120] as follows:
Phvll. sauva~.,ci
Leu-Met-Tyr-Tyr-Thr-Leu-Pro-Arg-Pro-VaI-NH~
C.~loITyr-Pro)
C.~clol Phe-Leu)
Phvll. hurmeistcri
Cyclo(Trp-Lys)
The pharmacological study of these molecules is in prog-
I'~SS.
QUANTITATIVE DATA ON THE PEPTIDE CONTENT IN
PHYLLOMEDUSA SKIN
The content of five peptides in the fresh skin of eleven
phyllomedusid frogs belonging to the genera Phyllomedusa,
Pachymedusa and Agalychnis, collected from northern
Argentina to Mexico, is shown in Table 2.
It appears that the spectrum of peptides occurring in the
skin of the various species was similar with, however, con-
siderable quantitative differences. Particularly high was the
peptide content in the skin of Phyllomedusa bicolor
(Amazonas), attaining for the four peptides considered a
total of 1.4--2.7 mg/g.
DISCUSSION
Data presented in this paper show that Phyllomedusa skin
contains an extraordinarily rich collection of active peptides,
astonishing both for the variety and concentration of these
molecules. Eliminating possible artifacts, as many as 19
peptides (including the diketopiperazines) have been isolated
and sequenced from methanol extracts.
Phyllocaerulein, phyllomedusin, phyllokinin, sauvagine
and rohdei-litorin have obvious counterparts in mammalian
tissues, whereas the position of the dermorphins and the
tryptophyllins is still uncertain. However, just these pep-
tides, together with the phyllolitorins, could represent
springboards for exciting and rewarding research.
It should be added that Phyllomedusa skin is possibly a
good material also for the identification of polyproteins or
prepropeptides by the recombinant mRNA technique. Such
research has been already carried out with success for caeru-
lein, xenopsin and TRH in the Xenopus laevis skin [13,26].
Peptides described in this paper do not exhaust the pep-
tide stock of Phyllomedusa skin. Not only do we await isola-
tion of several molecules already identified, but we are
aware that other molecules are escaping our attention be-
cause their activity lies beyond the limits of our screening
methods. The way out of this impasse is the systemic isola-
tion and sequence analysis of the numerous peaks of a prob-
able peptide nature appearing on HPLC tracings of crude or
semi-purified extracts of Phylhmledusa skin. We are pres-
ently engaged in this task.
The peptides of Phyllomedusa represent a pattern of am-
phibian skin peptides. We believe that it is difficult to find a
workshop of peptide, as well as of amine molecules com-
parable to the secretory syncytium defining the alveoli of the
cutaneous glands of amphibians. The reason why the peptide
coding is so productive and complex in this synctium is
obscure, as is the significance of this formidable array of
active molecules, apparently destined for external secretion.
 12
ACKNOWLEDGEMENT
Original research reported in this paper was supported through-
out by grants from the Consiglio Nazionale delle Ricerche. Roma
(Progetto finalizzato Chimica fine e secondaria).
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