RESUMEN
AIMS: TsTX-I scorpion toxin, also known as γ-toxin, is a ß-toxin which binds to site 4 of the sodium channel, shifting its activation potential. There are few studies about its pharmacological action in the central nervous system. The objective of this work was to determine the electroencephalographic, behavioral and histopathological effects of intrahippocampal injection of TsTX-I. MAIN METHODS: Rats were anesthetized and fitted with cannulae for injection into the hippocampus and with electrodes for cerebral recording. The animals were treated with Ringer solution, some doses of TsTX-I, DMSO 0.1% or veratridine. Behavioral and electrographic recordings were observed for 4 hours after the injection. After 7 days, the rats were perfused, and their brains removed for histological analysis. KEY FINDINGS: Increasing doses of the toxin evoked epileptic-like discharges, wet dog shakes, and in some cases hind limb paralysis and intense respiratory difficulty followed by death. The histopathological analysis demonstrated no cell loss. Animals injected with veratridine developed epileptiform activity in the electrographic recording and neuronal loss. SIGNIFICANCE: The results suggest that TsTX-I toxin may be responsible, at least in part, for the epileptic and behavioral effects observed with the crude venom, and although veratridine and TsTX-I act on Na-channel, the differences between them are remarkable, demonstrating that toxins can have different functional effects depending on the site of action in the channel. Thus, animal neurotoxins are often highly selective and may be useful for the identification of the sequence of events underlying neurotransmission.
Asunto(s)
Conducta Animal/efectos de los fármacos , Hipocampo/metabolismo , Venenos de Escorpión/toxicidad , Veratridina/toxicidad , Animales , Sitios de Unión , Relación Dosis-Respuesta a Droga , Electroencefalografía , Miembro Posterior , Inyecciones , Masculino , Parálisis/inducido químicamente , Unión Proteica , Ratas , Ratas Wistar , Venenos de Escorpión/administración & dosificación , Convulsiones/inducido químicamente , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismoRESUMEN
In the microcirculation, longitudinal conduction of vasomotor responses provides an essential means of coordinating flow distribution among vessels in a complex network. Spread of current along the vessel axis can display a regenerative component, which leads to propagation of vasomotor signals over many millimeters; the ionic basis for the regenerative response is unknown. We examined the responses to 10 s of focal electrical stimulation (30 Hz, 2 ms, 30 V) of mouse cremaster arterioles to test the hypothesis that voltage-dependent Na(+) (Na(v)) and Ca(2+) channels might be activated in long-distance signaling in microvessels. Electrical stimulation evoked a vasoconstriction at the site of stimulation and a spreading, nondecremental conducted dilation. Endothelial damage (air bubble) blocked conduction of the vasodilation, indicating an involvement of the endothelium. The Na(v) channel blocker bupivacaine also blocked conduction, and TTX attenuated it. The Na(v) channel activator veratridine induced an endothelium-dependent dilation. The Na(v) channel isoforms Na(v)1.2, Na(v)1.6, and Na(v)1.9 were detected in the endothelial cells of cremaster arterioles by immunocytochemistry. These findings are consistent with the involvement of Na(v) channels in the conducted response. BAPTA buffering of endothelial cell Ca(2+) delayed and reduced the conducted dilation, which was almost eliminated by Ni(2+), amiloride, or deletion of alpha(1H) T-type Ca(2+) (Ca(v)3.2) channels. Blockade of endothelial nitric oxide synthase or Ca(2+)-activated K(+) channels also inhibited the conducted vasodilation. Our findings indicate that an electrically induced signal can propagate along the vessel axis via the endothelium and can induce sequential activation of Na(v) and Ca(v)3.2 channels. The resultant Ca(2+) influx activates endothelial nitric oxide synthase and Ca(2+)-activated K(+) channels, triggering vasodilation.
Asunto(s)
Canales de Calcio Tipo T/fisiología , Canales de Calcio/fisiología , Endotelio Vascular/fisiología , Canales de Sodio/fisiología , Sistema Vasomotor/fisiología , Animales , Bupivacaína/farmacología , Señalización del Calcio/fisiología , Estimulación Eléctrica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microcirculación/fisiología , Óxido Nítrico/metabolismo , Canales de Potasio Calcio-Activados/fisiología , Bloqueadores de los Canales de Sodio/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología , Veratridina/farmacologíaRESUMEN
We have previously shown that veratrine, a mixture of alkaloids known as Veratrum alkaloids, produces skeletal muscle toxicity, and there is evidence that veratrine interferes with the energetics of various systems, including cardiomyocytes and synaptosomes. In this work, we explored the effects of veratrine and veratridine, a component of this mixture, in rat skeletal muscle mitochondria and compared the results with those seen in liver mitochondria. Veratrine and veratridine alkaloids caused a significant concentration-dependent decrease in the rate of state 3 respiration, respiratory control (RCR) and ADP/O ratios in isolated rat skeletal muscle mitochondria (RMM), but not in rat liver mitochondria (RLM) supported by either NADH-linked substrates or succinate. The oxygen consumption experiments showed that RMM were more susceptible to the toxic action of Veratrum alkaloids than RLM. The addition of veratrine (250 microg/ml) to RMM caused dissipation of the mitochondrial electrical membrane potential during succinate oxidation, but this effect was totally reversed by adding ATP. These results indicate that there are chemical- and tissue-specific toxic effects of veratrine and veratridine on mitochondrial respiratory chain complexes. Identification of the specific respiratory chain targets involved should provide a better understanding of the molecular mechanisms of the toxicity of these agents.
Asunto(s)
Potenciales de la Membrana/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Musculares/efectos de los fármacos , Músculo Esquelético/citología , Consumo de Oxígeno/efectos de los fármacos , Veratridina/farmacología , Veratrina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Mitocondrias Hepáticas/metabolismo , Mitocondrias Musculares/metabolismo , Ratas , Ratas WistarRESUMEN
The single and combined effects of carbamazepine and vinpocetine on the release of the excitatory amino acid neurotransmitter glutamate, on the rise in internal Na+ (Na(i), as determined with SBFI), and on the rise in internal Ca2+ (Ca(i), as determined with fura-2) induced by an increased permeability of presynaptic Na+ channels, with veratridine, or by an increased permeability of presynaptic Ca2+ channels with high K+, were investigated in isolated hippocampal nerve endings. The present study shows that carbamazepine and vinpocetine, both inhibit dose dependently the release of preloaded [3H]Glu induced by veratridine. However, carbamazepine is two orders of magnitude less potent than vinpocetine. The calculated IC(50)'s for carbamazepine and vinpocetine to inhibit veratridine-induced [3H]Glu release are 200 and 2 microM, respectively. Consistently 150 microM carbamazepine and 1.5 microM vinpocetine reduce the veratridine-induced rise in Na(i) in a similar extent. The single effects of carbamazepine and of vinpocetine on the presynaptic Na+ channel mediated responses, namely the rise in Na(i) and the release of Glu induced by veratridine, are additive. Responses that depend on the entrance of external Ca2+ via presynaptic Ca2+ channels, such as the release of [3H]Glu and the rise in Ca(i) induced by high K+, are insensitive to 300 microM carbamazepine and slightly reduced by 5 microM vinpocetine. It is concluded that the additive effects of carbamazepine, which is one of the most common antiepileptic drugs, and vinpocetine that besides its known neuroprotective action and antiepileptic potential is a memory enhancer, may perhaps be advantageous in the treatment of epileptic patients.
Asunto(s)
Carbamazepina/farmacología , Ácido Glutámico/metabolismo , Hipocampo/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Alcaloides de la Vinca/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Anticonvulsivantes/farmacología , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Hipocampo/metabolismo , Canales Iónicos/metabolismo , Masculino , Fármacos Neuroprotectores/farmacología , Técnicas de Cultivo de Órganos , Terminales Presinápticos/metabolismo , Ratas , Ratas Wistar , Sodio/metabolismo , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Veratridina/farmacologíaRESUMEN
Beta-Adrenoceptor antagonists may present receptor-independent mechanisms, such as blockade of voltage-gated sodium channels. This study aimed to investigate the effects of non-selective (propranolol), and selective beta1- (atenolol, metoprolol and betaxolol) and beta2-adrenoceptor (ICI 118,551) antagonists in the nitric oxide (NO)-mediated rabbit corpus cavernosum relaxations induced by either electrical field stimulation (EFS) or activators of voltage-gated sodium channels. The sodium channel blockers tetrodotoxin and saxitoxin abolished the relaxations induced by EFS or sodium channel activators of binding site-2 (aconitine and veratridine), site-3 (Ts3 toxin), site-4 (Ts1 toxin) and site-5 (brevetoxin-3). The beta-adrenoceptor antagonists failed to affect the relaxations induced by EFS, aconitine and veratridine. Relaxations induced by Ts3 and Ts1 toxins, as well as brevetoxin-3, were markedly reduced by prior addition of propranolol, betaxolol and ICI 118,551. During the established relaxation induced by Ts3 toxin, propranolol failed to restore the basal tone. In conclusion, beta-adrenoceptor antagonists may cause an allosteric inhibition at the binding site-3, -4 and -5 of voltage-gated sodium channels, leading to blockade of neural NO release.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Óxido Nítrico/metabolismo , Pene/efectos de los fármacos , Agonistas de los Canales de Sodio , Aconitina/farmacología , Animales , Atenolol/farmacología , Betaxolol/farmacología , Sitios de Unión , Estimulación Eléctrica , Técnicas In Vitro , Proteínas de Insectos , Masculino , Metoprolol/farmacología , Relajación Muscular/efectos de los fármacos , Neurotoxinas/farmacología , Pene/metabolismo , Pene/fisiología , Propanolaminas/farmacología , Propranolol/farmacología , Conejos , Saxitoxina/farmacología , Venenos de Escorpión/farmacología , Tetrodotoxina/farmacología , Veratridina/farmacologíaRESUMEN
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
The participation of voltage-sensitive Na+ channels (VSSC) on the changes on internal (i) Na+, K+, Ca2+, and on DA, Glu, and GABA release caused by different concentrations of 4-AP was investigated in striatum synaptosomes. TTX, which abolished the increase in Na(i) (as determined with SBFI), induced by 0.1 mM 4-AP only inhibited by 30% the rise in Na(i) induced by 1 mM 4-AP. One millimolar 4-AP markedly decreased the fluorescence of the K+ indicator dye PBFI but 0.1 mM 4-AP did not. Like 1 mM 4-AP, ouabain decreased PBFI fluorescence and increased a considerable fraction of Na(i) in a TTX-insensitive manner. In contrast with the different TTX sensitivity of the rise in Na(i) induced by 0.1 and 1 mM 4-AP, the rise in Ca(i) (as determined with fura-2) induced by the two concentrations of 4-AP was markedly inhibited by TTX, as well as by omega-agatoxin in combination with omega-conotoxin GVIA, indicating that only the TTX-sensitive fraction of the rise in Na(i) induced by 4-AP is linked with the activation of presynaptic Ca2+ channels. It is concluded that the TTX-sensitive fraction of neurotransmitter release evoked by 4-AP is released by exocytosis, and the TTX insensitive fraction involves reversal of the neurotransmitters transporters. This contrasts with the exocytosis evoked by high K+ that is unchanged by TTX and with the neurotransmitter-transporter-mediated release evoked by veratridine, which is highly TTX sensitive and does not require activation of Ca2+ channels.
Asunto(s)
4-Aminopiridina/farmacología , Cuerpo Estriado/metabolismo , Canales de Sodio/metabolismo , Sodio/metabolismo , Sinaptosomas/metabolismo , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Cuerpo Estriado/efectos de los fármacos , Electrofisiología , Técnicas In Vitro , Activación del Canal Iónico , Masculino , Neurotransmisores/metabolismo , Concentración Osmolar , Ouabaína/farmacología , Potasio/metabolismo , Ratas , Ratas Wistar , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/fisiología , Sinaptosomas/efectos de los fármacos , Tetrodotoxina/farmacología , Veratridina/farmacologíaRESUMEN
A number of neurotoxins from venoms of invertebrates and plants are ligands for voltage-gated Na+ channels and are useful tools for studying Na+ channel function and structure. Using whole-cell recordings from vagal afferent nodose neurons, we studied neurotoxins that target Na+ channels. We asked whether Ts3 (an alpha-scorpion toxin) and/or veratridine (a lipid-soluble toxin), could modify the TTX-resistant Na+ current generated by vagal afferent nodose neurons. Nodose TTX-resistant current was not affected by Ts3, whereas Ts3 slowed inactivation of the current generated by TTX-sensitive current component. We found that veratridine inhibited the TTX-resistant Na+ currents on rat nodose neurons. Interestingly, veratridine-modified Na+ channels developed a persistent current that accounted for the large tail current observed. We propose that veratridine modifies TTX-resistant Na+ channels through a mechanism distinct from its actions on other voltage-gated Na+ channels.
Asunto(s)
Neuronas Aferentes/metabolismo , Neurotoxinas/toxicidad , Venenos de Escorpión/toxicidad , Canales de Sodio/efectos de los fármacos , Sodio/metabolismo , Veratridina/toxicidad , Animales , Transporte Biológico Activo , Transporte Iónico/efectos de los fármacos , Masculino , Neurotoxinas/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Venenos de Escorpión/metabolismo , Canales de Sodio/metabolismo , Tetrodotoxina , Nervio Vago/metabolismo , Veratridina/metabolismoRESUMEN
OBJECTIVES: To investigate the capacity of voltage-gated Na(+) channel activators such as batrachotoxin, aconitine, veratridine, Ts1 (formerly Tityus gamma-toxin), and brevetoxin-3 to induce relaxation of rabbit isolated corpus cavernosum (RbCC) and the pharmacologic mechanisms underlying this phenomenon. The voltage-gated Na(+) channels of the corpus cavernosum are essential for erectile function. A number of biologic toxins exert their effects by modifying the properties of these channels. METHODS: Male New Zealand white rabbits were anesthetized with pentobarbital sodium. Strips of RbCC were transferred to 10-mL organ baths containing oxygenated and warmed Krebs solution. The RbCC strips were connected to force-displacement transducers, and changes in isometric force were recorded using a PowerLab 400 data acquisition system. Corporeal smooth muscle was precontracted submaximally with phenylephrine (10 micromol/L). RESULTS: The binding site-2 (batrachotoxin, aconitine, and veratridine) and binding site-5 (brevetoxin-3) voltage-gated Na(+) channel activators caused slow-onset RbCC relaxations, and the binding site-4 activator Ts1 produced transitory relaxations followed by a return to baseline. The Na(+)channel blockers tetrodotoxin and saxitoxin (0.1 micromol/L each) abolished the relaxations induced by these agonists. Similarly, the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester (100 micromol/L) markedly reduced the relaxations and l-arginine (1 mmol/L) restored the relaxations. The soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxidiazolo[4,3-alpha] quinoxalin-1-one (10 micromol/L) reduced the relaxations, and the phosphodiesterase type 5 inhibitor sildenafil (100 nmol/L) significantly potentiated the relaxations by all activators. CONCLUSIONS: Our results indicate that the relaxations evoked by selective activators of voltage-gated Na(+) channels are mediated by the release of nitric oxide from nitrergic nerves and the activation of the nitric oxide-cyclic guanosine monophosphate pathway in the smooth muscle cells of erectile tissue.
Asunto(s)
GMP Cíclico/metabolismo , Músculo Liso/efectos de los fármacos , Óxido Nítrico/metabolismo , Pene/fisiología , Agonistas de los Canales de Sodio , Aconitina/farmacología , Animales , Arginina/farmacología , Batracotoxinas/farmacología , Sitios de Unión , GMP Cíclico/antagonistas & inhibidores , Guanilato Ciclasa/antagonistas & inhibidores , Técnicas In Vitro , Proteínas de Insectos , Contracción Isométrica/efectos de los fármacos , Masculino , Toxinas Marinas/farmacología , Músculo Liso/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Neurotoxinas/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Oxocinas/farmacología , Erección Peniana/efectos de los fármacos , Piperazinas/farmacología , Purinas , Conejos , Venenos de Escorpión/farmacología , Citrato de Sildenafil , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismo , Sulfonas , Veratridina/farmacologíaRESUMEN
The effects of TsTx-V, an alpha-toxin isolated from Tityus serrulatus venom, on electrical activity and insulin secretion by rodent pancreatic islet cells were studied. TsTx-V (5.6 microg/ml) depolarized mouse pancreatic beta-cells, diminished the membrane input resistance and increased the duration of the active phase of glucose-induced electrical activity. Similar results were observed with the Na(+) channel agonist veratridine (110 microM). Both agents potentiated glucose (8.3 mM)-induced insulin secretion in rat islet. In the presence of TsTx-V or veratridine, insulin secretion increased 2- and 1.4-fold over basal values, respectively (P<0.001). The Na(+) channel antagonist tetrodotoxin (6 microM) significantly decreased glucose- and TsTx-V-induced insulin secretion (P<0.001). TsTx-V also stimulated insulin secretion at low glucose concentrations (2.8 mM) whereas the beta-toxin, Ts-gamma (gamma toxin), also obtained from Tityus serrulatus venom, significantly reduced TsTx-V-induced secretion at sub- and suprathreshold concentrations of glucose. These results are consistent with a model whereby Na(+) channels participate in glucose-induced electrical activity. Alteration in the activity of these channels changes the length of time during which the beta-cell depolarizes, thereby altering the secretory behavior of the cell. The construction of a three-dimensional model for TsTx-V revealed a conserved core containing an alpha-helix and three beta-strands, with minor differences when compared with toxins from other scorpion venoms.
Asunto(s)
Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Escorpiones/química , Agonistas de los Canales de Sodio , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Conductividad Eléctrica , Glucosa/farmacología , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ratones , Conformación Molecular , Neurotoxinas/genética , Neurotoxinas/farmacología , Filogenia , Ratas , Venenos de Escorpión/genética , Canales de Sodio/fisiología , Tetrodotoxina/farmacología , Veratridina/farmacologíaRESUMEN
This study evaluates the cardiovascular and respiratory effects evoked by hypertonic sodium chloride solution (HSS) and the possible interactions of these effects with scorpion toxin (TX) or veratridine (V). Groups 1 (1 mL/kg, rapid), 2 (4 mL/kg, rapid), and 3 (4 mL/kg, slow) were used for comparison of HSS administered by rapid or slow injection. HSS (4 mL/kg) was injected after bilateral vagotomy (group 4) or administration of atropine (group 5). In groups 6 (1 mL/kg in bolus), 7 (4 mL/kg in bolus), and 8 (4 mL/kg/60 s), HSS was injected 20 min after the administration of TX (250 microg/kg). In group 9, two doses of V (25 microg/kg, i.v.) were injected 10 min apart. Concomitantly with the second dose of V, HSS (4 mL/kg) was injected into the jugular vein. HSS administered by rapid injection (1 mL/kg) resulted in hypotension, hyperventilation, and a slight decrease in heart rate. However, when HSS was administered after TX, only bradypnea was observed. HSS (4 mL/kg, rapid) induced a rapid and marked fall in blood pressure, bradycardia, and apnea. However, when HSS was administered after TX, a more pronounced bradycardia and a smaller reduction in mean arterial pressure were observed. Slow injection of HSS (60 s) evoked hypotension, hyperventilation, and bradycardia. The same dose injected after TX resulted in bradypnea and a smaller reduction in blood pressure. The HSS-induced hypotension was attenuated by previous administration of atropine or by vagotomy, whereas bradycardia was prevented by previous injection of atropine, but not by bilateral vagotomy. Like vagotomy, atropinization prevented the apnea and bradypnea produced by HSS (4 mL/kg in bolus). V evoked a slight bradycardia, hypotension, and apnea. These effects were potentiated when V was injected concomitantly with HSS. The effects of HSS are dependent on both volume and speed of injection, and are affected by previous injection of TX or concomitant injection of V.
Asunto(s)
Sistema Cardiovascular/efectos de los fármacos , Sistema Respiratorio/efectos de los fármacos , Solución Salina Hipertónica/administración & dosificación , Venenos de Escorpión/administración & dosificación , Veratridina/administración & dosificación , Animales , Atropina/administración & dosificación , Presión Sanguínea/efectos de los fármacos , Gasto Cardíaco/efectos de los fármacos , Interacciones Farmacológicas , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Ratas , Respiración/efectos de los fármacos , Vagotomía , Resistencia Vascular/efectos de los fármacosRESUMEN
The modulation of neurotransmitter release by calcium channels is well established, yet, sodium channels were regarded mainly as charge carriers. Many lines of evidence suggest a more fine-tuning role played by sodium channels. Using rat cerebrocortical isolated nerve endings (synaptosomes) and two toxins that have separate sites of action on sodium channels and provoke distinct changes in channel kinetics, we were able to show that depending on the rate of increase in channel conductance, the outcome in terms of neurotransmitter release and calcium channel types coupled to that event are different. Mainly, our study focused on veratridine, an alkaloid from lilaceous plants that binds to sodium channel toxin site 2, and tityustoxin, a toxin purified from the venom of the Brazilian yellow scorpion Tityus serrulatus that binds to site 3. Veratridine induces a slower increase in intrasynaptosomal sodium and calcium concentrations, slower depolarization, delayed exocytosis and a slower and predominantly calcium-independent glutamate release, when compared to tityustoxin.Thus, we have used these two toxins to investigate the events that start with sodium entry and culminate with the release of glutamate in isolated nerve endings (synaptosomes) from rat cerebral cortex. With that in mind we measured intrasynaptosomal free sodium concentration [Na(+)](i), intrasynaptosomal free calcium concentration [Ca(2+)](i), membrane potential, exocytosis and glutamate release using fluorescent probes.
Asunto(s)
Ácido Glutámico/metabolismo , Neurotoxinas/toxicidad , Venenos de Escorpión/toxicidad , Canales de Sodio/metabolismo , Veratridina/toxicidad , Animales , Calcio/análisis , Calcio/metabolismo , Canales de Calcio/metabolismo , Exocitosis/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Ratas , Ratas Wistar , Sodio/análisis , Sodio/metabolismo , Canales de Sodio/efectos de los fármacos , SinaptosomasRESUMEN
The effect of vinpocetine, a nootropic drug with anti-ischemic potential, on the release of DA and its main metabolite, DOPAC, was investigated in striatum isolated nerve endings under resting and depolarized conditions. Vinpocetine does not modify the baseline release of DA or the exocytotic release of DA evoked by high K(+), but inhibits the release of DA evoked by veratridine reversal of the DA transporter. In addition to these results, which confirm the vinpocetine selective blockade of voltage-sensitive presynaptic Na(+) channels (VSSC) previously reported [Neurochem. Res. 24 (1999) 1585], vinpocetine increases DOPAC release either under resting, veratridine or high K(+) depolarized conditions. This latter effect, which does not involve VSSC, was characterized. The parallel determination of the released and retained catecholamine concentrations revealed that vinpocetine increases DOPAC release at the expense of internal DA in a dose-dependent manner (low microM range). In contrast to vinpocetine, the selective MAO-A inhibitor, clorgyline, increases DA and decreases DOPAC formation. The combined action of vinpocetine and clorgyline does not indicate, however, that the activation of MAO is the mechanism responsible for the increase in DOPAC caused by vinpocetine. Reserpine, although more potent and efficient than vinpocetine, qualitatively exerts the same pattern of changes on DA and DOPAC concentrations. It is concluded that, in addition to the inhibition of presynaptic VSSC permeability, which selectively inhibits the transporter-mediated release of all neurotransmitters, vinpocetine increases DOPAC by impairing the vesicular storage of DA. Our results indicate that the cytoplasm extravesicular DA is metabolized by MAO to DOPAC. Most of the DOPAC formed is exported to the extracellular medium.
Asunto(s)
Ácido 3,4-Dihidroxifenilacético/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Dopamina/metabolismo , Glicoproteínas de Membrana , Proteínas de Transporte de Membrana , Neostriado/efectos de los fármacos , Proteínas del Tejido Nervioso , Terminales Presinápticos/efectos de los fármacos , Sinaptosomas/efectos de los fármacos , Alcaloides de la Vinca/farmacología , Inhibidores de Captación Adrenérgica/farmacología , Animales , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Clorgilina/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas/fisiología , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Inhibidores de la Monoaminooxidasa/farmacología , Neostriado/metabolismo , Potasio/farmacología , Terminales Presinápticos/metabolismo , Ratas , Ratas Wistar , Reserpina/farmacología , Bloqueadores de los Canales de Sodio , Canales de Sodio/metabolismo , Sinaptosomas/metabolismo , Veratridina/farmacologíaRESUMEN
Ibogaine has aroused expectations as a potentially innovative medication for drug addiction. It has been proposed that antagonism of the NMDA receptor by ibogaine may be one of the mechanisms underlying its antiaddictive properties; glutamate has also been implicated in ibogaine-induced neurotoxicity. We here report the effects of ibogaine on [3H]glutamate release and uptake in cortical and cerebellar synaptosomes, as well as in cortical astrocyte cultures, from mice and rats. Ibogaine (2-1000 microM) had no effects on glutamate uptake or release by rat synaptosomes. However, ibogaine (500-1000 microM) significantly inhibited the glutamate uptake and stimulated the release of glutamate by cortical (but not cerebellar) synaptosomes of mice. In addition, ibogaine (1000 microM) nearly abolished glutamate uptake by cortical astrocyte cultures from rats and mice. The data provide direct evidence of glutamate involvement in ibogaine-induced neurotoxicity.
Asunto(s)
Astrocitos/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/farmacocinética , Ibogaína/farmacología , Sinaptosomas/metabolismo , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Calcio/farmacología , Células Cultivadas , Corteza Cerebral/citología , Masculino , Ratones , Ratones Endogámicos , Ratas , Ratas Wistar , Sinaptosomas/efectos de los fármacos , Tetrodotoxina/farmacología , Tritio , Veratridina/farmacologíaRESUMEN
During the last decades it has been shown that trophic molecules released by target, afferent and glial cells play a pivotal role controlling neuronal cell death. Trophic molecules are able to inhibit this regressive event during development as well as during degenerative diseases. One of the mechanisms involved in the control of neuronal survival by afferent cells requires the release of trophic molecules stimulated by electrical activity. It has been demonstrated that veratridine (a depolarizing agent that keeps the Na+ channels opened) induces an increase in neuronal survival. In the present work we show that 3 microM veratridine induced a two-fold increase on the survival of retinal ganglion cells after 48 h in culture. The veratridine effect was inhibited by 50 microM amiloride (an inhibitor of Ca2+ channels), 25 microM benzamil (an inhibitor of Na+ channels), 30 microM dantrolene and 7.5 microM caffeine (both inhibitors of Ca2+ release from the endoplasmatic reticulum) and 10 microM BAPTA-AM (an intracellular Ca2+ chelator). However, 5 microM nifedipine (a selective inhibitor of voltage-dependent L-type Ca2+ channels) and 100 microM MK 801 (an inhibitor of NMDA receptors) did not block the veratridine effect. On the other hand, treatment with 10 microM genistein (an inhibitor of tyrosine kinase enzymes), 20 microM fluorodeoxyuridine (an inhibitor of cell proliferation) or 10 microM atropine (an antagonist of muscarinic receptors) completely abolished the effect of veratridine. Taken together, our results indicate that veratridine increases the survival of rat retinal ganglion cells through mechanisms involving Na+ influx, intracellular Ca2+ release, activation of tyrosine kinase enzymes and cellular proliferation. They also indicate that cholinergic activity plays an important role in the veratridine effect.
Asunto(s)
Muerte Celular/efectos de los fármacos , Células Ganglionares de la Retina/citología , Veratridina/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Cafeína/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Quelantes/farmacología , Dantroleno/farmacología , Diuréticos/farmacología , Maleato de Dizocilpina/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Relajantes Musculares Centrales/farmacología , Nifedipino/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Ratas , Ratas Endogámicas , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
Glutamate concentration increases significantly in the extracellular compartment during brain ischaemia and anoxia. This increase has an important Ca(2+)-independent component, which is due in part to the reversal of glutamate transporters of the plasma membrane of neurons and glia. The toxin phoneutriatoxin 3-4 (Tx3-4) from the spider Phoneutria nigriventer has been reported to decrease the evoked glutamate release from synaptosomes by inhibiting Ca(2+) entry via voltage-dependent Ca(2+) channels. However, we report here that Tx3-4 is also able to inhibit the uptake of glutamate by synaptosomes in a time-dependent manner and that this inhibition in turn leads to a decrease in the Ca(2+)-independent release of glutamate. No other polypeptide toxin so far described has this effect. Our results suggest that Tx3-4 can be a valuable tool in the investigation of function and dysfunction of glutamatergic neurotransmission in diseases such as ischaemia.
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Ácido Glutámico/metabolismo , Neuropéptidos/farmacología , Neurotoxinas/farmacología , Péptidos/farmacología , Arañas , Sinaptosomas/efectos de los fármacos , omega-Conotoxinas , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Sistema de Transporte de Aminoácidos X-AG , Animales , Unión Competitiva , Transporte Biológico/efectos de los fármacos , Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Ácido Glutámico/farmacología , Hipocampo/citología , Cloruro de Potasio/antagonistas & inhibidores , Cloruro de Potasio/farmacología , Ratas , Ratas Wistar , Sinaptosomas/metabolismo , Veratridina/antagonistas & inhibidores , Veratridina/farmacologíaRESUMEN
In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 microM carbachol was used, the estimated IC50 value for kainate was 0.2 microM and the maximal inhibition of approximately 50% was obtained with 1 microM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 microM veratridine, but not 50 mM KCl, inhibited approximately 65% of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 +/- 38.0 to 2044.5 +/- 299.9 cpm/mg protein, retinal response decreased to 861.6 +/- 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.
Asunto(s)
Carbacol/antagonistas & inhibidores , Agonistas de Aminoácidos Excitadores/farmacología , Ácido Kaínico/farmacología , Agonistas Muscarínicos/farmacología , Fosfatidilinositoles/metabolismo , Receptores Muscarínicos/metabolismo , Retina/embriología , Retina/metabolismo , Veratridina/farmacología , Animales , Embrión de Pollo , Agonistas de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/farmacología , Ácido Kaínico/metabolismo , Cloruro de Potasio , Receptores Muscarínicos/efectos de los fármacos , Retina/efectos de los fármacos , Canales de SodioRESUMEN
Four subtypes of GABA carriers (GAT1-GAT4) that transport GABA in a sodium-dependent manner were identified so far. In this report, the sodium-dependent release of GABA was investigated in cultured chick retinal cells. Opening of voltage-sensitive sodium channels by veratridine or activation of non-NMDA glutamate receptors induced the release of GABA from cultured cells. The release of GABA was calcium-independent, but could be completely prevented by the substitution of sodium chloride by lithium or choline chloride in the extracellular medium, suggesting that GABA release could be triggered by multiple mechanisms that led to the flux of sodium into these cells. Pharmacological experiments revealed that, while GABA uptake was almost completely inhibited by the GAT-1 blockers NNC-711 (50 microM) or nipecotic acid (1 mM), the release of this amino acid was inhibited by NNC-711, but not by nipecotic acid. The incubation with beta-alanine (10 mM), a GAT-2/GAT-3 inhibitor, blocked 50% of GABA uptake but had no effect on the release. Our data suggest that sodium-dependent GABA release from cultured chick retina cells is mediated by a GAT-1 like transporter that shows some, but not all, the pharmacological properties of the GAT-1 carrier.
Asunto(s)
Proteínas Portadoras/fisiología , Ácido Glutámico/farmacología , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana , Transportadores de Anión Orgánico , Retina/metabolismo , Veratridina/farmacología , Ácido gamma-Aminobutírico/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Proteínas Transportadoras de GABA en la Membrana Plasmática , Retina/citología , Retina/embriología , Tetrodotoxina/farmacología , TritioRESUMEN
In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 muM carbachol was used, the estimated IC50 value for kainate was 0.2 muM and the maximal inhibition of ~50 percent was obtained with 1 muM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 muM veratridine, but not 50 mM KCl, inhibited ~65 percent of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 + 38.0 to 2044.5 + 299.9 cpm/mg protein, retinal response decreased to 861.6 + 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.
Asunto(s)
Animales , Embrión de Pollo , Carbacol/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Ácido Kaínico/farmacología , Agonistas Muscarínicos/farmacología , Fosfatidilinositoles/metabolismo , Receptores Muscarínicos/metabolismo , Retina/embriología , Veratridina/farmacología , Agonistas de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/farmacología , Ácido Kaínico/metabolismo , Cloruro de Potasio , Receptores Muscarínicos/efectos de los fármacos , Retina/efectos de los fármacos , Canales de SodioRESUMEN
Toxin-gamma (Tgamma) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time-dependent increase in the release of norepinephrine and epinephrine from bovine adrenal medullary chromaffin cells. Tgamma was approximately 200-fold more potent than veratridine judged from EC50 values, although the maximal secretory efficacy of veratridine was 10-fold greater than that of Tgamma (1.2 vs. 12 microg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca2+ concentration ([Ca2+]o), when 30 microM veratridine plus 0.45 microM Tgamma were used. Tgamma (0.45 microM) doubled the basal uptake of 45Ca2+, whereas veratridine (100 microM) tripled it. Again, a drastic synergism in enhancing Ca2+ entry was seen when Tgamma and veratridine were combined; this was particularly pronounced at 5 mM [Ca2+]o. Veratridine induced oscillations of cytosolic Ca2+ concentration ([Ca2+]i) in single fura 2-loaded cells without elevation of basal levels. In contrast, Tgamma elevated basal [Ca2+]i levels, causing only small oscillations. When added together, Tgamma and veratridine elevated the basal levels of [Ca2+]i without causing large oscillations. Tgamma shifted the current-voltage (I-V) curve for Na+ channel current to the left. The combination of Tgamma with veratridine increased the shift of the I-V curve to the left, resulting in a greater recruitment of Na+ channels at more hyperpolarizing potentials. This led to enhanced and more rapid accumulation of Na+ in the cell, causing cell depolarization, the opening of voltage-dependent Ca2+ channels, and Ca2+ entry and secretion.