RESUMEN
Reserpine (RES) is an irreversible inhibitor of VMAT2 used to study Parkinson's disease (PD) and screening for antiparkinsonian treatments in rodents. Recently, the repeated treatment with a low dose of reserpine was proposed as a model capable of emulating progressive neurochemical, motor and non-motor impairments in PD. Conversely, compared to Wistar rats, Spontaneously Hypertensive Rats (SHR) are resistant to motor changes induced by repeated treatment with a low dose of RES. However, such resistance has not yet been investigated for RES-induced non-motor impairments. We aimed to assess whether SHR would have differential susceptibility to the object recognition deficit induced by repeated low-dose reserpine treatment. We submitted male Wistar and SHR rats to repeated RES treatment (15 s.c. injections of 0.1 mg/kg, every other day) and assessed object memory acquisition and retrieval 48 h after the 6th RES injection (immediately before the appearance of motor impairments). Only RES Wistar rats displayed memory impairment after reserpine treatment. On the other hand, untreated SHR rats displayed object recognition memory deficit, but RES treatment restored such deficits. We also performed immunohistochemistry for tyrosine hydroxylase (TH) and α-synuclein (α-syn) 48 h after the last RES injection. In a different set of animals submitted to the same treatment, we quantified DA, 5-HT and products of lipid peroxidation in the prefrontal cortex (PFC) and hippocampus (HPC). SHR presented increased constitutive levels of DA in the PFC and reduced immunoreactivity to TH in the medial PFC and dorsal HPC. Corroborating the behavioral findings, RES treatment restored those constitutive alterations in SHR. These findings indicate that the neurochemical, molecular and genetic differences in the SHR strain are potentially relevant targets to the study of susceptibility to diseases related to dopaminergic alterations.
Asunto(s)
Trastornos del Conocimiento/inducido químicamente , Dopamina/metabolismo , Hipocampo , Enfermedad de Parkinson Secundaria/inducido químicamente , Corteza Prefrontal , Reconocimiento en Psicología/efectos de los fármacos , Reserpina/farmacología , Tirosina 3-Monooxigenasa/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Ratas , Ratas Endogámicas SHR/metabolismo , Ratas Wistar/metabolismo , Reserpina/administración & dosificación , Transducción de Señal/efectos de los fármacosRESUMEN
Aiming to extend the knowledge about the diversity of bradykinin-potentiating peptides (BPPs) and their precursor proteins, a venom gland cDNA library from the South American rattlesnake (Crotalus dursissus terrificus, Cdt) was screened. Two novel homologous cDNAs encoding the BPPs precursor protein were cloned. Their sequence contain only one single longer BPP sequence with the typical IPP-tripeptide, and two short potential BPP-like molecules, revealing a unique structural organization. Several peptide sequences structurally similar to the BPPs identified in the precursor protein from Cdt and also from others snakes, were chemically synthesized and were bioassayed both in vitro and in vivo, by means of isolated smooth muscle preparations and by measurements of blood pressure in anaesthetized rats, respectively. We demonstrate here that a pyroglutamyl residue at the N-terminus with a high content of proline residues, even with the presence of a IPP moiety characteristic of typical BPPs, are not enough to determine a bradykinin-potentiating activity to these peptides. Taken together, our results indicate that the characterization of the BPPs precursor proteins and identification of characteristic glutamine residues followed by proline-rich peptide sequences are not enough to predict if these peptides, even with a pyroglutamyl residue at the N-terminus, will present the typical pharmacological activities described for the BPPs.
Asunto(s)
Antihipertensivos/aislamiento & purificación , Venenos de Crotálidos/química , Crotalus/metabolismo , Oligopéptidos/aislamiento & purificación , Precursores de Proteínas/aislamiento & purificación , Glándulas Salivales/metabolismo , Secuencia de Aminoácidos , Animales , Antihipertensivos/síntesis química , Antihipertensivos/farmacología , Secuencia de Bases , Presión Sanguínea/efectos de los fármacos , Clonación Molecular , Cobayas , Íleon/efectos de los fármacos , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Oligopéptidos/síntesis química , Oligopéptidos/genética , Oligopéptidos/farmacología , Precursores de Proteínas/síntesis química , Precursores de Proteínas/genética , Precursores de Proteínas/farmacología , Ratas , Ratas Wistar , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Relación Estructura-ActividadRESUMEN
Bradykinin potentiating peptides (BPPs) from Bothrops jararaca venom were first described in the middle of 1960s and were the first natural inhibitors of the angiotensin-converting enzyme (ACE). BPPs present a classical motif and can be recognized by their typical pyroglutamyl (Pyr)/proline rich sequences presenting, invariably, a proline residue at the C-terminus. In the present study, we describe the isolation and biological characterization of a novel BPP isolated from the skin secretion of the Brazilian tree-frog Phyllomedusa hypochondrialis. This new BPP, named Phypo Xa presents the sequence Pyr-Phe-Arg-Pro-Ser-Tyr-Gln-Ile-Pro-Pro and is able to potentiate bradykinin activities in vivo and in vitro, as well as efficiently and competitively inhibit ACE. This is the first canonical BPP (i.e. Pyr-Aaa(n)-Gln-Ile-Pro-Pro) to be found not only in the frog skin but also in any other natural source other than the snake venoms.
Asunto(s)
Anuros/metabolismo , Bradiquinina/metabolismo , Oligopéptidos/aislamiento & purificación , Secuencia de Aminoácidos , Inhibidores de la Enzima Convertidora de Angiotensina/aislamiento & purificación , Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Anuros/genética , Presión Sanguínea/efectos de los fármacos , Bradiquinina/farmacología , Sinergismo Farmacológico , Femenino , Cobayas , Íleon/efectos de los fármacos , Técnicas In Vitro , Masculino , Oligopéptidos/genética , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Ratas , Ratas Wistar , Piel/metabolismoRESUMEN
1. The effects of the main component of the Tityus serrulatus scorpion venom, toxin TsTX-I, were studied on the contractility and release of neurotransmitters in the rat vas deferens. Since TsTX-I is known to act on sodium channels, we used veratridine, another sodium channel agent, for comparison. 2. Toxin TsTX-I induced concentration-dependent contractions with an EC(50) value of 47.8+/-0.1 nM and a maximum effect of 84.4+/-10.4% of that for BaCl(2). 3. Contractions by TsTX-I were abolished by denervation or tetrodotoxin (0.1 microM), showing that the toxin effects depend on the integrity of sympathetic nerve terminals. 4. To check for the presence of a noradrenergic component, experiments were conducted after removal of adrenergic stores in nerve terminals by reserpinization (10 mg kg(-1), 24 h prior to experiments) or blockade of alpha(1) adrenoceptors by prazosin (30 microM), showing that these procedures did not modify the response to TsTX-I, and therefore that adrenoceptors were not involved in contractions. 5. To check for the presence of a purinergic component, experiments were carried out after blockade of P(2X) receptors by suramin (0.1 mM) or desensitization by alpha,beta-methylene-ATP (30 microM). These agents greatly abolished the contractile response to TsTX-I (about 83% by desensitization and 96% by suramin), showing the involvement of purinergic receptors. 6. The release of noradrenaline and purinergic agents (ATP, ADP, AMP and adenosine) was detected by HPLC. Together, the total release of purines in the presence of TsTX-I was about 42 times higher than in the control group. In contrast, TsTX-I did not modify the overflow of noradrenaline, showing that the release was selective for purines. 7. The release of purinergic agents was reduced by the N-type calcium channel blocker omega-conotoxin GVIA (1 microM) and by the P/Q-type blocker omega-conotoxin MVIIC (1 microM), showing that the effects of TsTX-I are calcium-dependent. 8. The results show that TsTX-I produced a selective release of purines from postganglionic sympathetic nerves in the rat vas deferens.
Asunto(s)
Adenosina Trifosfato/metabolismo , Contracción Muscular/efectos de los fármacos , Venenos de Escorpión/farmacología , Escorpiones/metabolismo , Sistema Nervioso Simpático/efectos de los fármacos , Conducto Deferente/efectos de los fármacos , Animales , Bloqueadores de los Canales de Calcio/farmacología , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Masculino , Músculo Liso/efectos de los fármacos , Músculo Liso/inervación , Músculo Liso/metabolismo , Neurotransmisores/metabolismo , Ratas , Ratas Wistar , Receptores Adrenérgicos/metabolismo , Receptores Purinérgicos/metabolismo , Venenos de Escorpión/aislamiento & purificación , Simpatectomía , Sistema Nervioso Simpático/metabolismo , Factores de Tiempo , Conducto Deferente/inervación , Conducto Deferente/metabolismo , Veratridina/farmacologíaRESUMEN
Tityus serrulatus venom (Tsv)-induced pulmonary edema can occur in severe envenomation and the mechanisms involved are not completely understood. Therefore, we studied the effect of pharmacological modulation of the mast cell activation and the histamine antagonism on airways edema (investigated by Evans blue dye extravasation) and measured 5-hydroxytryptamine (5-HT) concentration in bronchoalveolar lavage fluid (BALF) in rats envenomed by Tsv. Additionally, the in vitro effect of Tsv on mast cells was studied using histological method and 5-HT release from mesenteric and peritoneal mast cells. We found that i.v. injection of Tsv increase vascular permeability in trachea, upper and lower bronchi and in lung parenchyma. This was not affected by ketotifen, a mast cell "stabilizer," or by pretreatment with pyrilamine (histamine H1 receptor antagonist). Moreover, 5-HT was not found in BALF of envenomed rats. In vitro experiments showed that Tsv did not induce mast cell degranulation nor release of 5-HT by mesenteric or peritoneal mast cells, in sharp contrast to preparations challenged by a mast cell activator, compound 48/80. In conclusion, our results show that Tsv causes strong edema in rat airways which is independent of mast cell activation and show that mast cells are not directly activated by Tsv.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Mordeduras y Picaduras de Insectos/patología , Mastocitos/efectos de los fármacos , Sistema Respiratorio/efectos de los fármacos , Venenos de Escorpión/toxicidad , Animales , Líquido del Lavado Bronquioalveolar/citología , Degranulación de la Célula/efectos de los fármacos , Azul de Evans , Indicadores y Reactivos , Mordeduras y Picaduras de Insectos/fisiopatología , Cetotifen/farmacología , Masculino , Mastocitos/fisiología , Pirilamina/farmacología , Ratas , Ratas Wistar , Sistema Respiratorio/irrigación sanguínea , EscorpionesRESUMEN
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.