A proenkephalin A-derived peptide analogous to bovine adrenal peptide E from frog brain: Purification, synthesis, and behavioral effects

A peptide derived from the posttranslational processing of proenkephalin A was isolated from an extract of the brain of the European green frog Rana ridibunda and its primary structure established as: Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg¹⁰-Pro-Glu-Trp-Trp-Gln-Asp-Tyr-Gln-Lys-Arg²⁰-Tyr-Gly-Gly-Phe-Met. The structure was confirmed by chemical synthesis. The peptide represents an amphibian equivalent of bovine adrenal peptide E [preproenkephalin A (206–230)-peptide] but the sequence contains two amino acid substitutions (Met¹⁵ → Gln and Leu²⁵ → Met) compared with the mammalian peptide. The data support previous hypotheses that the Leu-enkephalin sequence is not present in preproenkephalin A of amphibians. Intracerebroventricular injections of frog peptide E (10 and 100 ng) in mice had no significant effect on horizontal locomotor activity. The peptide, in doses up to 1 μg, had no effect on latency of escape jumping in the hot plate test and the peptide (100 ng) did not modify responses (paw licking, rearing, and escape jumping) in morphine-treated mice.     

Isolation and sequence determination of frog C-type natriuretic peptide

A new bioactive peptide was isolated from frog brain using a bioassay for chick rectum relaxant activity. Amino acid sequence of this peptide was determined to be Gly-Tyr-Ser-Arg-Gly-Cys-Phe-Gly-Val-Lys-Leu-Asp-Arg-Ile-Gly-Ala-Phe-Ser- Gly- Leu-Gly-Cys, in which two cysteines were linked by a disulfide bond. The peptide was found to belong structurally to the natriuretic peptide family and to exert diuretic-natriuretic activity as well as hypotensive activity when injected into rats. The peptide showed a high homology to recently identified porcine C-type natriuretic peptide (CNP) and a pharmacological spectrum highly similar to porcine CNP. Thus, the peptide was designated frog C-type natriuretic peptide (frog CNP). Frog CNP may participate in the central control of body fluid homeostasis, since its tissue concentration is high in brain.     

The primary structure and tissue distribution of an amphibian neuropeptide Y.

Neuropeptide Y (NPY) has been isolated and sequenced from brain extracts of the European common frog, Rana temporaria. Plasma desorption mass spectroscopy of the purified peptide indicated a molecular mass of 4243.3 Da which was in agreement with that deduced from the sequence (4243.7 Da), incorporating a C-terminal amide. The primary structure of frog NPY was established as: YPSKPDNPGEDAPAEDMAKYYSALRHYINLITRQRY-NH2. Frog NPY contains a single, highly-conservative amino acid substitution (Lys for Arg at residue 19) with respect to human NPY. NPY immunoreactivity was localised exclusively in nerves within the brain, pancreas and gastrointestinal tract and reverse-phase HPLC of extracts of these tissues resolved a single immunoreactive peptide of identical retention time in each case. The primary structure of NPY has therefore been highly-conserved over a considerable evolutionary time-span.     

Effects of neurotransmitters upon the discharge of secretory product from the cutaneous glands of the red-spotted newt.

The effects of sympathetic and parasympathetic agonists and antagonists on discharge of secretory product by the granular and mucous glands were examined in the red-spotted newt, Notopthalmus viridescens viridescens. Observations were made also on the South African clawed toad. Xenopus laevis, the grass frog, Rana pipiens, and the crested newt, Triturus cristatus. In contrast to the granular glands of the South African clawed toad and the grass frog, which were stimulated by alpha-adrenergic agents, those of the red-spotted newt discharge in response to acetylcholine, either in vitro when added to the Holtfreter's solution in which explants were incubated, or in vivo when injected subcutaneously. Granular glands of the crested newt were also dischared in response to subcutaneous injection of acetylcholine. Stimulation of the granular glands by acetylcholine was blocked by atopine but not by tubocurarie, indicating that the cholinergic receptors are muscarinic rather than nicotinic. The mucous glands of the red-spotted newt, on the other hand, did not discharge in response to either acetylcholine or to adrenergic agents.     

Isolation and primary structure of urotensin II from the brain of a tetrapod, the frog Rana ridibunda.

A peptide related to urotensin II has been isolated in pure form from an extract of the brain of the European green frog, Rana ridibunda. The primary structure of the peptide was established as Ala-Gly-Asn-Leu-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val and this sequence was confirmed by chemical synthesis. Frog urotensin II contains an additional amino acid residue compared with fish urotensin II peptides but the structure of the cyclic region of the molecule has been fully conserved. The data show that urotensin II is not confined to the caudal neurosecretory system of fish but is present in the central nervous system of a tetrapod.     

Primary structures of the bombesin-like neuropeptides in frog brain show that bombesin is not the amphibian gastrin-releasing peptide

Peptides displaying gastrin-releasing peptide/bombesin-like immunoreactivity were isolated in pure form from an extract of the brain of the European green frog, Rana ridibunda. The primary structure of the more abundant peptide was established as: Gly-Ser-His-Trp-Ala-Val-Gly-His-Leu-Met. NH2. This sequence shows one substitution (Ser for Asn) compared with mammalian gastrin-releasing peptide (18-27) (neuromedin C). The extract also contained gastrin-releasing peptide but bombesin was absent. The data indicate that bombesin is not the amphibian counterpart of gastrin-releasing peptide.     

Intragastric administration of cyclo(Leu-Gly) inhibits the development of tolerance to the analgesic effect of morphine in the rat

The effect of intragastric administration of cyclo(Leu-Gly), a cyclic dipeptide derived from melanotropin release inhibiting factor (Pro-Leu-Gly-NH2), on the development of tolerance to the analgesic effect of morphine in the rat was determined. The tolerance to morphine in the rat was induced by subcutaneous implantation of four morphine pellets during a 3-day period. The rats which served as controls were implanted with placebo pellets. The analgesic response to a challenge dose of morphine was determined by the tail-flick test. The tail-flick latencies were determined before and then every 30 min for 180 min. The analgesic response was computed by determining the area under the time-response curve. Implantation of morphine pellets resulted in the development of tolerance as evidenced by decreased analgesic response to morphine in morphine pellet implanted rats as compared to placebo pellet implanted rats. Chronic intragastric administration of cyclo(Leu-Gly) (4 to 16 mg/kg) inhibited the development of tolerance to morphine. A dose of 8 mg/kg of cyclo(Leu-Gly) completely blocked the tolerance to morphine. The study provides for the first time evidence that intragastric administration of a cyclic peptide can inhibit the development of tolerance to morphine, and that effective neuropeptides and their analogs can be developed as potential drugs to inhibit opiate-induced tolerance.     

Long-lasting antinociceptive effect of DAMGO chloromethyl ketone in rats

Previously, the opioid peptide Tyr-D-Ala-Gly-(NMe)Phe-CH2Cl (DAMCK) has been shown to bind irreversibly to mu opioid receptors in vitro. In the present work, the antinociceptive effect of DAMCK has been evaluated. Rats treated systemically with DAMCK (1-100 pg/kg) displayed a dose-dependent increase in tail-flick analgesia that peaked by 15 min, then stayed about the same until 60 min, followed by some decrease over time. Higher doses of DAMCK (10 ng/kg-100 microg/kg) produced a near-maximal antinociceptive effect that remained stable for 4 h. Significant antinociception was still detected 8 h, but not 24 h postinjection. In comparison, the parent peptide DAMGO (Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol) reached maximal effect by about 30 min, followed by a rapid cessation of its antinociceptive response. Naloxone administered before DAMCK antagonized the antinociceptive response of DAMCK, indicating that it was mediated via opioid receptors. Naloxone administered 45 min after DAMCK attenuated the tail-flick response to some extent, but a substantial part (40-60% depending on the peptide concentration and evaluation time) remained unaffected. Central administration of DAMCK also elicited time- and concentration-dependent, profound, opioid receptor mediated, apparently irreversible antinociception.     

Isolation and primary structure of an amphibian neurotensin.

Using a radioimmunoassay system employing an antiserum which recognises the common C-terminal tripeptide (YIL) of neurotensin (NT) and neuromedin N (NN), immunoreactivity was identified in extracts of brain (65.8 pmol/g), small intestine (44.2 pmol/g) and rectum (13.2 pmol/g) of the European common frog (Rana temporaria). No immunoreactivity was detected in extracts of stomach and skin. Reverse-phase HPLC analysis of each tissue extract resolved a single immunoreactive peptide with identical retention time in each case. The immunoreactive peptide was isolated by reverse-phase HPLC from brain extracts and an N-terminal pyroglutamyl residue was successfully removed enzymatically. The molecular mass of des(pyroglutamyl) frog NT, determined by plasma desorption mass spectroscopy, was 1440 Da. The primary structure of this peptide was determined by gas-phase sequencing and the calculated molecular mass, 1440.7 Da, was in close agreement with that derived by mass spectroscopy. The full primary structure of frog NT was established as: QSHISKARRPYIL. When compared with bovine NT, frog NT exhibits five amino acid substitutions in the N-terminal region, whereas the C-terminal hexapeptide sequence (RRPYIL), which mediates the classical biological effects of NT, is completely conserved. Amphibia thus possess a tridecapeptide NT which is analogous to that of higher vertebrates and considerable constraints on the primary structure of the C-terminal biologically-active core have existed for a vast evolutionary time span.     

Isolation, localization, and cloning of a kainic acid binding protein from frog brain

Excitatory amino acids (EAA) are major neurotransmitters in the vertebrate central nervous system. EAA receptors have been divided into three major subtypes on the basis of electrophysiological and ligand binding studies: N-methyl-D-aspartate, kainate, and quisqualate receptors. To understand their molecular properties, we undertook a project aimed at isolation and cloning of these receptor subtypes. We purified a kainate binding protein (KBP) from frog brain, in which kainate binding sites are about fortyfold more abundant than in rat brain, using domoic acid affinity chromatography, and made monoclonal and polyclonal antibodies to the purified protein. These antibodies immunoprecipitate the frog KBP but not KBPs from other species. Immunocytochemical analyses show that KBP has a synaptic and extrasynaptic localization in frog optic tectum, with most labeling being extrasynaptic. The cDNA encoding frog brain KBP was isolated by screening a frog brain cDNA library with oligonucleotide probes that were based on the amino acid sequence of the purified protein. The deduced amino acid sequence of the KBP has a hydrophobic profile similar to those of other ligand-gated ion channel subunits, such as the nicotinic acetylcholine receptor, the GABAA receptor, and the glycine receptor. Frog brain KBP is very similar (36% amino acid identity to the carboxyl half) to rat brain kainate receptor, suggesting that these two proteins evolved from a common ancestor. The function of KBP in frog brain remains a major question. Preliminary results showed that Xenopus laevis oocytes injected with KBP RNA did not produce a detectable electrophysiological response when perfused with kainate. These results suggest that additional subunits may be required to form a functional receptor or that KBP is not functionally related to a neurotransmitter receptor.     

PD-sauvagine: a novel sauvagine/corticotropin releasing factor analogue from the skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor

Sauvagine is a potent and broad-spectrum biologically active peptide of 40 amino acid residues originally isolated from the skin of the South American frog, Phyllomedusa sauvagei. Since its discovery, no additional sauvagine structures have been reported. Following the discovery of sauvagine, peptides with similar primary structures/activities were identified in mammalian brain [corticotropin-releasing factor (CRF) and urocortin]. Here, we report the identification of a second sauvagine from the Mexican giant leaf frog, Pachymedusa dacnicolor, which displays primary structural features of both sauvagine and CRF. A cDNA encoding the peptide precursor was "shotgun" cloned from a cDNA library constructed from lyophilised skin secretion by 3'- and 5'-RACE reactions. From this, the primary structure of a 38-mer peptide was deduced and this was located in reverse phase HPLC fractions of skin secretion and both its mass and structure were confirmed by mass spectrometry. The biological activities of synthetic replicates of PD-sauvagine and sauvagine were compared using two different mammalian smooth muscle preparations and the novel peptide was found to be more potent in both. Bioinformatic analyses of PD-sauvagine revealed that it shared different regional sequence identities with both sauvagine and CRF.     

Identification of a peptide arising from the specific post-translation processing of secretogranin II

The biological role of secrotogranin II is unknown but it has been suggested that the protein may function as a precursor of one or more biologically active neuroendocrine peptides. We have isolated a 33 amino acid-residue peptide from the brain of the frog Rana ridibunda that shows strong (82%) homology with human presecretogranin II-(182-204)-peptide. This region of secretogranin II has also been very strongly conserved in the rat and bovine proteins. Analysis of the nucleotide sequence of the mammalian secretogranin II cDNAs indicates that the peptide sequence is flanked by two Lys-Arg dibasic residue processing sites. It is proposed, therefore, that this fragment represents a specific product of the post-translational processing of secretogranin II and, by analogy with peptides derived from chromogranin A, may be important in the regulation of neurosecretion.     

Kassinatuerin-1: a peptide with broad-spectrum antimicrobial activity isolated from the skin of the hyperoliid frog, Kassina senegalensis

Kassinatuerin-1 (GFMKYIGPLI(10)PHAVKAISDL(20)I.NH(2)) was isolated in high yield (75 nmol/g) from an extract of the skin of a Hyperoliid frog, the African running frog Kassina senegalensis and its sequence was confirmed by total synthesis. The peptide inhibited growth of the gram-negative bacterium Escherichia coli (minimum inhibitory concentration, MIC = 4 microM), the gram-positive bacterium Staphylococcus aureus (MIC = 8 microM), and the yeast Candida albicans (MIC = 70 microM). A structurally related peptide, kassinatuerin-2 (FIQYLAPLI(10)PHAVKAISDL(20)I.NH(2)) was also isolated in high yield (96 nmol/g) from the extract but was devoid of antimicrobial activity against these microrganisms. Kassinatuerin-1 may be classified with other linear, cationic antimicrobial peptides that can potentially adopt an amphipathic alpha-helical conformation but it contains almost no amino acid sequence identity with previously characterized bioactive peptides from frog skin.     

Neurotrophic activity of brain extracts in forelimb regeneration of the urodele, Triturus.

The loss in protein synthesis which the regenerating forelimb of the newt suffers after denervation can be recovered by infusing into it an extract of newt soluble brain protein. Moreover, the synthesis of basic protein shows a greater response to the active brain principle than does that of acidic protein. The active agent of the nervous tissue is destroyed by heat and trypsin digestion. Extracts of liver and spleen, similarly prepared, do not evoke recovery of lost protein synthesis. Synaptosomal extracts of the frog brain also cause recovery of protein synthesis in the denervated regenerate, demonstrating the likelihood that the active agent is not species-specific within these amphibians, that it is a constituent of the neuronal fraction of nervous tissue, and that it is present in axonal terminals. Additional experiments showed that the nervous agent is likely a basic protein, and that the amount of protein infused is of the order of only 1.0% of the total regenerate protein. The significance of the findings is discussed in relation to the nature of the effect on protein synthesis and the nature of the active principle.     

A novel myotropic peptide from the skin secretions of the tree frog, Polypedates pingbianensis

A novel myotropic peptide, polypedatein, was purified and characterized from the skin secretions of the tree frog, Polypedates pingbianensis. Its primary structure, TLLCKYFAIC, was determined by Edman degradation and mass spectrometry. Polypedatein was subjected to bioassays including myotropic, antimicrobial, and serine protease inhibitory activities, which are related with many amphibian skin bioactive peptides. It was found to elicit concentration-dependent contractile effects on isolated rat ileum. cDNA clones encoding the precursor of polypedatein were isolated by screening a skin cDNA library of P. pingbianensis and then sequenced. The amino acid sequence deduced from the cDNA sequences matches well with the result from Edman degradation. BLAST search revealed that the sequence of polypedatein did not show similarity to known protein or peptide sequences. Especially, polypedatein does not contain conserved structural motifs of other amphibian myotropic peptides, such as bradykinins, bombesins, cholecystokinin (CCK), and tachykinins, indicating that polypedatein belongs to a novel amphibian myotropic peptide family. The signal peptide of the precursor encoding polypedatein shows significant sequence identity to that of other amphibian skin defensive peptides, such as antimicrobial peptides, bradykinins, lectins, and serine protease inhibitors, suggesting that polypedatein belongs to a novel amphibian myotropic peptide family. Polypedatein is also the first bioactive peptide from the genus of the frog, Polypedates.     

Molecular cloning of a cDNA encoding atypical antimicrobial and cytotoxic Brevinin-1Ja from the skin of the Japanese brown frog, Rana japonica

Antimicrobial peptides (AMPs) are important components of the host innate defense system against pathogenic microbial invasion in many organisms. In the present study, we cloned by RT-PCR a cDNA from total RNA prepared from the skin of the Japanese brown frog Rana japonica. The cDNA directs the synthesis of a novel, non-C-terminally alpha-amidated peptide composed of 21 amino acid residues (FLGSLIGAAIPAIKQLLGLKK). The putative peptide showed limited sequence similarity to atypical acyclic brevinin-1OK family AMPs originally isolated from the skin of the Ryukyu brown frog (R. okinavana), which lacks the COOH-terminal cyclic domain commonly observed in typical brevinin-1 groups, but that contains a C-terminally alpha-amidated residue. Although it is unclear whether the peptide, designated brevinin-1Ja, is produced in R. japonica skin, a synthetic replicate of the peptide showed differential growth-inhibiting activity against the Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli (minimal inhibitory concentrations: 15 microM and 119 microM, respectively), and produced cell death of mammalian COS7 cells (LD50=28 microM).     

The Ca2+ increase by the sperm factor in physiologically polyspermic newt fertilization: its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca(2+) increase occurs as a Ca(2+) wave at each sperm entry site in the polyspermic egg. Some Ca(2+) waves are preceded by a transient spike-like Ca(2+) increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca(2+) wave was induced by a sperm factor derived from sperm cytoplasm after sperm-egg membrane fusion. The Ca(2+) increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca(2+) store for the Ca(2+) wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

A comparison of structure-activity relationships within alpha-MSH on melanophores of Anolis carolinensis and Rana pipiens

We have compared the structure-function relationship of the tridecapeptide alpha-melanocyte stimulating hormone (alpha-MSH) on the melanophores of the lizard Anolis carolinensis and the frog Rana pipiens by determining the melanosome-dispersing potency of 15 shorter peptide sequences and 8 substituted alpha-MSH analogues. Major differences were found between the lizard and the frog in their response to alpha-MSH peptide fragments and analogues. In Anolis, the sequence Ser-Tyr-Ser- is not as important for the pigmentary response as in Rana since alpha-MSH-(4-13) was nearly as potent (89%) as alpha-MSH-(1-13) (100%), whereas in Rana alpha-MSH-(4-13) potency was reduced to 7.5%. In addition, loss of potency due to removal of residues Pro and Val was more marked in Rana (alpha-MSH-(1-11) = 0.1%) than in Anolis (alpha-MSH-(1-11) = 1%), suggesting that this C-terminal sequence is necessary for pigmentary activity in the frog melanophore. These results together with those of other peptide fragments and analogues have led us to define the minimal pigmentary sequence of alpha-MSH as alpha-MSH-(4-12) in Anolis in contrast to alpha-MSH-(1-13) in Rana. This suggests that Anolis and Rana alpha-MSH receptors recognise different message amino acids of the alpha-MSH peptide sequence even though the final response (melanosome dispersion) is the same.     

An antimicrobial peptide from the skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae)

A 25 amino-acid-residue, C-terminally alpha-amidated peptide with antimicrobial activity, which has been termed fallaxin, was isolated in high yield from the norepinephrine-stimulated skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae). The amino acid sequence of the peptide (Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Ile-Ala-Gly-His-Leu-Ala-Ser-Lys-Val-Met-Asn-Lys-Leu.NH2) shows structural similarity with members of the ranatuerin-2 family previously isolated from the skins of frogs of the genus Rana that are only distantly related to the Leptodactylidae. This observation is consistent with the hypothesis that many frog skin antimicrobial peptides are related evolutionarily, having arisen from multiple duplications of an ancestral gene that existed before the radiation of the different families. Fallaxin inhibited the growth of reference strains of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) but with relatively low potency (MIC> or =20 microM) and was inactive against the Gram-positive bacterium (Staphylococcus aureus) and the yeast Candida albicans. The hemolytic activity of fallaxin was very low (HC50>200 microM). A second peptide, comprising residues (1-22) of fallaxin, was also isolated from the skin secretions but this component was inactive against the microorganisms tested.     

Role of catecholamine neurons in the reticular lateral nuclei in regulating sensitivity to pain during exposure to reflex stimuli

Experiments were made on rats in which the effects of catecholaminergic neuronal systems of lateral reticular A-1 nuclei were eliminated with 6-OHDA. The latency of pain reactions tested by the hot-plate and tail-flick tests remained unchanged after operation. After auricular electric acupuncture the rats manifested no changes in the above reactions as compared with the initial level, which evidences that A-1 nuclei play an important role in the mechanisms of analgesia under consideration. Stimulation of the small pelvis organs (SSPO) entailed a short-term and significant inhibition of the analgetic effect as regards the control which also points to the involvement of A-1 nuclei into activation of antinociceptive processes. Besides, during SSPO, there was a significant elevation of the response measured by the tail-flick test as compared to the initial level of the pain reaction.