RESUMEN
Multiple types of high-voltage-activated Ca(2+) channels trigger neurotransmitter release at the mammalian central synapse. Among them, the omega-conotoxin GVIA-sensitive N-type channels and the omega-Aga-IVA-sensitive P/Q-type channels mediate fast synaptic transmission. However, at most central synapses, it is not known whether the contributions of different Ca(2+) channel types to synaptic transmission remain stable throughout postnatal development. We have addressed this question by testing type-specific Ca(2+) channel blockers at developing central synapses. Our results indicate that N-type channels contribute to thalamic and cerebellar IPSCs only transiently during early postnatal period and P/Q-type channels predominantly mediate mature synaptic transmission, as we reported previously at the brainstem auditory synapse formed by the calyx of Held. In fact, Ca(2+) currents directly recorded from the auditory calyceal presynaptic terminal were identified as N-, P/Q-, and R-types at postnatal day 7 (P7) to P10 but became predominantly P/Q-type at P13. In contrast to thalamic and cerebellar IPSCs and brainstem auditory EPSCs, N-type Ca(2+) channels persistently contribute to cerebral cortical EPSCs and spinal IPSCs throughout postnatal months. Thus, in adult animals, synaptic transmission is predominantly mediated by P/Q-type channels at a subset of synapses and mediated synergistically by multiple types of Ca(2+) channels at other synapses.
Asunto(s)
Química Encefálica/fisiología , Canales de Calcio Tipo N/fisiología , Sistema Nervioso Central/crecimiento & desarrollo , Transmisión Sináptica/fisiología , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Factores de Edad , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo R/fisiología , Sistema Nervioso Central/química , Sistema Nervioso Central/fisiología , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/fisiología , Glicinérgicos/farmacología , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Estricnina/farmacología , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/fisiología , omega-Agatoxina IVA/farmacología , omega-Conotoxinas/farmacologíaRESUMEN
Isolated muscle fibers from the motor legs of the crab Trichodactilus dilocarcinus were submitted to strong hyperpolarizing currents of varied intensities which produced tension during the current pulse. Threshold for tension was obtained with intensities of about 0.2 x 10(-5) A, changing E(m) to ca. -150 mV (starting from a resting potential ofca. -80 mV). At the closure of the anodic square pulse, a second phase of tension usually appeared superimposed upon the one obtained during hyperpolarization. The first phase of tension increased with the increase of Ca(++) concentration in the bath. Sr(++) produced the same type of mechanical output as Ca(++). When added to the normal Ca(++) concentration, Ba(++) and Mn(++) in low concentrations (up to 21.5 mM) also increased the tension of this phase, but at higher concentrations they blocked both phases while Mg(++) did not alter the tension. Of all the divalent cations employed, only Sr(++) is capable of developing tension as a substitute for Ca(++) in the external media. Procaine administered in a dosage (5 x 10(-3) W/V)which would suppress the contracture due to caffeine (10 mM), did not modify the tension developed during the hyperpolarization. The preceding data indicate that the Ca(++) required for tension during hyperpolarization comes from sites which would differ from those usually postulated for tension due to depolarization in the muscle fibers of other crustaceans (American crayfish). Furthermore, the external source of Ca(++) appears to be one mainly implicated in the induction of tension due to inward current pulses.
Asunto(s)
Braquiuros/fisiología , Contracción Muscular , Animales , Bario/fisiología , Cafeína/farmacología , Calcio/fisiología , Estimulación Eléctrica , Magnesio/fisiología , Manganeso/fisiología , Potenciales de la Membrana , Contracción Muscular/efectos de los fármacos , Procaína/farmacología , Estroncio/fisiologíaRESUMEN
Spinal nucleus of bulbocavernosus and its target musculature, the bulbocavernosus and levator ani muscles, are sexually dimorphic, and their sexual differentiation depends on plasmatic levels of testosterone. Electrophysiological and immunocytochemical studies have demonstrated that at mammalian adult neuromuscular junctions only P/Q-type Ca2+ channels (Ca(v2.1)), mediate evoked transmitter release. Here we report that N-type Ca2+ channel (Ca(v2.2)) blocker omega-Conotoxin GVIA, as well as Ca(v2.1) blocker omega-Agatoxin IVA, significantly reduced quantal content of transmitter release by approximately 80% and approximately 70% respectively at levator ani muscle of the adult rats, indicating that neuromuscular transmission is jointly mediated by both types of channels. In these synapses, we also observed that castration and restitution of plasmatic testosterone in rats resulted in changes in the sensitivity to omega-Conotoxin GVIA. Castration induced, whereas testosterone treatment avoided, functional loss of Ca(v2.2), as mediators of transmitter release in these synapses. Strikingly, the expression and localization of alpha1B subunits, which form the pore of the Ca(v2.2) channel, were similar at control, gonadectomized and gonadectomized testosterone-treated rats, suggesting that testosterone may regulate the coupling mechanisms between Ca(v2.2) and transmitter release at the neuromuscular junctions of these sexually dimorphic motoneurons.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Testosterona/farmacología , Animales , Animales Recién Nacidos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N , Diafragma/citología , Diafragma/efectos de los fármacos , Interacciones Farmacológicas , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Potenciales Evocados/efectos de la radiación , Inmunohistoquímica/métodos , Masculino , Orquiectomía/métodos , Diafragma Pélvico , Radioinmunoensayo/métodos , Ratas , Ratas Sprague-Dawley , Receptores Colinérgicos/metabolismo , omega-Agatoxina IVA/farmacología , omega-Conotoxina GVIA/farmacologíaRESUMEN
Transmitter release evoked by nerve stimulation is highly dependent on Ca2+ entry through voltage-activated plasma membrane channels. Calcium influx may be modified in some neuromuscular diseases like Lambert-Eaton syndrome and amyotrophic lateral sclerosis. We studied the pharmacologic sensitivity of the transmitter release process to different calcium channel blockers in normal human muscles and found that funnel web toxin and omega-Agatoxin-IVA, both P-type calcium channel blockers, blocked nerve-elicited muscle action potentials and inhibited evoked synaptic transmission. The transmitter release was not affected either by nitrendipine, an L-type channel blocker, or omega-Conotoxin-GVIA, an N-type channel blocker. The pharmacologic profile of neuromuscular transmission observed in normal human muscles indicates that P-like channels mediate transmitter release at the motor nerve terminals.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Unión Neuromuscular/fisiología , Neurotransmisores/metabolismo , Potenciales de Acción/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Humanos , Músculos Intercostales , Unión Neuromuscular/efectos de los fármacos , Nitrendipino/farmacología , Péptidos/farmacología , Poliaminas/farmacología , Valores de Referencia , Músculos Respiratorios , Venenos de Araña/farmacología , Transmisión Sináptica/efectos de los fármacos , Tubocurarina/farmacología , omega-Agatoxina IVA , omega-Conotoxina GVIARESUMEN
We investigated the role of the immune system in the pathogenesis of amyotrophic lateral sclerosis (ALS) by studying the long-term consequences of ALS immunoglobulin (Ig) application on the levator auris muscle of the mouse. We applied Ig from seven ALS patients, four disease controls, and a pool of normal Ig (6 mg of Ig in 2 weeks) by subcutaneous injection; removed the muscles 4 to 12 weeks after the beginning of treatment; and recorded both spontaneous and evoked release of transmitter. None of the control Ig induced changes in transmitter, whereas five of seven ALS Ig induced a significant increase in the rate of spontaneous release, and all ALS Ig produced significant changes in the quantal content of evoked release. In muscles treated with one of the ALS Igs, synaptic activity was completely absent. Cholinesterase and silver staining demonstrated intact neuromuscular junctions in the control Ig-treated muscles and also in many areas of ALS Ig-treated muscles. Axonal degeneration and denervation were present in most muscles treated with ALS Ig. There was complete denervation when no synaptic activity could be recorded. Thus, ALS Ig appears to lead to long-lasting effects at the neuromuscular junction, and such effects may be an early stage in the immune-mediated pathogenesis of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/inmunología , Inmunoglobulinas/fisiología , Neuronas Motoras/inmunología , Adulto , Anciano , Animales , Estimulación Eléctrica , Potenciales Evocados/fisiología , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Neuronas Motoras/fisiología , Músculos/inmunología , Músculos/fisiología , Unión Neuromuscular/fisiologíaRESUMEN
Individual skeletal muscle fibers in newborn vertebrates are innervated at a single endplate by several motor axons. During the first postnatal weeks, the polyneuronal innervation decreases in an activity-dependent process of synaptic elimination by axonal competition. Because synaptic activity depends strongly on the influx of calcium from the external media via presynaptic voltage-dependent calcium channels, we investigate the relationship between calcium channels, synaptic activity and developmental axonal elimination. We studied how several calcium channel blockers affect (after 1 h of incubation) the total number of functional axons per muscle fiber (poly-innervation index) of the Levator auris longus muscle of 6-day-old rats. We determined the poly-innervation index by gradually raising the stimulus amplitude and recorded the recruitment of one or more axons that produced a stepwise increment of the endplate potential.The L-type channel blocker nitrendipine (1 microM) increased the mean poly-innervation index (35.79% +/- 3.91; P<0.05). This effect was not washed out with normal Ringer, although the poly-innervation index returned to the control value when high-calcium Ringer (5 mM) was used. The P-type channel blocker omega-agatoxin-IVA (100 nM) also increased the number of recruitable endplate potentials (27.49% +/- 1.78; P<0.05), whereas N-type channel blocker omega-conotoxin-GVIA (1 microM) was ineffective (P>0.05). However, neither nitrendipine nor omega-agatoxin-IVA modified the poly-innervation index on high-calcium Ringer (P>0.05 in both cases). A more intense inhibition of calcium influx (by the sequential use of two calcium channel blockers) did not recruit any additional silent synapses. Moderately increasing the magnesium ions (by 500 microM) in the physiological solution produces a synaptic recruitment (36.78% +/- 2.1; P<0.05) similar to that with L- and P-type calcium channel blockers incubation. This magnesium effect was not washed with normal Ringer but a Ringer that is high in calcium can reverse it. The recruited endings were identified by selective activity-dependent loading with styryl dyes. Rhodaminated alpha-bungarotoxin-labeled acetylcholine receptors were present in the postsynaptic counterpart. Based on these findings we suggest that, before their complete retraction, functionally silent nerve terminals can be manifested or recovered if calcium influx is reduced by a calcium channel blocker or if external magnesium is increased. The normal activation of this calcium-dependent silencing mechanism during development may be related to the final loss of the supernumerary axons.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Diferenciación Celular/fisiología , Neuronas Motoras/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Unión Neuromuscular/crecimiento & desarrollo , Plasticidad Neuronal/fisiología , Terminales Presinápticos/metabolismo , Animales , Animales Recién Nacidos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Colorantes Fluorescentes , Magnesio/farmacología , Neuronas Motoras/citología , Neuronas Motoras/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/inervación , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
Using intracellular recording, we studied how several muscarinic antagonists affected the evoked endplate potentials in singly and dually innervated endplates of the levator auris longus muscle from 3 to 6-day-old rats. In dually innervated fibers, a second endplate potential (EPP) may appear after the first one when we increase the stimulation intensity. The lowest and highest EPP amplitudes are designated "small-EPP" and "large-EPP," respectively. In singly innervated endplates and large-EPP, we found an inhibition of acetylcholine release by M1-receptor antagonists pirenzepine and MT-7 (more than 30%) and M2-receptor antagonists methoctramine and AF-DX 116 (more than 40%). The small-EPP was also inhibited by both M2-receptor antagonists methoctramine (approximately 70%) and AF-DX 116 (approximately 40%). However, the small-EPP was enhanced by M1-receptor antagonists pirenzepine (approximately 90%) and MT-7 (approximately 50%). The M4-receptor selective antagonists tropicamide and MT-3 can also increase the small-EPP amplitude (75% and 120%, respectively). We observed a graded change from a multichannel involvement (P/Q- N- and L-type voltage-dependent calcium channels) of all muscarinic responses (M1-, M2- and M4-mediated) in the small-EPP to the single channel (P/Q-type) involvement of the M1 and M2 responses in the singly innervated endplates. This indicates the existence of a progressive calcium channels shutoff in parallel with the specialization of the adult type P/Q channel. In conclusion, muscarinic autoreceptors can directly modulate large-EPP generating ending potentiation, and small-EPP generating ending depression through their association with the calcium channels during development.
Asunto(s)
Autorreceptores/fisiología , Canales de Calcio/fisiología , Unión Neuromuscular/crecimiento & desarrollo , Receptores Muscarínicos/fisiología , Animales , Animales Recién Nacidos , Autorreceptores/antagonistas & inhibidores , Placa Motora/efectos de los fármacos , Placa Motora/crecimiento & desarrollo , Antagonistas Muscarínicos/farmacología , Unión Neuromuscular/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
We studied the effect of several calcium channel blockers (omega-Conotoxin-GVIA, 1 and 3microM; omega-Agatoxin-IVA, 100nM; Nitrendipine, 1 and 10microM) on evoked transmitter release at singly and dually innervated endplates of the levator auris longus muscle from three- to six-day-old rats. In dually innervated fibers, a second endplate potential may appear after the first one when we increase the stimulation intensity. The lowest and highest endplate potential amplitudes are designated "small endplate potential" and "large endplate potential", respectively. The percentage of doubly innervated junctions remains almost constant throughout the age range examined. Nevertheless, the percentage of junctions innervated by three or more terminal axons drops, whereas the singly innervated junctions increase. Therefore, between postnatal days 3 and 6, roughly half the neuromuscular junctions may experience the final process of axonal elimination. The synaptic efficacy of the large endplate potential in dual junctions, measured as the mean amplitude of the synaptic potential and mean quantal content, was the same as in the junctions that had become recently mono-innervated in the same postnatal period. In singly innervated fibers, the endplate potential size was strongly reduced by both the P/Q-type voltage-dependent calcium channel blocker omega-Agatoxin-IVA (79.17+/-4.02%; P < 0.05) and the N-type voltage-dependent calcium channel blocker omega-Conotoxin-GVIA (56.31+/-7.80%; P < 0.05), whereas endplate potential amplitude was not significantly changed by the L-type voltage-dependent calcium channel blocker Nitrendipine. In dually innervated fibers, the P/Q-type voltage-dependent calcium channel blocker omega-Agatoxin-IVA and L-type voltage-dependent calcium channel blocker Nitrendipine increased the size of the small endplate potential (161.29+/-47.87% and 109.32+/-11.03%, respectively; P < 0.05 in both cases) and reduced the large endplate potential (74.42+/-15.32% and 70.91+/-10.04%, respectively; P < 0.05 in both cases). The N-type voltage-dependent calcium channel blocker omega-Conotoxin-GVIA significantly increased the small endplate potential in the first few minutes after toxin application (at 10min: 90.23+/-17.38%; P < 0.05). This increase was not maintained, while the large endplate potential was strongly inhibited (69.25+/-7.5%; P < 0.05). In conclusion, in the dually innervated endplates of the newborn rat, presynaptic calcium channel types can have different roles in transmitter release from each of the two inputs, which suggests that nerve terminal voltage-dependent calcium channels are involved in neonatal synaptic maturation.
Asunto(s)
Envejecimiento/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Placa Motora/fisiología , Unión Neuromuscular/fisiología , Neurotransmisores/metabolismo , Animales , Animales Recién Nacidos , Canales de Calcio/efectos de los fármacos , Estimulación Eléctrica , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Placa Motora/efectos de los fármacos , Músculo Esquelético/inervación , Unión Neuromuscular/efectos de los fármacos , Nitrendipino/farmacología , Ratas , Ratas Sprague-Dawley , Sinapsis/fisiología , omega-Agatoxina IVA/farmacología , omega-Conotoxina GVIA/farmacologíaRESUMEN
The involvement of different types of voltage-dependent calcium channels in nerve-evoked release of neurotransmitter was studied during recovery from neuromuscular paralysis produced by botulinum toxin type A intoxication. For this purpose, a single subcutaneous injection of botulinum toxin (1 IU; DL50) on to the surface of the mouse levator auris longus muscle was performed. The muscles were removed at several time-points after injection (i.e. at one, two, three, four, five, six and 12 weeks). Using electrophysiological techniques, we studied the effect of different types of calcium channel blockers (nitrendipine, omega-conotoxin-GVIA and omega-agatoxin-IVA) on the quantal content of synaptic transmission elicited by nerve stimulation. Morphological analysis using the conventional silver impregnation technique was also made. During the first four weeks after intoxication, sprouts were found at 80% of motor nerve terminals, while at 12 weeks their number was decreased and the nerve terminals were enlarged. The L-type channel blocker nitrendipine (1 microM) inhibited neurotransmitter release by 80% and 30% at two and five weeks, respectively, while no effects were found at later times. The N-type channel blocker omega-conotoxin-GVIA (1 microM) inhibited neurotransmitter release by 50-70% in muscles studied at two to six weeks, respectively, and had no effect 12 weeks after intoxication. The P-type channel blocker omega-agatoxin-IVA (100 nM) strongly reduced nerve-evoked transmitter release (>90%) at all the time-points studied. Identified motor nerve terminals were also sensitive to both nitrendipine and omega-conotoxin-GVIA. This study shows that multiple voltage-dependent calcium channels were coupled to transmitter release during the period of sprouting and consolidation, suggesting that they may be involved in the nerve ending functional recovery process.
Asunto(s)
Toxinas Botulínicas Tipo A/envenenamiento , Canales de Calcio/fisiología , Neuronas Motoras/fisiología , Terminaciones Nerviosas/fisiología , Neurotransmisores/metabolismo , Animales , Electrofisiología , Masculino , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/patología , Recuperación de la FunciónRESUMEN
We have compared the effect of calcium channel blockers on the potassium-evoked release of tritium-labeled acetylcholine and on preganglionic spike-evoked synaptic transmission in the rat superior cervical ganglion. Transmitter release at the nerve terminals is mediated by the influx of calcium through voltage-gated calcium channels. While four types of voltage-gated calcium channels (T, L, N and P) have been identified in neurons, it is not clear which may actually be involved in excitation-secretion coupling. Release of tritiated acetylcholine evoked by sustained depolarization in high (40 mM) extracellular potassium decreased markedly in the absence of calcium or the presence of cadmium. High potassium-evoked release was substantially inhibited by the P-type channel blockers, purified from funnel-web spider toxin, and omega-agatoxin-IVA, and by the N-type channel blocker omega-conotoxin-GVIA, but was unaffected by the L-type channel blocker nitrendipine. In contrast, postganglionic compound action potentials synaptically triggered by preganglionic stimulation were strongly blocked by funnel-web spider toxin and slightly blocked by a high concentration of omega-agatoxin-IVA, but were unaffected by either omega-conotoxin-GVIA, nitrendipine or a low concentration of omega-agatoxin-IVA. Thus, at the superior cervical ganglion, funnel-web spider toxin-sensitive calcium channels play a dominant role in transmitter release evoked by transient, spike-mediated depolarization, but other types of voltage-gated calcium channels in addition to the funnel-web spider toxin-sensitive channel mediate the transmitter release that is evoked by sustained high potassium depolarization.
Asunto(s)
Acetilcolina/metabolismo , Potenciales de Acción/efectos de los fármacos , Canales de Calcio/fisiología , Ganglios Simpáticos/fisiología , Animales , Arginina/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Poliaminas/farmacología , Ratas , Ratas Wistar , Espermidina/farmacologíaRESUMEN
Voltage-dependent calcium channels (VDCC) have a key role in neuronal function transforming the voltage signals into intracellular calcium signals. They are composed of the pore-forming alpha(1) and the regulatory alpha(2)delta, gamma and beta subunits. Molecular and functional studies have revealed which alpha(1) subunit gene product is the molecular constituent of each class of native calcium channel (L, N, P/Q, R and T type). Electrophysiological and immunocytochemical studies have suggested that at adult mouse motor nerve terminal (MNT) only P/Q type channels, formed by alpha(1A) subunit, mediate evoked transmitter release. The generation of alpha(1A)-null mutant mice offers an opportunity to study the expression and localization of calcium channels at a synapse with complete loss of P/Q calcium channel. We have investigated the expression and localization of VDCCs alpha(1) and beta subunits at the wild type (WT) and knockout (KO) mouse neuromuscular junction (NMJ) using fluorescence immunocytochemistry. The alpha(1A) subunit was observed only at WT NMJ and was absent at denervated muscles and at KO NMJ. The subunits alpha(1B), alpha(1D) and alpha(1E) were also present at WT NMJ and they were over- expressed at KO NMJ suggesting a compensatory expression due to the lack of the alpha(1A). On the other hand, the beta(1b), beta(2a) and beta(4) were present at the same levels in both genotypes. The presence of other types of VDCC at WT NMJ indicate that they may play other roles in the signaling process which have not been elucidated and also shows that other types of VDCC are able to substitute the alpha(1A) subunit, P/Q channel under certain pathological conditions.
Asunto(s)
Canales de Calcio Tipo L/biosíntesis , Canales de Calcio Tipo N/biosíntesis , Canales de Calcio/biosíntesis , Proteínas de Transporte de Catión , Proteínas del Tejido Nervioso/biosíntesis , Unión Neuromuscular/metabolismo , Animales , Canales de Calcio/deficiencia , Canales de Calcio/genética , Canales de Calcio Tipo L/deficiencia , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo N/deficiencia , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo R , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genéticaRESUMEN
1. The effects of the voltage-dependent calcium channel (VDCC) blockers omega-agatoxin IVA (omega-AgaIVA), omega-conotoxin GVIA (omega-CgTx), omega-conotoxin MVIIC (omega-MVIIC) and omega-conotoxin MVIID (omega-MVIID) were evaluated on transmitter release in the mouse diaphragm preparation. The effects of omega-AgaIVA and omega-MVIIC were also evaluated on the perineurial calcium and calcium-dependent potassium currents, ICa and IK(Ca), respectively, in the mouse levator auris preparation. 2. The P- and Q-type VDCC blocker omega-AgaIVA (100 nM) and P- Q- and N-type channel blockers omega-MVIIC (1 microM) and omega-MVIID (3 microM) strongly reduced transmitter release (> 80-90% blockade) whereas the selective N-type channel blocker omega-CgTx (5 microM) was ineffective. 3. The process of release was much more sensitive to omega-MVIIC (IC50 = 39 nM) than to omega-MVIID (IC50 = 1.4 microM). After almost completely blocking transmitter release (quantal content approximately 0.3% of its control value) with 3 microM omega-MVIIC, elevating the external [Ca2+] from 2 to 10 mM induced an increase of approximately 20 fold on the quantal content of the endplate potential (e.p.p.) (from 0.2 +/- 0.04 to 4.8 +/- 1.4). 4. Nerve-evoked transmitter release in a low Ca(2+)-high Mg2+ medium (low release probability, quantal content = 2 +/- 0.1) had the same sensitivity to omega-AgaIVA (IC50 = 16.8 nM) as that in normal saline solutions. In addition, K(+)-evoked transmitter release was also highly sensitive to the action of this toxin (IC50 = 11.5 nM; 100 nM > 95% blockade). The action of omega-AgaIVA on transmitter release could be reversed by toxin washout if the experiments were carried out at 31-33 degrees C. Conversely, the effect of omega-AgaIVA persisted even after two hours of toxin washout at room temperature. 5. Both the calcium and calcium-dependent potassium presynaptic currents, ICa and IK(Ca), respectively, were highly sensitive to low concentrations (10-30 nM) of omega-AgaIVA. The ICa and the IK(Ca) were also strongly reduced by 1 microM omega-MVIIC. The most marked difference between the action of these two toxins was the long incubation times required to achieve maximal effects with omega-MVIIC. 6. In summary these results provide more evidence that synaptic transmission at the mammalian neuromuscular junction is mediated by Ca2+ entry through P- and/or Q-type calcium channels.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Unión Neuromuscular/efectos de los fármacos , Neurotoxinas/farmacología , Neurotransmisores/metabolismo , Péptidos/farmacología , Venenos de Araña/farmacología , Animales , Canales de Calcio/efectos de los fármacos , Masculino , Ratones , Unión Neuromuscular/fisiología , Potasio/farmacología , Canales de Potasio/efectos de los fármacos , omega-Agatoxina IVA , omega-Conotoxina GVIARESUMEN
Mammalian neuromuscular transmission is resistant to L and N type calcium channel blockers but very sensitive to a low molecular weight funnel web spider venom toxin, FTX, which selectively blocks P type calcium channels. To further characterize the calcium channels involved in neuromuscular transmission we studied the effect of omega Agatoxin (omega-Aga-IVA) a polypeptide P type channel blocker from the same spider venom. We show that omega-Aga-IVA is a potent and irreversible inhibitor of the presynaptic Ca2+ currents and of acetylcholine release induced by electrical stimulation or by K+ depolarization. This provides further evidences that transmitter release at the mammalian neuromuscular junction is mediated by P type Ca2+ channels.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Neurotransmisores/metabolismo , Terminales Presinápticos/metabolismo , Venenos de Araña/farmacología , Acetilcolina/metabolismo , Animales , Canales de Calcio/efectos de los fármacos , Estimulación Eléctrica , Potenciales Evocados/efectos de los fármacos , Masculino , Ratones , Placa Motora/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Nervio Frénico/fisiología , Terminales Presinápticos/efectos de los fármacos , Músculos Respiratorios/efectos de los fármacos , Músculos Respiratorios/fisiología , omega-Agatoxina IVARESUMEN
The effect of omega-conotoxin GVIA (omega-CgTx) was studied on spontaneous, K(+)-induced and electrically evoked neurotransmitter release at the neuromuscular junction of mouse diaphragm. omega-CgTx decreased the frequency and amplitude of basal and K(+)-induced miniature end plate potentials. This effect was abolished by raising the extracellular Ca2+ concentration. omega-CgTx had no effect on the quantal content of the electrically evoked release in this preparation.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Unión Neuromuscular/metabolismo , Neurotransmisores/metabolismo , Péptidos Cíclicos/farmacología , Animales , Calcio/metabolismo , Calcio/fisiología , Canales de Calcio/efectos de los fármacos , Estimulación Eléctrica , Potenciales Evocados/efectos de los fármacos , Técnicas In Vitro , Iontoforesis , Masculino , Ratones , Placa Motora/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Potasio/farmacología , omega-Conotoxina GVIARESUMEN
The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the release process was studied in presence of different extracellular Ca2+ concentrations, in the mouse phrenic-diaphragm preparation. Hemidiaphragms were incubated for 2 h at room temperature in the presence or absence of TPA. TPA increased the basal frequency of miniature end plate potentials (mepp's) in a dose-dependent manner, resulting in a maximal increase of 280% at a concentration of 0.5 microM. An inverse relationship between extracellular Ca2+ concentration and TPA effect was observed: at high extracellular concentrations of Ca2+ the action of TPA decreased significatively, while at low Ca2+ concentrations the effect of TPA was remarkably augmented. The highest effect of TPA was obtained when tested in a calcium-free medium. TPA also increased mepp frequency stimulated by 10 mM K+. As at basal conditions, the effect of TPA was higher at lower concentrations of extracellular calcium. The results suggest that the effect of stimulation of PKC on neurotransmitter release at the mice neuromuscular junction is not exerted at the level of calcium influx to the nerve terminal. Moreover the action of calcium and TPA seems to be superimposed. The effect of K+ on neurotransmitter release could be explained not only by depolarization of the nerve terminal but by increasing the pool of activable PKC.
Asunto(s)
Calcio/farmacología , Unión Neuromuscular/fisiología , Neurotransmisores/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Animales , Técnicas In Vitro , Cinética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Unión Neuromuscular/efectos de los fármacos , Nervio Frénico/fisiología , Potasio/farmacologíaRESUMEN
Clinical and experimental evidence support an autoimmune etiopathogenesis for amyotrophic lateral sclerosis (ALS). We have shown that local application of ALS-IgG onto nerve terminals induces dysfunction in transmission at the neuromuscular junction. It has been established that IgG and other circulating serum proteins can be taken up by motor nerve terminals, being immunolocalized in the soma where they accumulate following retrograde axonal transport. In the present study, we investigated the presence of human ALS and control IgG in the soma of mouse motoneurons. IgG was applied onto motor nerve terminals of mice by subcutaneous injections on the left levator auris longus muscle which is innervated by a branch of the facial nerve. After several injections, sections of the brainstem containing the facial nuclei were immunoprocessed to detect human IgG. For all IgG tested, motoneuron labeling was significantly more intense in the facial nucleus ipsilateral to the site of injection. In ALS-IgG-treated animals, ipsilateral labeling was significantly stronger than that found on the ipsilateral side of control IgG-treated animals. Our results are compatible with the concept that motoneurons preferentially take up, transport and/or accumulate ALS-IgG. Uptake of pathogenic antibodies by motoneuron terminals may play a role in the pathogenesis of motoneuron disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/inmunología , Inmunoglobulina G/metabolismo , Neuronas Motoras/inmunología , Terminaciones Nerviosas/inmunología , Unión Neuromuscular/inmunología , Adulto , Anciano , Animales , Estudios de Casos y Controles , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos , Persona de Mediana EdadRESUMEN
Potassium-induced contractures were measured isometrically in slow fibres of gastrocnemius muscle from the frog Leptodactylus ocellatus. Optical isomers of verapamil and of D 600 decreased the tension of K-contractures with the following characteristics: 1. 90 min exposures of the muscles to the (-)isomers of the drugs were more effective in decreasing tension of 40 mM KCl-contractures when successive challenges to 40 mM KCl were made each 10-15 min than without challenges during the incubation time. 2. In contrast to the depressing effect of (-)isomers of verapamil and D 600, the decrease of K-contractures by 1 mM EGTA in "Ca-free" solutions was independent on the history of 40 mM KCl-contractures. 3. The threshold concentration of K to cause contractures was the lower the lower the Ca-concentration. This relationship was little affected by (-)verapamil at concentrations of the drug which depressed by 50% the tension of 40 mM KCl-contractures in 1.8 mM CaCl2. 4. Verapamil and its methoxy-derivative D 600 were equipotent in depressing 40 mM KCl-contractures. Their optical (-)isomers were 4 to 5 times more potent than their corresponding (+)isomers. 5. K-tension curves in the presence of 6.1 muM (-)verapamil in 1.8 mM CaCl2 were similar to curves in 0.18 mM CaCl2 without the drug. 6. K-tension curves in 10 mM CaCl2 were shifted by (-)verapamil in nearly parallel manner towards higher K-concentrations. 7. The stereoselective decrease of K-contractures by verapamil and D 600 may be due to blockade of inward Ca-flux and to retardation of the reavailability of Ca2+ for release during partial depolarizations with K.
Asunto(s)
Galopamilo/farmacología , Contracción Muscular/efectos de los fármacos , Potasio/antagonistas & inhibidores , Verapamilo/análogos & derivados , Verapamilo/farmacología , Animales , Calcio/metabolismo , Calcio/farmacología , Ácido Egtácico/farmacología , Técnicas In Vitro , Músculos/efectos de los fármacos , Potasio/farmacología , Estereoisomerismo , Factores de TiempoRESUMEN
Calcium enters the cytoplasm mainly via voltage-activated calcium channels (VACC), and this represents a key step in the regulation of a variety of cellular processes. Advances in the fields of molecular biology, pharmacology and electrophysiology have led to the identification of several types of VACC (referred to as T-, N-, L-, P/Q- and R-types). In addition to possessing distinctive structural and functional characteristics, many of these types of calcium channels exhibit differential sensitivities to pharmacological agents. In recent years a large number of toxins, mainly small peptides, have been purified from the venom of predatory marine cone snails and spiders. Many of these toxins have specific actions on ion channels and neurotransmitter receptors, and the toxins have been used as powerful tools in neuroscience research. Some of them (omega-conotoxins, omega-agatoxins) specifically recognize and block certain types of VACC. They have common structural backbones and some been synthesized with identical potency as the natural ones. Natural, synthetic and labeled calcium channel toxins have contributed to the understanding of the diversity of the neuronal calcium channels and their function. In particular, the toxins have been useful in the study of the role of different types of calcium channels on the process of neurotransmitter release. Neuronal calcium channel toxins may develop into powerful tools for diagnosis and treatment of neurological diseases.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Neuronas/efectos de los fármacos , Toxinas Biológicas/farmacología , Secuencia de Aminoácidos , Animales , Bloqueadores de los Canales de Calcio/farmacología , Toxinas Marinas/farmacología , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Ponzoñas/químicaRESUMEN
Ca(v)2.1 channels (P/Q-type) play a prominent role in controlling neurotransmitter release. Transgenic mice in which the α1A pore-forming subunit of Ca(v)2.1 channels is ablated (KO) provide a powerful tool to study Ca(v)2.1 function in synaptic transmission in vivo. Whole-cell patch clamp was used to measure inhibitory glycinergic postsynaptic currents (IPSCs) from the lateral superior olive (LSO). Comparing wild-type (WT) and KO mice, we investigated the relevance of P/Q-type calcium channels at a glycinergic synapse mediated by multiple types of Ca(2+) channels, in opposition to synapses where only this type of Ca(2+) channels are in charge of transmitter release. We found that in KO mice, N-type and L-type Ca(2+) channels control synaptic transmission, resulting in a functional but reduced glycinergic transmitter release. Pair pulse facilitation of synaptic currents is retained in KO mice, even when synaptic transmission is driven by either N or L-type calcium channels alone, in contrast with lack of this phenomenon in other synapses which are exclusively mediated by P/Q-type channels. Thus, pointing a difference between P/Q- and N-type channels present in single or multiple types of calcium channels driven synapses. Significant alterations in short-term synaptic plasticity were observed. KO mice exhibited a stronger short term depression (STD) of IPSCs during repetitive stimulation at high frequency and recovered with a larger time constant compared to WT mice. Finally, transmitter release at the LSO synapse from KO mice was strongly modulated by presynaptic GTP-binding protein-coupled receptor γ-aminobutyric acid type B (GABA(B)).