Half a century of comparative research on biogenic amines and active peptides in amphibian skin and molluscan tissues

Camp. Biochem. Physiol. Printed in Great Britain

Vol. 79C.

No. I. pp. l-7. 1984 c

0306-4492/84 $3.00 + 0.00 1984 Pergamon Press Ltd

HALF A CENTURY OF COMPARATIVE RESEARCH ON BIOGENIC AMINES AND ACTIVE PEPTIDES IN AMPHIBIAN SKIN AND MOLLUSCAN TISSUES VITTORIO

Institute of Medical Pharmacology,

ERSPAMER

1st University of Rome, Citti Universitaria, 00100 Roma, Italy. Telephone: 06/4940094 (Receiced 15 November 1983)

As I entered the University of Pavia in 1935, I had simultaneously the chance of obtaining, by examination, a place as a pupil in the Ghislieri College, and of being accepted, as intern, in the Institute of Comparative Anatomy and Physiology which, at that time belonged to the Faculty of Medicine. This allowed me to overcome the financial obstacles which were opposed to my aspiration for medical studies, and to have my first contact with Dr Vialli, who exerted a decisive influence on the entire course of my scientific life. The Institute of Comparative Anatomy and Physiology was, under the direction of professor Edoardo Zavattari (188331972) succeeded in 1935 by Professor Maffo Vialli (1897-1983) one of the most active research laboratories of the University of Pavia, which produced not only several professors in Biological Sciences, but also some outstanding clinicians. After a training in histology and histochemistry, which was at its very beginning, I was given my M.D. thesis on the enterochromaffin cell system in vertebrates. Research was carried out with enthusiasm, enlivened by the constant interest and advice of my teachers. It was shown that the enterochromaffin cells were normal constituents of the digestive tract throughout the entire vertebrate phylum. Cells possessing exactly the same histochemical characteristics were soon found also in the ascidian intestine, the posterior salivary glands of Octopoda, the hypobranchial body of Muricidae, and finally the cutaneous glands of amphibians. Starting from materials rich in enterochromaffin or enterochromaffin-like cells, we obtained, already in 1937, acetone extracts which give the same colour reactions as the specific granules of the enterochromaffin cells. We named the substance responsible for the reactions enteramine to indicate its origin and its probable chemical structure. After taking my degree in medicine, I remained as assistant in the same Institute for a couple of years. In 1937 I was persuaded by Pietro Di Mattei, Professor of Pharmacology at the University of Pavia, to move with him to Rome, as first assistant, changing not only university, with major teaching responsibilities, but also discipline. To make the switch from morphology to pharmacology less abrupt I spent a semester in Berlin, in the Institute of Pharmacology directed by Professor Wolfgang Heubner (1877-1957). I learned there the

elementary pharmacological methods I used with great profit throughout the course of my research. Finally, in the Autumn of 1938 I went to Rome, with some perplexity and anxiety. I was leaving the university in which I was educated, and was leaving Professor Vialli, to whom I was deeply bound by feelings of esteem and filial love. In fact not only did Professor Vialli familiarize me with histochemical methods but, far more important, he made me aware of the incomparable value of comparative research. Biochemical problems which may be very complex in mammals are sometimes considerably simpler in lower vertebrates and invertebrates; isolation and structure studies of minute amounts of active substances in mammals may require exceptional skill and the availability of sophisticated, expensive apparatus; isolation of the same substances from tissues of lower animals, in which they occasionally occur in large amounts, could be within the range of our technical possibilities. In keeping with this firm belief I did not abandon in Rome my trend of research but, encouraged by Professor Di Mattei, pursued it even with greater eagerness. Unfortunately, we were at the eve of World War II and immediately I had to face a long period of enormous difficulties owing to drastic shortage of reagents, experimental animals and food for them, and the impossibility of substituting and completing the limited, old equipment of the Institute. For a number of years, which could have been among the most productive of my life, I was constrained to very limited activity, until complete inaction. I remember that at that time I had at my disposal for research on isolated smooth muscle preparations a one-speed drum, the clockwork movement of which was eventually reinforced by an elastic band, and a water bath of sheet copper which was kept at constant temperature by a small, hand-regulated gas flame. In spite of this situation I managed to start with the study of the pharmacological actions of enteraminerich tissue extracts on vascular and extravascular smooth muscle. The isolated rat oestrous-uterus was suggested, already in 1940, as a preparation of choice in the qualitative and quantitative bioassay of enteramine. After the war was over, things rapidly began to improve. In 1947 I was appointed Professor of Phar-

2

VITTORIO ERSPAMER

macology at the University of Bari (a good place for collecting marine materials) and research was taken up with renewed enthusiasm. At that time I also spent brief but repeated periods of work at the Marine Zoological Station of Naples, where I met Drs F. Ghiretti, A. Monroy, G. Reverberi and others. In Bari and Naples I made the definitive decision to dedicate all my future activity to the study of active compounds in molluscan tissues and in amphibian skin, which already for some years had begun to attract my attention as a formidable store-house of indolealkylamines, possibly strictly related to enteramine (1946). Two main lines of research were then cultivated in parallel: biogenic amines and active polypeptides, often occurring together in the same materials. The second line had a definitive prevalence in the last decade. When I moved from Bari to Parma (Institute of Pharmacology) in 1956 and then to Rome (Institute of Medical Pharmacology) in 1967 my research trends remained unchanged and there was no difficulty in persuading several excellent collaborators to join me. The outlines of the study of our active compounds were always the same: identification of the compound by colour reactions and/or bioassay; semipurification by column chormatography or gel filtration (more recently also by HPLC); preliminary pharmacological study of the eluates; complete purification and elucidation of the structure; synthesis; extensive pharmacological study of the synthetic compound; synthesis and pharmacological study of analogues. For structure studies and synthetic work, which were beyond our competence and technical facilities, I established a close, fruitful collaboration with the Farmitalia Carlo Erba Research Laboratories,

&r”‘-r, H

Tryptamine

trimethyl

Milan, a collaboration that still lasts after 30 years. Among the most valid collaborators in these laboratories I wish to remember Drs B. Asero and 0. Benati for biogenic amines, Drs A. Anastasi, P. C. Montecucchi, L. Gozzini and Professors B. Camerino and R. de Castiglione for the peptides. But let me proceed to a summary of our research. In 1949-1951 Rapport demonstrated that serotonin, originating in serum by the disruption of platelets, was 5-hydroxytryptamine and in 1952 we found that enteramine, isolated from extracts of Discoglossus pictus skin and posterior salivary glands of Octopus vulgaris, had exactly the same structure of serotonin, i.e. it was 5-hydroxytryptamine (5-HT). The occurrence of 5-HT and related indolealkylamines was studied in our laboratory, starting from 1948, in approximately 500 amphibian species (skin extracts) and 100 molluscan species (extracts of posterior salivary glands and hypobranchial body). Not only the very wide distribution of 5-HT and the occurrence of related, in part hitherto undescribed, indolealkylamine molecules emerged from this study, but also the presence of several novel imidazolealkylamines, phenylalkylamines and choline esters. The new molecules are listed below, together with some representative structural formulae. Indolealkylamines Tryptamine-trimethylammonium; N-methyl-5HT; 5-methoxytryptamine (5-MT); N-methyl-5-MT; NJ’-dimethyl-5-MT; 5-HT 0-sulphate; bufotenine 0 -sulphate; bufotenidine 0 -sulphate; bufoviridine. Bufoviridine was the first example of a I-sulphonated derivative of indolealkylamines.

) 3 3 N, N- Dimethyl - 5 - MT

- ammonium

AOsH Bufotenine

Bufowldine (Bufotemne I - sulphonic acid)

Trypargine

0

- sulphate

Comparative

research

on biogenic

In this place also tryppargine (1981) should be remembered, an interesting tetrahydrocarboline derivative, possibly originating from the condensation of tryptamine with arginine, which has been isolated from extracts of the skin of the African frog Kassina senegalensis. Other indolealkylamines have been traced, but their structure has not yet fully elucidated (tryptamine 1sulphonic acid?; 0 -methylbufotenine 1-sulphonic acid?). The status of our knowledge up to 1965 on “‘5-Hydroxytryptamine and Related Indolealkylamines” was presented in the big monographic volume XIX of the Heffter’s Handbook of Experimental Pharmacology, edited by myself in 1966. Imidazoleulk~~lamines Spina~amine; 6-methylspinaceamine; N-acetylhistidine; imidazolepropionylhistamine; urocanylhistamine. All these compounds are still in need of a definite pharmacological collocation.

amines

3

and active peptides

It may be seen that the array of new amine molecules so far identified by our group is constituted by 10 indolealkylamines, 5 imidazolealkylamines, 3 phenylalkylamines and 2 choline esters. It should be added that our materials were found to be rich also in a variety of other already known biogenic amines, belonging to the four families above: tryptamine, bufotenine, bufotenidine, dehydrobufotenine, bufothionine; histamine, N-acetylhistamine, N-methylhistamine, N,N-dimethylhistamine; tyramine; acetylcholine, senecioylcholine. The study of biogenic amines in molluscan tissues and amphibian skin has been pursued in our laboratory with determination because we are convinced that data obtained in these materials may be largely valid also for mammals. Amphibian skin especially, owing to the variety and abundance of amines which it contains, is extraordinarily suitable for the detection of new amines and new metabolic pathways. Virtually every theoretically conceivable amine

f:

~CH=CH-CO-NH-CH,-CH2~--,-=,

N+,,N H

HN+N

Urocanyl . histamine

H::;F~Y C H* Spinaceamine

CHOH-CH, f NH, Octopamine

Phenylalkylamines Octopamine; leptodactyline; candicine (first traced in vegetables). Octopamine has found ample renown as putative “fatse” neurotransmitter in vertebrates and as “true” transmitter in a number of invertebrates. Leptodactyline, in turn, exhibited very potent nicotinic effects and a remarkable neuroIt was the first muscular blocking activity. m-tyramine ever identified in nature. Choline esters Murexine and dihydromurexine. Both compounds are provided with intense neuromuscular blocking and nicotinic activity. +

HN~CH=CH--CO-OCH,-CH,-N(CH,),

Leptodactyline

derivative may be traced in amphibian skin. It is only a matter of screening more and more species. It is obvious that, once the possibility of the biosynthesis of a new amine or metabolite is demonstrated in amphibian skin or molluscan tissues, it will be much easier to check its presence also in mammalian tissues or fluids under normal and pathological conditions. A pertinent example is that of octopamine. Finally, it should not be passed over in silence that the spectra of biogenic amines and peptides assessed in the ditrerent amphibian families and genera have sometimes proved to be of considerable value in solving taxonomic and evolutionary problems.

“k-cl---

CH2--CH,-CO-OCH2--CH?-_---N(CHs)3

dN

v Mure.wle

Dihydmmurexine

+

VITTORIOERSPAMER

4

Vittorio Erspamer.

As already stated, the second important line of research cultivated by our group was that on active peptides. The first impact with polypeptides was a matter of serendipity. During the study of biogenic amines in extracts of posterior salivary glands of the Mediterranean octopod Eledone ~0~~~~~~ (1949) I found that these extracts displayed an extraordinarily potent hypotensive action in dogs and rabbits and potently stimulated several smooth muscle preparations. This effect could not be ascribed to any of known active substances. First I believed that e~e~oi~~n was a choline ester, like murexine found in the hypobranchial body of Muricidae, but later on, when the spectrum of activity of eledoisin and its behaviour towards proteolytic enzymes appeared very similar to that of substance P, its polypeptide nature became evident. Times were not yet mature for structure studies. However, after a long pause, research on eledoisin was taken up again, under the stimulus of the success achieved in the amino acid sequence determination of bradykinin. In 1962, as a first result of a fruitful collaboration with Dr Ada Anastasi, destined to last for years, we succeeded in establishing the primary structure of eledoisin, soon followed by a thorough pharmacological study of the peptide on vascular and extravascular smooth muscle and on salivary, lachrymal and pancreatic secretions. In the same year, quite unexpectedly and again as a result of a biological screening for biogenic amines, I found that methanol extracts of the skin of the South American frog Ph~valaemus biligonigerus dis-

played a potent eledoisin-like activity. The peptide responsible for this activity was isolated and sequenced in 1964. Synthesis of ph~is~l~emin was the premise for its extensive pharmacological study, in which more than one hundred synthetic analogues of physalaemin and eledoisin were involved. At this point of our research on peptides, serendipity went off the scene, and a systematic collection of amphibians, all over the world, was undertaken with the precise purpose of investigating the occurrence in their skin of peptides, and other active molecules. Among the collaborators in this undertaking a particular place should be reserved to my friends Dr Jose M. Cei, Professor of Biology at the University of Mendoza, Argentina, and Dr Robert Endean, reader in Zoology at the University of Queensland, Brisbane, Australia. The first, an authentic field-zoologist, travelled far and wide through South and Central America, from Patagonia to Mexico, to collect frogs, the other was responsible for frog collection in Australia and Papua New Guinea. Altogether more than 200 amphibian species came from Dr Cei and 100 from Dr Endean. Other zoologists who collaborated with us in the collection and classification of frogs were Dr J. Visser (Zoology Dept., University of Natal, Petermaritzburg, South Africa), Dr Angel C. Alcal& (Dept. of Biology, Silliman University, Dumaguete City, Philippines), Dr William C. Duellman, Museum of Natural History, University of Kansas, Lawrence, Kansas), and Dr P. Y. Berry (School of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia). Finally, large amounts of some more common amphibians were purchased from Dr W. De Rover, Den Dolder, Holland. In regular succession numerous peptides, belonging to eight distinct families, have been isolated, sequenced and mostly reproduced by synthesis during the past twenty years. Their structures are shown below. I was very fortunate to have enjoyed throughout my research on polypeptides, the skilful and enthusiastic collaboration of many people. Some of them are currently engaged in independent lines of research on biogenic amines and polypeptides. It is a privilege to remember here G. Bertaccini (Professor of Pharmacology, University of Parma), G. De Caro (Professor of Pharmacology, University of Camerino), M. Impicciatore (Professor of Pharmacology and Pharmacognosy, University of Parma), M. Roseghini, L. Negri and G. Improta (Associate Professors in Pharmacology, University of Rome) and last, but not least, P. Melchiorri (Professor of Pharmacology, University of Rome), who at present is certainly one of the most qualified research workers in the field of polypeptides. Among people of our University who became interested in the clinical study of frog peptides a prominent place is held by the groups of Professors A. Torsoli (Chair of Gastroenterology) and V. Speranza (VI. Surgical Clinic). All the peptides listed above, with the exception of the tryptophyllins and the miscellaneous peptides, have been the object of extensive pharmacological studies by ourselves and a number of research work-

5

Comparative research on biogenic amines and active peptides Table

1.

Tachykinins Pyr-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leo-Met-NH, Pyr-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leo-Met-NH? Pyr-Asn-Pro-Asn-Arg-Phe-Ile-Gly-Leo-Met-NH? Pyr-Pro-Asp-Pro-Am-Ala-Phe-Tyr-Gly-Leo-Met-NH* Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-Val-Gly-Leu-Met-NH, Pyr-Ala-Asp-Pro-Lys-Thr-Phe-Tyr-Gly-Leo-Met-NH2 Asp-Glu-Pro-Lys-Pro-Asp-Gln-Phe-Val-Gly-Leu-Met-NH~ Asp-Pro-Pro-Asp-Pro-Asp-Arg-Phe-Tyr-Gly-Met-Met-NH, Ltradykinins Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ile-Tyr(HSO,) Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg Cam&ins Pyr-Gln-Asp-Tyr(HSO,)-Thr-Gly-Trp-Met-Asp-Phe-NH* Pyr-Glu--Tyr(HSO,)-Thr-Gly-Trp-Met-Asp-Phe-NH1 Pyr-Asn-Asp-Tyr(HSO,)-Leu-Gly-Trp-Met-Asp-Phe-NH~ Bombesins Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH~ Pyr-Gly-Arg-Leu-Gly-Thr-Gln-Trp-Ala-Val-G~y-His-Leu-Met-NH~ Gin-Trp-Ala-Val-Gly-His-Phe-Met-NH, PYr Glu(OMe)-‘?rp-Ala-Val-His-Phe-Met-NH, P;r Glu(OEt)-Trp-Ala-Val-Gly-His-Phe-Met-NH, PYr Lea-Trp-Ala-Val-Gly-Ser-Phe-Met-NH, PYr --Leo-Trp-Ala-Val-Gly-Ser-Leu-Met-NH, PYr Angiotensins Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe Sauoagine (1979) Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-Leu-Arg-Lys-Met-Ile-Glu-IleGlu-Lys-Gln-Glu-Lys-GIu-Lys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Leu-Asp-Thr-Ile-NH~ Dermorphins Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser_NH2 Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser.NH, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser Tryptophyllins (1983-1984) Pyr-Pro-Trp-Met-NH2 Pyr-Pro-Trp-Val-NH, Phe-Pro-Pro-TIP-Met-NH, Phe-Pro-Pro-Trp-Val-NH, Phe-Pro-Pro-Trp-Leo-NH, Val-Pro-Pro-Leo-Gly-Trp-Met Miscellaneous peptides Leu-Met-Tyr-Tyr-Thr-Leu-Pro-Arg-Pro-Val-NH1 (1981) Cyclo(Trp-Lys) (1981) Cyclo(Trp-Pro) (I 98 1) Cyclo(Phe-Leo) (1981) *Isolated

in collaboration

with the Nakajima

Eledoisin ( 1962) Physalaemin (1964) Phyllomedusin (1970) Uperolein (I 975) Kassinm (1977) Lys’,Thr’-Physalaemin (1980) Gl?,Pro’-Kassinin (1981)* Hylambatin (1981)’ Phyllokinin (1966) Hyp’-Bradykinin (1979)* Caerulein (1967) Phyllocaerulein (1969) Asn’,Leu’-Caerulein (1977) Bombesin (1971) Alytesin (1971) Litorin (1975) Glu(OMe)‘-Litorin (1977) Glu(OEt)‘-Litorin (1980) Phyllolitorin (1983) Leux-Phyllolitorin (1983) Crinia

angiotensin

II (1979)*

Dermorphin (1981) Hyp’-Dermorphin (1981) Deamidated Dermorphin (1981) Deamidated Hyp’-Dermorphin (198 I)

group (Hiroshima-Tokyo).

ers all over the world. More than 2000 papers have appeared on frog skin peptides, and caerulein, tachykinins and bombesins are usual topics in congresses and symposia dealing with brain and intestinal peptides. Frog skin peptides have aroused a general interest since it has become evident that they regularly have counterparts in mammalian tissues, especially gastrointestinal tract and brain, where they are represented by identical or analogous molecules. Sometimes identification of mammalian peptides (bradykinins, angiotensins, substance P, gastrincholecystokinin) preceded that of the amphibian counterparts. However, in the case of substance P, elucidation of its primary structure occurred 8 and 9 years later than that of physalaemin and eledoisin, respectively, and frog tachykinins decisively helped in demonstrating that mammalian tachykinins are represented not only by substance P, but also by physalaeminand kassinin-like molecules, among which is neuromedin K (1983). Similarly, elucidation of the structure of CCK-33 was simultaneous to that of the decapeptide caerulein, which may be considered the forerunner of the

octapeptide CCK-8, probably the most active and disposable cholecystokinin form. Other times the identification of amphibian peptides heralded the discovery of analogous peptides in mammalian tissues. Typical examples were the bombesins and sauvagine. Several years after the discovery of bombesin and alytesin, three heptacosapeptides were isolated and sequenced, in succession, from porcine, avian and canine gastrointestinal tract. Dog intestine contained, in addition to bombesin-27, also its fragments 5-27 and 18-27. All the above peptides possessed the same C-terminal nonapeptide as bombesin, which is essential and sufficient for full’ biological activity. At the same time, bombesin-like peptides of the same molecular size as the frog peptide were repeatedly described in the CNS, where they act as typical neuropeptides, interfering in thermoregulation, glucoregulation and food intake. This is not all: it has been found quite recently (1983) that neuromedin B, a peptide isolated from porcine spinal cord, shows a C-terminal amino acid sequence identical to that of ranatensin C, a bombesin-like peptide of the ranatensimlitorin subfamily.

6

VITTORIOERSPAMER

Thus, it appears that the different subfamilies of the bombesin family also have distinct counterparts in mammalian tissues, as do the tachykinins. Sauvagine, in turn, has anticipated (1979) the discovery (1981) of the ovine hypothalamic corticotropin-releasing factor (CRF), with which it has in common 20 amino acids, with 12 additional amino acids, out of the 41 constituting the molecule, representing single base exchanges. The activity spectrum of sauvagine is essentially similar to that of CRF, with some important differences, however. For example, unlike CRF, the amphibian peptide inhibits PRL, TSH and GH release from the anterior pituitary, suggesting the possibility that mammalian hypothalamus may contain, in addition to CRF, another peptide more similar to sauvagine, possessing a broader spectrum of activity on the adenohypophysis. Dermorphin has been so far unequivocally demonstrated only in amphibian skin. Its unique position among all other natural opioid peptides is determined by the amino acid composition of its N-terminal pentapeptide, which sharply differs from that of the enkephalins, and by the puzzling presence, at position 2, of a D-amino acid (D-Ala). To our knowledge, this is the first example of the occurrence of a D-amino acid in a peptide molecule of animal origin. We firmly believe that dermorphin pre-exists as such in the living skin and is neither an artifact arising from total interconversion of L-Ala2 dermorphin into dermorphin during extraction or isolation procedures nor a metabolic product of bacterial or viral contaminants of the skin. Dermorphin possesses an extraordinarily potent opioid activity, both peripherally and centrally. Professor F. Lembeck (personal communication) has recently shown that the efficiency of dermorphin in inhibiting the peristaltic reflex in an intra-arterially infused preparation of guinea-pig ileum exceeded by 50-500 times that of dynorphin, the enkephalins and morphine. On the other hand, no natural peptide could compete with dermorphin in its analgesic action on the CNS, by intracerebroventricular injection. Again, the potency of dermorphin was more than 1000 times greater than that of dynorphin, the enkephalins and morphine, and 15 times greater than that of /I-endorphin. We are convinced that dermorphin-like peptides occur also in the mammalian brain. Data in favour of this assumption have already been collected, but decisive evidence is still lacking. Should future research definitively remove present uncertainties, dermorphin would further complicate the already intricate problems concerning brain opioid peptides. The tryptophyllins, exceptionally identified in skin extracts of Phyllomedusa rhodei, not by bioassay but by colour reactions on paper chromatograms, are molecules which are in the first stages of pharmacological investigation, but seem to reveal some interwith a Immunostaining properties. esting tryptophyllin-antiserum raised in our laboratory seems to be positive in cells of the frog and mammalian anterior pituitary. In addition to those isolated and studied by our groups, eight other amphibian peptides, belonging to the bradykinin and bombesin (ranatensin) families

have been described, in independent research, by Nakajima and coworkers, adding to the already crowded array of these molecules in amphibian skin. Apart from biogenic amines and peptides, amphibian skin contains other kinds of active molecules. It is sufficient to remember the Salamandra alkaloids, the toad bufodienolides, finally the fantastic set of more than 200 alkaloids discovered and isolated by Daly’s group, in a year-long, dedicated field and laboratory work, from the poisonous frogs of the Phyllobates and Dendrobates genera. The last surprise coming from some of our Australian frogs (Pseudophryne corroborree, Ps. nicholsi and especially Ps. coriacea) was the finding, by my wife and myself, that their skin contained, in addition to amines and active peptides, a compound or, more likely, a mixture of compounds which should be included in the group of Daly’ alkaloids, probably in the pumiliotoxin A-class. Actually, Daly has recently demonstrated (paper in press) the occurrence of allopumiliotoxin B (323 B) and another pumiliotoxin (an isomer of 267 Me) in skin extracts of Pseudophryne semimarmorata. In the mixture present in the skin of Ps. coriacea one alkaloid seems to be responsible for most, if not all, the bioactivity displayed by the crude extract. The active principle has been obtained by HPLC, in a pure form, but in amounts insufficient for structure studies. Available data suggest that its concentration in the skin does not exceed 300,ng per g dry tissue ( = 60-80~~ per g wet tissue). If this estimate is correct then the alkaloid would be different from, and considerably more potent than, the pumiliotoxins hitherto studied from a pharmacological point of view. Awaiting the frog collections, which are in progress, will enable us to have at disposal sufficient material for elucidation of structure, a number of experiments have been carried out, with a puritied extract of Ps. coriacea skin, on isolated smooth and skeletal muscle preparations and on small, intact animals. Results were as follows: (1) LDSO in the mouse, by subcutaneous injection, was 100 mg kg-‘, expressed in terms of dry frog skin. (2) The extract potently stimulated both spontaneously contracting and electrically driven gastrointestinal smooth muscle preparations and isolated, electrically driven, vas deferens preparations; conspicuously increased and prolonged response to electical stimulation of different nerve-skeletal muscle preparations; potently enhanced, up to spasm, the in vitro and in vitro leach helicoid musculature; remarkably increased amplitude and even more, frequency, of spontaneously beating isolated mammalian auricles; conspicuously affected rat blood pressure, causing both a fleeting fall and a more persistent elevation of pressure. (3) By intracerebroventricular injection in the cat the extract caused a series of autonomic and behavioural changes. Most of the above effects were blocked by tetrodotoxin. We believe that the active alkaloid acts, directly or indirectly, via Ca2 + translocation, mainly to release amine or peptide transmitters from the nerve terminals. Release of noradrenaline has been

Comparative

research

on biogenic

unequivocally demonstrated from the rabbit vas deferens preparation. It would be not surprising, owing to the high potency of the compound and its lack of tachyphylaxis (present only in the case of exhaustion of the transmitter), if a similar substance were present also in mammalian peripheral and central nervous system, where it could act as a modulator of transmitter release. This is, in a panoramic overview, the work I have carried out with my collaborators, during the last fifty years, sometimes under relatively easy, at other times under difficult, conditions. A work, which has always been gratifying and which has given results surpassing by far our boldest expectations. It was a long-lasting game, which made fleeting the months and years, and was the only solace in the moment of agony when I tragically lost my eighteen-year old daughter Maria Luisa, heart of my heart. My wife, Giuliana Falconieri, now Associate Professor of

amines

and active peptides

I

Applied Pharmacology at the University of Rome, was fortunately near me not only in the tragedy but, since then even more than before, in the laboratory work. Nearly all personal research that I carried out during the last ten years, was made possible by her continuous, gifted collaboration. Before concluding let me look at my mild, gracious frogs with sympathy and love for the many years we have spent together. Their names, aspect, relationships, distribution and life history have become familiar to me. And similarly let me remember with nostalgia my collection trips of molluscs in Australia (Great Barrier Reef), the Philippines, and South Africa, where I came into contact with the wonderful, spectacular marine life, on uncontaminated coasts. wish to express my deep gratitude to Professor G. A. Kerkut for the appreciation of my scientific work shown by his invitation to write this semiautobiographic article, overcoming my obvious perplexity. Acknowledgement-I