Parallel bioassay of 39 tachykinins on 11 smooth muscle preparations. Structure and receptor selectivity/affinity relationship.

Parallel bioassay on smooth muscle preparations demonstrated that: all TKs having a neutral or basic residue at position 7 from the C-terminus show a clear-cut preference for the NK1 TK receptor, reinforced by the presence of the aromatic doublet Phe-Phe or Phe-Tyr (aromatic TKs); all aliphatic TKs (Phe-Ile/Val) having an acidic residue at position 7 show a clear-cut preference for NK2/NK3 receptors, generally without selectivity for a single receptor. However, in aromatic TKs having the same acidic residue, the preference for NK2/NK3 receptors is weakened, with a more or less pronounced co-preference for the NK1 receptor. Amino acid substitutions in the C-terminal tripeptide may influence receptor affinity.

Effects of natural tachykinins on porcine lower urinary tract smooth muscle.

1. The aim of our study was to ascertain the possible differences and/or similarities in natural tachykinin activity in vitro on lower urinary tract of large-sized animals as compared with data obtained in laboratory animals. 2. Besides tachykinins normally present in mammals, namely substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), we tested non-mammalian tachykinins, such as eledoisin (ELED), physalaemin (PHYS), kassinin (KASS) and PG-kassinin II (PG-KASS II). 3. NKA, KASS and ELED were found to be the most potent peptides in contracting detrusor strips from porcine bladder. In particular, NKA showed a pD2 of 7.14, whereas KASS and ELED showed pD2 values of 7.20 and 7.22, respectively. The activity of NKB and PG-KASS II corresponded to 72.4 and 55.0% respectively of that of NKA. SP and PHYS activity corresponded to only 2% of that of NKA. 4. NKA (pD2 7.92) was the most active peptide in contracting bladder neck tissues as well. ELED and KASS were found to have lower, similar pD2 values (7.62 and 7.70, respectively), whereas NKB and PG-KASS II were much less active (pD2 7.12 and 6.74, respectively). Moreover, SP and PHYS showed an activity range lower than 2% of that of NKA. 5. The reported results confirm that, on pig vesical neck and detrusor, NK1 receptors represent a minority as compared with NK2 and NK3 receptors. By contrast, the presence of NK2 receptors is demonstrated by a greater potency of NKA. The presence of NK3 receptors both on detrusor and neck is evidenced by NKB activity and by results achieved with PG-KASS II.

Neuropeptide Y release by pumiliotoxin-B in the electrically-stimulated mouse vas deferens: an immunohistochemical study.

Morphologic and immunohistochemical studies were conducted to ascertain whether pumiliotoxin-B (PTX-B), an indolizine alkaloid from the skin of the Neotropical dendrobatid frog, Dendrobates pumilio, affects the anatomic and immunohistochemical features of the electrically stimulated mouse vas deferens preparations. PTX-B, at a concentration of 1 microM, consistently decreased the density pattern of neuropeptide Y (NPY)-immunoreactive nerve fibers contained within the circular muscular layer. The alkaloid also induced striking morphologic changes. It enlarged the lumen of the vasa and relaxed the muscular wall. Pretreatment with prazosin or haloperidol affected neither the release of NPY nor the morphologic changes; pretreatment with tetrodotoxin and guanethidine abolished NPY release and prevented the PTX-B-induced morphologic changes. PTX-B had no appreciable effect on the density and distribution pattern of nerve fibers immunostained for vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, enkephalin, pancreatic polypeptide, 5-hydroxy-tryptamine and tyrosine hydroxylase.

Transmitter release and uptake evoked by the amphibian skin alkaloid, pumiliotoxin-B (PTX-B), in the electrically stimulated mouse vas deferens preparation (MVD).

Upon electrical stimulation three transmitters are known to be released from the adrenergic nerve terminals of the isolated MVD preparation: two motor transmitters (noradrenaline (NA) and ATP) acting synergistically to provoke twitch contraction, and an inhibitory transmitter, the peptide NPY. The frog alkaloid pumiliotoxin-B (PTX-B) displayed two opposite effects on the electrically stimulated MVD: at low concentrations (0.1-0.3 microM) it caused twitch depression, at higher concentrations (0.5-2 microM) there was a potent twitch stimulation. Transmitters and/or receptors involved in the depressive effect could not be clearly identified, although interference with NPY is possible. On the other hand, the potent twitch stimulation caused by PTX-B may be due to exaggerated release of the same transmitters (NA and ATP) involved in twitch stimulation produced by electrical stimulation. Opening by PTX-B of the Na+ channels on the membrane of the adrenergic nerve terminals causes activation of the amine pump facilitating re-uptake of not only endogenous NA but also of exogenous catecholamines.

[D-Leu2]deltorphin, a 17 amino acid opioid peptide from the skin of the Brazilian hylid frog, Phyllomedusa burmeisteri.

A novel 17 amino acid peptide, having a D-leucine in position 2 of its sequence, has been isolated from methanol extracts of the skin of the Brazilian frog, Phyllomedusa burmeisteri. The sequence of the peptide is: Tyr-D-Leu-Phe-Ala-Asp-Val-Ser-Thr-Ile-Gly-Asp-Phe-Phe-His-Ser-Ile-NH2. It displays a poor affinity for delta-opioid binding sites, both in the periphery and in the central nervous system. However, the shorter synthetic amidated analogue (1-10) possess both on the central and peripheral delta binding sites an agonistic potency equalling in affinity and exceeding in selectivity that of the enkephalins. The shorter amidated analogue (1-7) is virtually inactive on opioid binding sites in the periphery, but displays a clear-cut affinity for both delta and mu binding sites on rat brain membranes. To date six different D-amino acid residues have been found, always in position 2 of the sequence, in as many as 11 natural peptide molecules of animal origin.

Structure-activity relationships of the delta-opioid-selective agonists, deltorphins.

Deltorphins are naturally occurring peptides with high affinity and selectivity for delta-opioid receptors. They share with dermorphin, another mu-selective opioid agonist, the same N-terminal tripeptide Tyr-D-Xaa-Phe, where D-Xaa is a D-Ala or a D-Met residue. This common sequence appears to be essential for the best fitting of the peptides to both mu- or delta-opioid sites. We studied the changes in receptor affinity and selectivity and in biological potency of deltorphins due to shortening of the sequence, C-terminal deamidation or single amino acid substitutions. The results support the view that a code addressing the molecule towards delta-opioid sites is expressed in the C-terminal region of these peptides. This addressing domain confers high delta-selectivity to the ligand in the following two ways: (i) increased affinity for delta-sites; (ii) decreased affinity for mu-sites. The sequence of the C-terminal tripeptide appears to be responsible for the high delta-affinity of the molecules. Negatively charged side chains inhibit mu-binding and enhance delta-selectivity.

Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites.

Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.

Biogenic amines and active peptides in extracts of the skin of thirty-two European amphibian species.

1. Extracts prepared from fresh or dried skins of 32 European amphibian species were submitted to chemical (colour reactions) and biological screening to determine the occurrence and contents of biogenic amines and peptides active on smooth muscle preparations and blood pressure. 2. Only indolealkylamines were detectable in the skins. They were represented by tryptamine, 5-hydroxytryptamine, and its N-methylated, cyclized and sulphoconjugated derivatives. 3. The peptide families identified in the extracts were as follows: bombesins (bombesin and alytesin), bradykinins (bradykinin, bradykinin 1-8, bradykinin 1-7), chemotactic peptides (RECP I, II and III), bombinins and TRH. Bombesins, bombinins and TRH (thyrotropin-releasing hormone) were isolated from skin extracts of discoglossid frogs; chemotactic peptides and again TRH from extracts of ranid frogs. 4. Further research will certainly lengthen the list of active peptides in the skin of European amphibians, as is the case with Australian, American and African amphibians.

Parallel bioassay of 27 bombesin-like peptides on 9 smooth muscle preparations. Structure-activity relationships and bombesin receptor subtypes.

Thirteen natural bombesin-like peptides and 14 synthetic analogues were submitted to parallel bioassay on 9 smooth muscle preparations in order to determine their relative potency, in comparison to bombesin and litorin. The natural peptides of the bombesin subfamily showed a uniformly high or moderate potency on all preparations. However, synthetic bombesins of shorter chain length (hepta- and octapeptides) manifested a good potency only on the rat uterus preparation. Among the peptides of the litorin and phyllolitorin subfamilies, only litorin and ranatensin presented a full spectrum of potency, equalling or even surpassing that of bombesin. All other natural and synthetic members of the two subfamilies showed a sharply dissociated spectrum of potency on the different smooth muscle preparations. The only exception was the rat urinary bladder and, in part, the chicken intestine, on which the peptides displayed a uniformly high potency, comparable to, or even greater than that of bombesin. The present results help to explain structure/activity relationships and suggest the probable existence, in the periphery, of multiple bombesin receptor subtypes.

Biogenic amines and active peptides in the skin of fifty-two African amphibian species other than bufonids.

1. Extracts prepared from dried or fresh skins of 52 African amphibian species, other than bufonids, were subjected to chemical (colour reactions) and biological screening, to determine occurrence and contents of aromatic biogenic amines and peptides active on smooth muscle preparations and blood pressure. 2. Only indolealkylamines were detectable in the skins. They were represented by 5-hydroxytryptamine, its N-methylated derivatives and tryptamine. The indolealkylamines considered included the alkaloid trypargine, a carboline derivative resulting from the condensation of tryptamine with arginine. 3. The peptide families identified in skin extracts of the African frogs were as follows: caeruleins (caerulein, [Asn2, Leu5] caerulein), tachykinins (kassinin, [Glu2, Pro5] kassinin, hylambatin), bradykinins [( Hyp3] bradykinin), xenopsin, thyrotropin releasing hormone, peptide PYLa and the magainins I and II. The last five peptides have been so far identified only in the skin of Xenopus laevis, together with their precursors. 4. Since numerous other peptide molecules await isolation, elucidation of structure, and definition of possible biological activities, the array of peptides occurring in the skin of African amphibians, as in that of Australian and American amphibians, is destined to increase.

Litorin-like peptides in the skin of five phyllomedusa species other than Phyll.sauvagei and Phyll.rohdei.

Methanol extracts of the skin of five Phyllomedusa species, other than Phyll. sauvagei and Phyll.rohdei, were estmined to check their content in litorin-like peptides. Extracts of Phyll.bicolor and Pachymedusa dacnicolor did not contain appreciable amounts of litorin-like activity. However, extracts of Phyll. burmeisteri presented a typical phyllolitorin-like activity and extracts of Phyll.hypochondrialis an activity mimicking that of rohdei-litorin. A particular position is taken by extracts of Phyll.trinitatis, which displayed a litorin-like activity differing, in parallel bioassay, from that of all known natural litorins.

A potent factor in extracts of the skin of the Australian frog, Pseudophryne coriacea--II. Stimulation of the leech helical muscle in vitro and in vivo.

Extracts of the skin of Pseudophryne coriacea displayed a powerful stimulant action on the leech helical muscle, both in vitro and in vivo. In the isolated dorsal muscle, the extract caused the appearance of vigorous phasic movements, accompanied by rapid increase in tonus, up to intense spasm. Hyoscine, physostigmine, hexamethonium, tubocurarine and alpha-bungarotoxin did not affect the response to the extract; tetrodotoxin, nifedipine and 5-hydroxytryptamine (5-HT) produced a partial blockade. In the intact animal, the extract at first potently stimulated the musculature, evoking the appearance of a succession of incoordinated, spastic movements, with twisting and rolling of the body of the animal. Stimulation was followed by paralysis and death. It is suggested that the pumiliotoxin-like alkaloid of Pseudophryne coriacea, responsible for these effects, acts directly on the helical muscle.

A potent factor in extracts of the skin of the Australian frog, Pseudophryne coriacea--III. Potentiation of contractions elicited in avian and mammalian isolated skeletal muscle preparations by direct and indirect electrical stimulation.

Extracts of the skin of the Australian frog Pseudophryne coriacea displayed a striking potentiating effect on contractions evoked in isolated skeletal muscle preparations of mammals (phrenic nerve diaphragm) and birds (chick biventer cervicis and semispinalis muscles) by indirect and direct electrical stimulation. There was both a conspicuous increase in the amplitude of the twitch, up to 10-fold, and a remarkable prolongation of the duration of the twitch. The effect was dose- and frequency-dependent. In the presence of the extract, fusion of twitches after tetanic stimulation occurred earlier. No tachyphylaxis upon repeated stimulation by the extract was observed and the response to large doses persisted, declining slowly, for hours. These effects must be ascribed to an alkaloid related in structure to pumiliotoxin B. Response to the extract of Pseudophryne coriacea by indirectly-stimulated preparations was potentiated by physostigmine and blocked by tubocurarine and alpha-bungarotoxin, demonstrating that in these preparations the extract acted pre-synaptically to facilitate the release of acetylcholine from motor nerve endings. However, the extract of Pseudophryne coriacea displayed equally potent effects in directly stimulated preparations, insensitive to physostigmine and to blockers of nicotinic acetylcholine receptors, indicating a direct action on the skeletal muscle. It is suggested that, like pumiliotoxin B, the Pseudophryne coriacea alkaloid may interfere in the regulation of calcium channels in both nerve and muscle fibres.

Active peptides in the skins of two hundred and thirty American amphibian species.

Extracts prepared from dried or fresh skins of more than 200 American amphibian species were subjected to biological screening in order to determine occurrence and contents of peptides active on smooth muscle preparations, systemic blood pressure and, subordinately, external secretions, anterior pituitary and the central nervous system. The peptide families identified in skin extracts were as follows: caruleins (caerulein, phyllocaerulein), tachykinins (physalaemin, phyllomedusin), bombesins (phyllolitorin, [Leu8]phyllolitorin, rohdeilitorin), bradykinins (phyllokinin and others), sauvagine, dermorphins (dermorphin, [Hyp6]dermorphin), tryptophyllins (numerous peptides) and, finally, miscellaneous peptides. None of the above peptide families showed a widespread distribution, but all were restricted to particular amphibian genera or stocks. The hylid frogs of the Phyllomedusinae family occupy a unique position, as their skin displayed the greatest variety and abundance of active peptides ever found in any amphibian is destined to increase because numerous other peptide molecules await isolation, elucidation of structure and definition of possible biological activities.

Indole-, imidazole- and phenyl-alkylamines in the skin of one hundred and forty American amphibian species other than bufonids.

Extracts prepared from dried or fresh skins of 140 American amphibian species, other than bufonids, were subjected to chemical and biological screening in order to determine the presence and concentrations of aromatic biogenic amines. The most frequent and abundantly occurring amine category was that of indolealkylamines, represented by their prototype 5-hydroxytryptamine and its N-methylated derivatives. Conjugated and cyclized indolealkylamines, typical for the toad skin, were apparently lacking. Phenylalkylamines were represented by two quaternary ammonium bases: leptodactyline and, very rarely, candicine. Leptodactyline was particularly abundant in leptodactylid frogs of the genus Leptodactylus. Histamine occurred in trace amounts in different species, in large amounts only in some Leptodactylus species of the "pachypus" section. On the other hand, N-methylated histamines and cyclized histamines (spinaceamines) were confined to the skin of Leptodactylus pentadactylus labyrinthicus. The possible taxonomical and evolutionary significance of amphibian skin amines is pointed out.

A potent factor in extracts of the skin of the Australian frog, Pseudophryne coriacea. Apparent facilitation of transmitter release in isolated smooth muscle preparations.

Extracts of the skin of the Australian frog Pseudophryne coriacea (PsC) displayed potent stimulant effects on isolated smooth muscle preparations of intestine and similar effects on electrically-stimulated vas deferens preparations. These effects must be ascribed to an alkaloid, related in structure to the pumiliotoxins, a class of alkaloid compounds occurring in the skin of neotropical poison frogs. On the basis of results obtained with antagonists and blocking agents, it is suggested that the extract has a pre-synaptic, neurogenic point of attack and that it acts to facilitate the release of transmitters from nerve endings. Acetylcholine is the most important agent involved in the response to the extract by the intestinal muscle and noradrenaline in the response by vas deferens preparations. However, release of other aminergic or peptidergic transmitters may participate, positively or negatively, in the response.

Rohdei-litorin: a new peptide from the skin of Phyllomedusa rohdei.

The bombesin-litorin family of peptides is characterized by the common amino acid sequence-Gly-His-X-Met-NH2 at the C-terminus, where X is a hydrophobic or aromatic residue. A new member of this family, rohdei-litorin, has been isolated from amphibian skin and its structure shown to be: Glp-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH2. This new peptide displayed a greater affinity than other members of the family for rat urinary bladder receptors. A litorin-like peptide, with high affinity for this kind of receptor, has already been described in mammalian spinal cord and named neuromedin B. Rohdei-litorin shares with neuromedin B the entire C-terminal octrapeptide and may be considered the amphibian counterpart of this mammalian neuropeptide.

Phyllomedusa skin: a huge factory and store-house of a variety of active peptides.

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 phyllocaerulein), the bradykinins (phyllokinin), the tachykinins (phyllomedusin), the bombesins (phyllolitorin, [Leu8]phyllolitorin, rohdei-litorin), sauvagine, the dermorphins (dermorphin, [Hyp6]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 Phyllomedusa peptides within their families is briefly discussed, dwelling upon some recent and controversial data.

Parallel bioassay of litorin and phyllolitorins on smooth muscle preparations.

Phyllolitorin and Leu8-phyllolitorin, two nonapeptide amides from the skin of the South American frog Phyllomedusa sauvagei, are representatives of a novel bombesin subfamily, characterized by the occurrence in their molecule of a serine residue substituting the usual histidine residue at position 3 from the C-terminus. In parallel bioassay on ten different smooth muscle preparations and rat blood pressure, phyllolitorin and Leu8-phyllolitorin were virtually equiactive, but the two peptides appeared remarkably less potent that litorin in all test preparations, except the rat urinary bladder. The shape of contractions produced by the phyllolitorins and promptness of cessation of their action upon washing seem to indicate a looser binding of these peptides to their receptors and/or a more rapid inactivation, in comparison to litorin.

Parallel bioassay of physalaemin and hylambatin on smooth muscle preparations and blood pressure.

Hylambatin, a novel tachykinin endecapeptide isolated from the skin of the African frog Hylambates maculatus, must be ascribed to the physalaemin subfamily. It differs structurally from all other known tachykinins mainly in having a methionine residue replacing the usual leucine residue at position 2 from the C-terminus. In parallel bioassay on a number of in-vitro and in-vivo test objects, hylambatin and physalaemin were nearly indistinguishable from each other, with few moderate quantitative differences.