Tachykinins and other biologically active peptides from the skin of the Costa Rican phyllomedusid frog Agalychnis callidryas.

Peptides present in a methanol extract prepared from skin of the Costa Rican frog Agalychnis callidryas of the Phyllomedusinae subfamily were studied by sequence analysis and pharmacological tests. Members of five different peptide families-tachykinins, bradykinins, caerulein, opioid peptides and sauvagine-were found. In particular, the extract contained a number of tachykinins with the following sequences: Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Ile-Gly-Leu-Met-NH2, Gly-Pro-Pro-Asp-Pro-Asp-Arg(Lys)-Phe-Tyr-Pro-Gly-Met-NH2, pGlu-Pro-Asp-Pro-Asp-Arg-Phe-Tyr-Pro-Gly-Met-NH2, Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Tyr-Pro-Val-Met. The latter three peptides have the unusual C-terminal sequence Pro-Gly(or Val)-Met-NH2 rather than Gly-Leu-Met-NH2 found in many other members of the tachykinin family. The observed amino acid substitutions may be the reason for the marked decrease in the biological activity observed in all in vitro and in vivo tests, even through the spectrum of tachykinin activities was retained. A kassinin-like peptide, with the sequence Gly-Pro-Pro-Asp-Pro-Asn-Lys-Phe-Ile-Gly-Leu-Met-NH2, was also found in the A. callidryas skin. While kassinin has a much higher affinity for NK-3 than for NK-1 receptors, the opposite is true for this A. callidryas peptide. The extract from A. callidryas skin also contained a new caerulein (pGlu-Asp-Tyr(HSO3)-Lys-Gly-Trp-Met-Asp-Phe-NH2) and a phyllokinin (Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg-Ile-Tyr), as well as the opioid peptides dermorphin and [Hyp6]dermorphin, both previously isolated from different Phyllomedusa species.

Choline esters and biogenic amines in the hypobranchial gland of 55 molluscan species of the neogastropod Muricoidea superfamily.

As many as 55 neogastropod molluscs, all belonging to the Muricoidea superfamily, have been investigated for occurrence and contents, in their hypobranchial gland (HG), of choline esters and, subordinately, biogenic amines. Very high amounts of esters, strictly localized in the median area of the HG, were found in all dye-secreting molluscs. The choline esters were represented by murexine, dihydromurexine and senecioylcholine. A fourth ester, acryloylcholine, occurred in the HG of a single, non dye-secreting mollusc. All the compounds displayed potent neuromuscular blocking actions in all examined vertebrate and invertebrate species, as well as potent nicotinic actions. Muscarinic effects were either lacking or unimportant. In addition to choline esters the HG occasionally contained known and hitherto unknown biogenic amines: tyramine, octopamine, 5-hydroxytryptamine, histamine, urocanylhistamine and imidazole-propionylhistamine. The interest of extending the search of bioactive compounds to carnivorous, predatory molluscs other than those described in this paper and, more, extensively, to any molluscan species provided with 'venomous' glands or apparatuses, is emphasized.

Pharmacological studies of 'sapo' from the frog Phyllomedusa bicolor skin: a drug used by the Peruvian Matses Indians in shamanic hunting practices.

The dried skin secretion from Phyllomedusa bicolor, 'sapo', is used by the Matses Indians of the Northern Peru, in shamanic rites mainly designed to improve luck in hunting. When rubbed into burned, exposed areas of the skin, the drug causes the prompt appearance of violent peripheral gastrointestinal and cardiovascular effects soon followed by remarkable central effects (increase in physical strength, heightening of senses, resistance to hunger and thirst, exalted capacity to face stress situations). All the peripheral and most of the central effects of 'sapo' can be ascribed to the exceptionally high content of the drug (up to 7% of its weight) in potently active peptides, easily absorbed through the burned, inflamed areas of the skin. The concentration in 'sapo' of the single peptides (phyllocaerulein, phyllomedusin, phyllokinin, demorphins and deltorphins) has been determined by bioassay, and peptide contents were correlated with the different symptoms of the 'sapo' intoxication.

Guinea-pig ileum (GPI) and mouse vas deferens (MVD) preparations in the discovery, discrimination and parallel bioassay of opioid peptides.

As many as 47 amphibian and mammalian, natural and non-natural opioid peptides have been examined in guinea-pig ileum (GPI) and mouse vas deferens (MVD) preparations. The great value of these extremely simple and accessible tissue models in the identification, isolation and purification of endogenous opioid peptides, in studying structure/activity relationships, and in determining selectivity of the peptide molecules for the various opioid receptors, especially delta- and mu-receptors, is emphasized.

Dermorphin-related peptides from the skin of Phyllomedusa bicolor and their amidated analogs activate two mu opioid receptor subtypes that modulate antinociception and catalepsy in the rat.

Three naturally occurring dermorphin-like peptides from the skin of the frog Phyllomedusa bicolor, the related carboxyl-terminal amides, and some substituted analogs were synthesized, their binding profiles to opioid receptors were determined, and their biological activities were studied in isolated organ preparations and intact animals. The opioid binding profile revealed a very high selectivity of these peptides for mu sites and suggested the existence of two receptor subtypes, of high and low affinity. The peptides tested acted as potent mu opioid agonists on isolated organ preparations. They were several times more active in inhibiting electrically evoked contractions in guinea pig ileum than in mouse vas deferens. When injected into the lateral brain ventricle or peritoneum of rats, the high-affinity-site-preferring ligand, [Lys7-NH2]dermorphin, behaved as a potent analgesic agent. By contrast, the low-affinity-site-preferring ligand, [Trp4,Asn7-NH2]dermorphin, produced a weak antinociception but an intense catalepsy.

Identification and characterization of two dermorphins from skin extracts of the Amazonian frog Phyllomedusa bicolor.

Skin extracts of South American hylid frogs of the subfamily Phyllomedusinae contain dermorphins and deltorphins, opioid heptapeptides highly selective for either mu or delta receptors. In all these peptides, a D-amino acid is present in the second position. The structure of the precursors for Ala-deltorphins was recently deduced from cloned cDNAs derived from skin of Phyllomedusa bicolor (Richter et al. (1990) Proc. Natl. Acad. Sci. USA 87, 4836-4839). From the amino acid sequence of these precursors, the existence of three peptides related to dermorphin could be predicted. From methanol extracts of skin of Ph. bicolor we have isolated two of these peptides, [Lys7]dermorphin-OH and [Trp4,Asn7]dermorphin-OH. The biological activity of these new dermorphins and their amidated counterparts is presented.

Purification and characterization of bioactive peptides from skin extracts of Rana esculenta.

The peptide fraction extracted by methanol from the skin of Rana esculenta, a species widely distributed in Western Europe, was investigated. The pharmacological activity found in the extract is attributable to the presence of authentic bradykinin, together with a shorter, partially active version of this molecule, des-Arg9-bradykinin. Also the bradykinin fragment 1-7 has been isolated, but it was inactive in our bioassay system. Moreover, a family of hydrophobic peptides has been purified and characterized, which appeared devoid of pharmacological activities when tested on smooth muscle preparations, but were provided with hemolytic activities.

Pumiliotoxin B-like alkaloid in extracts of the skin of the Australian myobatrachid frog Pseudophryne coriacea: effects on the systemic blood pressure of experimental animals and the rat heart.

Semi-purified extracts of the skin of the Australian myobatrachid frog Pseudophryne coriacea (PS)displayed striking, reversible and, in part, dose-dependent effects on the systemic blood pressure of the rat and other experimental animals, as well as on the rat heart. The blood pressure response in the rat consisted typically in an abrupt, short-lasting fall, followed by a conspicuous, more persistent rise. The fall in pressure was abolished by atropine and potentiated by physostigmine, indicating a cholinergic mechanism; rise was abolished by prazosin and guanethidine, suggesting a release of catecholamines from adrenergic nerve terminals in the vasculature. Tachyphylaxis was the obvious result of exhaustion of stores of catecholamines. On the heart, as shown in the electrocardiographic tracings, PS produced a variety of rhythm disorders, attributable both to the release of aminergic transmitters and to a direct effect on the myocardium. Whereas the pressure and electrocardiographic responses were virtually unaffected by calcium channel antagonists and agonists, all the effects of PS were sharply reduced or completely abolished by the sodium channel blocker tetrodotoxin. This suggests that activation of sodium channels may play a role in the mechanism of action of PS. However, the exact nature of ionic flux(es)influenced by the extract of frog skin remains to be established.

Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for delta opioid receptors.

With a cDNA library prepared from skin of Phyllomedusa sauvagei, the sequence of the precursor of dermorphin was elucidated recently. The sequence suggested the existence of another peptide, distantly related to dermorphin. Two variants of this peptide have now been synthesized containing either L- or D-methionine as the second amino acid. The peptide containing the D-methionine exhibited high-affinity and selectivity for delta opioid receptors in the mouse vas deferens and in rat brain homogenates. Moreover, using the synthetic peptide as marker, we could isolate small quantities of the corresponding natural peptide containing D-methionine as the second amino acid from skin extracts of Phyllomedusa sauvagei. The name deltorphin is proposed for this new peptide and its sequence is Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2.

Action of a semi-purified extract of Pseudophryne coriacea skin on the frog rectus abdominis muscle.

Extracts of the skin of the Australian frog Pseudophryne coriacea (PS) displayed a striking potentiating effect on contractions evoked in the isolated frog rectus abdominis muscle by direct electrical stimulation. There was both a conspicuous increase in twitch amplitude and a remarkable prolongation of twitch duration. High concentrations of PS also elicited an increase in tone. The effect on twitch was mainly due to direct stimulation of muscle fibres, the effect on tone to facilitation of acetylcholine release from the motor nerve terminals embedded in the musculature. Unlike the first effect, the second was blocked by tubocurarine and alpha-bungarotoxin. Tetrodotoxin inhibited both the basal and PS-stimulated twitch. Experiments with EGTA, a calcium chelating agent, suggest that activation of voltage-dependent calcium channels is involved in the response of the frog muscle to PS. Low concentrations of PS were ineffective on the non-stimulated muscle; high concentrations, on the contrary, generally caused an increase in tone. This was completely inhibited by the blockers of the nicotinic acetylcholine receptors, again suggesting a transmitter release from the nerve terminals.

Active peptides in the skins of one hundred amphibian species from Australia and Papua New Guinea.

Extracts prepared from the dried skins of approximately one hundred amphibian species from Australia and Papua New Guinea were subjected to biological screening in order to determine the nature and amounts of peptides active on smooth muscle preparations and systemic blood pressure present in these extracts. The most frequently and abundantly occurring peptides were those of the caerulein, bombesin and tachykinin peptide families represented, respectively, by caerulein; litorin, Glu(OMe)2-litorin and Glu(OEt)2-litorin; uperolein and Lys5-Thr6-physalaemin. Bradykinin-like peptides seem to have a rather diffuse distribution, in the species examined, but so far no peptide of this family has been isolated and sequenced. The only angiotensin-like peptide ever found in amphibian skin, crinia angiotensin II, has been isolated from skin extracts of a few species, belonging to the genera Crinia, Geocrinia, Ranidella and Litoria. The array of peptides occurring in amphibians from Australia and Papua New Guinea is destined to increase, because several apparently novel peptides have been identified in skin extracts by bioassay and radioimmunoassay.

Synthetic peptides related to the dermorphins. II. Synthesis and biological activities of new analogues.

A new series of analogues of the potent opiate-like peptides dermorphins (mainly tetra- and pentapeptides) were synthesized in order to better evaluate the structure-activity relationships. Relative potencies were referred to dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), the prototype of this class of frog skin peptides. Peripheral opioid activity (guinea-pig ileum and mouse vas deferens) was determined for all the dermorphin analogues. For a selected number of them also central analgesic (hot plate and tail-flick tests) and cataleptic activities were assayed in the rat by intracerebroventricular administration.

The brain-gut-skin triangle: new peptides.

New data on tachykinins and bombesins are displayed and the present situation of research on the novel amphibian skin peptides sauvagine and dermorphin is illustrated. The potent stimulant effect of sauvagine on ACTH and beta-endorphin release has been confirmed both in vivo and on columns of isolated and dispersed rat pituitary cells, and similarly the potent inhibitory effect on PRL and GH release, both in the rat and man. Particular emphasis is laid on the occurrence of sauvagine-like immunoreactivity in fish urophysis and in amphibian nervous structures, including the retina. It is suggested that the long-searched corticotropin releasing factor and PRL release-inhibiting factor may be a sauvagine-like peptide. Dermorphin, in its turn, has been found to cause, by intracerebroventricular injection, not only analgesia and catalepsy, but also conspicuous EEG and behavioral changes in the rabbit and chick, as well as a sharp reduction in gastric emptying time and gastric acid output in the rat, together with marked stimulation of PRL release.

Radioimmunoassay of dermorphin-like peptides in mammalian and non-mammalian tissues.

A selective RIA for D-Ala2-Dermorphin (Der), a natural peptide extracted from amphibian skin, has been developed using an antibody raised in rabbits against Der which has been coupled to BSA through its phenolic hydroxyl groups of tyrosine residues with 2,4-Dichloro-6-methoxy-1,3,5-triazine. The cross-reactivity of this antibody with dermorphin analogs, C- and N-terminal fragments of dermorphin molecule, some opioid and gastrointestinal peptides was tested. Der-like immunoreactivity has been identified in tissue extracts of rats, frog and cephalopoda. Der-like peptides were purified by passing methanol extracts of the tissues through a Sephadex G25 column (16 x 100 cm) eluted with 0.1 M acetic acid at 4 degrees C. Der-like immunoreactivity from neural tissue of Dosidicus gigas, Eledone moscata, and rat brain showed a good agreement with an authentic sample of synthetic dermorphin.

Sauvagine, a new polypeptide from Phyllomedusa sauvagei skin. Occurrence in various Phyllomedusa species and pharmacological actions on rat blood pressure and diuresis.

1. The occurrence of sauvagine, a new polypeptide from amphibian skin, and its actions on rat blood pressure and diuresis were studied. 2. Sauvagine was found to be present in the skin of all the 10 Phyllomedusa species so far studied, amounts ranging from a few micrograms to 240 micrograms per g fresh skin. 3. The polypeptide displayed in the rat an intense, long-lasting hypotensive action accompanied by tachycardia. Hypotension was not modified by either atropine or propranolol, excluding the participation of the autonomic nervous system in its production. Tachycardia, on the contrary, was partially inhibited by propranolol. 4. Hypotension is probably the main cause of the intense antidiuresis seen in hydrated rats following sauvagine administration. Reduction in urina volume was accompanied by a decrease in GFR and an increase in tubular NA+ reabsorption.

Parallel bioassay of physalaemin and kassinin, a tachykinin dodecapeptide from the skin of the African frog Kassina senegalensis.

1. Kassinin, a tachykinin dodecapeptide isolated from the skin of the African frog Kassina senegalensis was submitted to parallel bioassay with physalaemin, eledoisin and substance P, three major representatives of the tachykinin peptide family. Bioassay was carried out on blood pressure, salivary secretion and isolated or in situ smooth muscle preparations. 2. As expected, kassinin possessed the entire spectrum of biological activity peculiar to the tachykinins. However, among the examined tachykinins kassinin was the poorest stimulant of salivary secretion and the weakest hypotensive agent, while displaying very powerful stimulant effects on different smooth muscle preparations, especially on isolated preparations of urinary bladder. 3. Kassinin differed from the other tachykinins also for its more gradual and sustained action on several in situ and isolated preparations. The peptide most similar to kassinin in its spectrum of activity was eledoisin. 4. Emphasis is laid on the possibility to dissociate the effects of the tachykinins on different target systems through changes in the N-moiety of the tachykinin molecule.

Occurrence and polymorphism of bombesin-like peptides in the gastrointestinal tract of birds and mammals.

The gastrointestinal tract of mammals and birds, especially stomach and upper small intestine, contains bombesin-like peptides. This has been unequivocally demonstrated by radioimmunoassay and bioassay. Concentrations of bombesin-like activity may range from a few ng to 500-600 ng per g fresh tissue. Last values refer to the chicken proventriculus, which has been the object of a more thorough investigation. The bombesin-like peptide of the chicken proventriculus showed a marked heterogeneity. All forms probably stem from a pro-bombesin, a large precursor molecule which is insoluble in methanol, acetone, and even boiling water, but may be cleaved by acid hydrolysis. Methanol extracts contain at least two forms of the bombesin-like peptide; HCl extracts at least three forms; HCl extracts of the residue of methanol extraction at least four forms. Whereas some forms--for example, the methanol extractable forms--probably pre-exist in the tissue, other forms may be artefacts arising from acid treatment. The various forms may be distinguished from each other not only by their elution profile, but also by bioassay. In fact, though all forms show the activity spectrum characteristic for the amphibian bombesin-like peptides, they present considerable quantitative differences in activity. Pro-bombesin(s) probably occur also in the rat and guinea-pig stomach; similarly, a clear-cut heterogeneity is appreciable for the bombesin-like peptide of the human gastric mucosa.