C. Severini

Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for δ 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.

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 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.

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.

Reproducible withdrawal contractions of isolated guinea-pig ileum after brief morphine exposure : effects of clonidine and nifedipine

Abstract— Guinea‐pig ileum stored for 30 min in Krebs solution and then mounted in Tyrode solution gave reproducible contracture responses to naloxone after brief exposure to morphine. The preparation lasted for several hours and a variety of pharmacological tests could be made. Clonidine, an α2‐adrenoceptor agonist, and nifedipine, a calcium channel antagonist, both known to interfere with tolerance and physical dependence, inhibited naloxone withdrawal contractures in a dose related way. Their action seemed to be receptor‐mediated since yohimbine and Bay k 8644, respectively, reversed their inhibitory effect.

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.

Six novel tachykinin- and bombesin-related peptides from the skin of the Australian frog Pseudophryne güntheri

Six novel peptides belonging to the tachykinin and bombesin families were isolated and sequenced from extracts of the skin of the Australian myobatrachid frog Pseudophryne güntheri. One of these peptides (PG-L) was of the bombesin family and may be considered an N-elongation of the litorin/ranatensin molecule, with which it shares an identical spectrum of activity on isolated smooth muscle preparations. The other five peptides were of the tachykinin family with two of these peptides (PG-SPI and PG-SPII) related to substance P and three (PG-KI, PG-KII and PG-KIII) to kassinin. In contrast to the basic nature of substance P, the PG-SP peptides showed a clear acidic character and displayed a more potent and sustained action on isolated smooth muscle preparations and rat blood pressure than did substance P. Two of the three PG-K peptides were more potent than kassinin; PG-KIII was considerably less potent. PG-KI and PG-KII were also present in a deamidated, poorly active, form.

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.

A new opioid peptide predicted from cloned cDNAs from skin of Pachymedusa dacnicolor and Agalychnis annae

We have isolated a cDNA encoding a precursor of dermorphin from the skin of Pachymedusa dacnicolor. Besides four copies of this opioid peptide, the deduced sequence also contains the genetic information for a novel peptide Tyr‐Ile‐Phe‐His‐Leu‐Met‐Asp‐NH2. This differs from Met‐deltorphin by the presence of Ile at position 2. In a related precursor from the skin of Agalychnis annae, the sequence of this peptide is in the 3′‐untranslated region of the cloned cDNA. From earlier results we predict that in skin peptides the second residue is d‐allo‐Ile. We have synthesized this and related peptides with different d‐amino acids, and determined their δ agonist activity. The peptide with d‐nor‐Leu binds with high affinity to δ receptors, while that with d‐allo‐Ile is about 100 times less active.

Postnatal development of δ-opioid receptor subtypes in mice

The density and affinity of binding sites for the δ‐selective opioid ligands [3H]‐[d‐Ala2, Asp4]deltorphin (DELT‐I), [3H]‐[d‐Ala2Glu4]‐deltorphin (DELT‐II), [3H]‐[d‐Pen2,d‐Pen5]enkephalin (DPDPE), and [3H]‐naltrindole (NTI) were determined in whole brain from 10, 15, 25 and 60 day‐old C57BL mice. At all ages, the analyses of the homologous displacement curves, gave best fits to single rather than to multiple site models. The binding capacity (Bmax) labelled by [3H]‐NTI was about one half that labelled by [3H]‐DELT‐I, [3H]‐DELT‐II and [3H]‐DPDPE. In 25 and 60 day‐old mouse brain the DPDPE Bmax was 25% less than the deltorphin‐II Bmax. In saturation experiments, specific binding of [3H]‐DELT‐I on adult mouse brain homogenates was best fitted by a two‐site model (34% high affinity site, Kd=1.08 nm and 66% low affinity sites, Kd=39.9 nm). DPDPE produced a biphasic inhibition of specific [3H]‐DELTI‐I binding, from 15 days of age onwards. The relative percentage of high and low affinity sites was 72% and 28% in 15 day‐, 65% and 35% in 25 day‐ and 30% and 70% in 60 day‐old mice. In adult mouse brain labelled with [3H]‐DELT‐I, DELT‐II recognized 71% of high‐affinity and 29% of low‐affinity sites. DELT‐I and DPDPE produced monophasic inhibition of specific [3H]‐DELT‐II binding to brain homogenates of adult mice. These data suggest that a sub‐population of δ‐sites (probably the δ2‐subtype), recognized by DELT‐I, with high affinity for DELT‐II and low affinity for DPDPE develops from 25 days onward. In electrically stimulated mouse vas deferens (MVD) the rank order of potency of the three δ‐agonists was: DELT‐I> DELT‐II> DPDPE in 10 day‐old mice, and: DELT‐I= DELT‐II> DPDPE, from 25 days onward. During this time, the potency of DELT‐II increased about 15 fold whereas the potency of DELT‐I and DPDPE increased only 5 times. The higher efficacy of DELT‐II could depend on receptor maturation towards the δ2‐subtype.