RESUMO
CONTEXT: Thr6-bradykinin is a peptide found in the venom of social and solitary wasps. This kinin, along with other bradykinin-like peptides, is known to cause irreversible paralysis in insects by presynaptic blockade of cholinergic transmission. However, this activity has never been tested in mammals. OBJECTIVE: As such, the objective of this study was to evaluate the effect of Thr6-bradykinin on the cholinergic system of rats. MATERIALS AND METHODS: The peptide was isolated from the venom of the Neotropical social wasp Polybia occidentalis Olivier (Vespidae). After correct identification and quantification by ESI-MS and MS/MS, the peptide was tested in [14C]-choline uptake using rat cortical synaptosomes. Each uptake assay was accompanied by lactic acid dehydrogenase (LDH) activity measurement to evaluate synaptosome integrity in the presence of six increasing concentrations of BK or Thr6-BK (0.039, 0.156, 0.625, 2.500, 10.000 and 40.000 µM). RESULTS: Data revealed that neither BK nor Thr6-BK at any of the six concentrations tested (from 0.039 to 40.000 µM) affected [14C]-choline uptake in synaptosomes. Moreover, there was no increase in LDH in the supernatants, indicating that BK and Thr6-BK did not disrupt the synaptosomes. DISCUSSION AND CONCLUSION: In contrast to previous reports for the insect central nervous system (CNS), Thr6-BK had no effect on mammalian cholinergic transmission. Nevertheless, this selectivity for the insect CNS, combined with its irreversible mode of action may be relevant to the discovery of new sources of insecticides and could contribute to understanding the role of kinins in the mammalian CNS.
Assuntos
Bradicinina/metabolismo , Córtex Cerebral/metabolismo , Colina/metabolismo , Venenos de Vespas/metabolismo , Animais , Bradicinina/isolamento & purificação , Bradicinina/farmacologia , Radioisótopos de Carbono/metabolismo , Córtex Cerebral/efeitos dos fármacos , Colina/antagonistas & inibidores , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Masculino , Ratos , Ratos Wistar , Venenos de Vespas/isolamento & purificação , Venenos de Vespas/farmacologia , VespasRESUMO
Anxiety disorders are major health problems in terms of costs stemming from sick leave, disabilities, healthcare and premature mortality. Despite the availability of classic anxiolytics, some anxiety disorders are still resistant to treatment, with higher rates of adverse effects. In this respect, several toxins isolated from arthropod venoms are useful in identifying new compounds to treat neurological disorders, particularly pathological anxiety. Thus, the aims of this study were to identify and characterize an anxiolytic peptide isolated from the venom of the social wasp Polybia paulista. The peptide was identified as Polisteskinin R, with nominal molecular mass [M+H](+)=1301Da and primary structure consisting of Ala-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH. The anxiolytic effect was tested using the elevated plus maze test. Moreover, adverse effects on the spontaneous behavior and motor coordination of animals were assessed using the open field and rotarod tests. Polisteskinin R induced a dose-dependent anxiolytic effect. Animals treated with the peptide and diazepam spent significantly more time into the open arms when compared to the groups treated with the vehicle and pentylenetetrazole. No significant differences in spontaneous behavior or motor coordination were observed between the groups, showing that the peptide was well tolerated. The interaction by agonists in both known BK receptors induces a variability of physiological effects; Polisteskinin R can act on these receptors, inducing modulatory activity and thus, attenuating anxiety behaviors. The results of this study demonstrated that the compound Polisteskinin R exerted potent anxiolytic effects and its analogues are promising candidates for experimental pharmacology.
Assuntos
Ansiolíticos/uso terapêutico , Ansiedade/tratamento farmacológico , Bradicinina/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Venenos de Vespas/uso terapêutico , Animais , Ansiolíticos/efeitos adversos , Ansiolíticos/isolamento & purificação , Ansiedade/psicologia , Bradicinina/efeitos adversos , Bradicinina/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/métodos , Masculino , Fragmentos de Peptídeos/efeitos adversos , Fragmentos de Peptídeos/isolamento & purificação , Ratos , Ratos Wistar , Venenos de Vespas/efeitos adversos , Venenos de Vespas/isolamento & purificaçãoRESUMO
Bradykinin (BK) and its related peptides are widely distributed in venomous animals, including wasps. In fact, we have previously purified a novel BK-related peptide (BRP) named Cd-146 and the threonine(6)-bradykinin (Thr(6)-BK) from the venom of the solitary wasp Cyphononyx fulvognathus. Further survey of this same wasp venom extract allowed the structural characterization of two other novel BRPs, named here as fulvonin and cyphokinin. Biochemical characterization performed here showed that although the high primary structure similarity observed with BK, these wasp peptides are not good substrates for angiotensin I-converting enzyme (ACE) acting more likely as inhibitors of this enzyme. In pharmacological assays, only those more structurally similar to BK, namely cyphokinin and Thr(6)-BK, were able to promote the contraction of guinea-pig ileum smooth muscle preparations, which was completely blocked by the B(2) receptors antagonist HOE-140 in the same way as observed for BK. Only fulvonin was shown to potentiate BK-elicited smooth muscle contraction. Moreover, the 2 new wasp BRPs, namely fulvonin and cyphokinin, as well as Cd-146 and Thr(6)-BK, showed hyperalgesic effect in the rat paw pressure test after intraplantar injection. This effect was shown here to be due to the action of these peptides on BK receptors, since the hyperalgesia induced by both Cd-146 and fulvonin was blocked by B(1) receptor antagonist, while the effect of both cyphokinin and Thr(6)-BK was reversed by B(2) antagonist. This data give support to a better understanding of the function and targets of the kinin-related peptides widely found in several insect venoms.
Assuntos
Bradicinina/análogos & derivados , Bradicinina/fisiologia , Peptídeos/fisiologia , Venenos de Vespas/farmacologia , Sequência de Aminoácidos , Animais , Bradicinina/isolamento & purificação , Feminino , Motilidade Gastrointestinal/efeitos dos fármacos , Cobaias , Íleo/efeitos dos fármacos , Íleo/fisiologia , Masculino , Dados de Sequência Molecular , Medição da Dor/métodos , Peptídeos/isolamento & purificação , Coelhos , Ratos , Ratos Wistar , Venenos de Vespas/isolamento & purificação , VespasRESUMO
BACKGROUND AND PURPOSE: In this work, a neuroactive peptide from the venom of the neotropical wasp Polybia occidentalis was isolated and its anti-nociceptive effects were characterized in well-established pain induction models. EXPERIMENTAL APPROACH: Wasp venom was analysed by reverse-phase HPLC and fractions screened for anti-nociceptive activity. The structure of the most active fraction was identified by electron-spray mass spectrometry (ESI-MS/MS) and it was further assessed in two tests of anti-nociceptive activity in rats: the hot plate and tail flick tests. KEY RESULTS: The most active fraction contained a peptide whose structure was Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH, which corresponds to that of Thr(6)-BK, a bradykinin analogue. This peptide was given by i.c.v. injection to rats. In the tail flick test, Thr(6)-BK induced anti-nociceptive effects, approximately twice as potent as either morphine or bradykinin also given i.c.v. The anti-nociceptive activity of Thr(6)-BK peaked at 30 min after injection and persisted for 2 h, longer than bradykinin. The primary mode of action of Thr(6)-BK involved the activation of B(2) bradykinin receptors, as anti-nociceptive effects of Thr(6)-BK were antagonized by a selective B(2) receptor antagonist. CONCLUSIONS AND IMPLICATIONS: Our data indicate that Thr(6)-BK acts through B(2) bradykinin receptors in the mammalian CNS, evoking antinociceptive behaviour. This activity is remarkably different from that of bradykinin, despite the structural similarities between both peptides. In addition, due to the increased metabolic stability of Thr(6)-BK, relative to that of bradykinin, this peptide could provide a novel tool in the investigation of kinin pathways involved with pain.
Assuntos
Analgésicos/farmacologia , Bradicinina/análogos & derivados , Dor/tratamento farmacológico , Venenos de Vespas/química , Analgésicos/administração & dosagem , Animais , Bradicinina/administração & dosagem , Bradicinina/isolamento & purificação , Bradicinina/farmacologia , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Injeções Intraventriculares , Sistema Calicreína-Cinina , Masculino , Morfina/administração & dosagem , Morfina/farmacologia , Dor/fisiopatologia , Medição da Dor , Ratos , Ratos Wistar , Receptor B2 da Bradicinina/efeitos dos fármacos , Receptor B2 da Bradicinina/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Bradykinins were identified in three solitary wasp venoms. Purification and characterization of the venom extract of the scoliid wasp Megacampsomeris prismatica led to the identification of bradykinin and threonine(6)-bradykinin as the major peptide components. The survey of a number of extracts from solitary wasp venom by MALDI-TOF MS revealed that the venoms of two other scoliid wasps, Campsomeriella annulata annulata and Carinoscolia melanosoma fascinata, also contained Thr(6)-BK as one of the major components. Thus, this study showed the presence of bradykinins in some of the solitary wasp venoms. Moreover, it indicated that these peptides play a major role in their paralyzing action for prey capture because these bradykinins have been shown to block the synaptic transmission of the nicotinic acetylcholine receptor in the insect central nervous system.
Assuntos
Bradicinina/isolamento & purificação , Venenos de Vespas/química , Vespas , Animais , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Sistema Nervoso Central , Insetos , Espectrometria de Massas , Receptores Nicotínicos/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
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.
Assuntos
Oligopeptídeos/química , Oligopeptídeos/isolamento & purificação , Pele/química , Taquicininas/química , Taquicininas/isolamento & purificação , Animais , Anuros , Bioensaio , Bradicinina/análogos & derivados , Bradicinina/química , Bradicinina/isolamento & purificação , Bradicinina/metabolismo , Ceruletídeo/análogos & derivados , Ceruletídeo/química , Ceruletídeo/isolamento & purificação , Ceruletídeo/metabolismo , Costa Rica , Cassinina/análogos & derivados , Cassinina/química , Cassinina/isolamento & purificação , Cassinina/metabolismo , Oligopeptídeos/metabolismo , Peptídeos Opioides , Taquicininas/metabolismoRESUMO
Venom of the spider Scaptocosa raptoria was fractionated by chromatography on Sephadex G-10 followed by HPLC, and a bradykinin potentiating peptide, BPP-S, was obtained in pure form. The amino acid sequence of this undecapeptide is presented. Peptide BPP-S significantly potentiates the effects of bradykinin on smooth muscle, and inhibits the angiotensin-converting enzyme (ACE) in vitro.
Assuntos
Bradicinina/metabolismo , Venenos de Crotalídeos/metabolismo , Músculo Liso/efeitos dos fármacos , Oligopeptídeos/isolamento & purificação , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Bothrops , Bradicinina/química , Bradicinina/isolamento & purificação , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/química , Relação Dose-Resposta a Droga , Contração Muscular/efeitos dos fármacos , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologiaRESUMO
1. Bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg9- bradykinin and des-Arg1- bradykinin were separated by capillary zone electrophoresis in an apparatus constructed in our laboratory which utilizes a novel N2 pulsed laser-induced fluorescence detector. 2. Detection limits of 1.2 fmol for fluorescamine-derivatized bradykinin and 90 attomol for O-phthaldiadehyde-derivatized bradykinin were achieved. 3. This powerful analytical tool is described and its successful application to the measurements of bradykinin after enzymatic release from blood is documented