RESUMEN
High-voltage-gated calcium channels have pivot role in the cellular and molecular mechanisms of various neurological disorders, including epilepsy. Similar to other calcium channels, P/Q-type calcium channels (Cav2.1) are also responsible for vesicle release at synaptic terminals. Up to date, there are very limited reports showing the mechanisms of Cav2.1 in epileptogenesis. In the present study, we investigated the anticonvulsive and neuroprotective effects of ω-agatoxin IVA, a specific Cav2.1 blocker, in a chemical kindling model of epileptogenesis. Righting reflex and inclined plane tests were used to assess motor coordination. Electroencephalography was recorded for electrophysiological monitoring of seizure activity in freely moving rats. Immunohistochemical analyses were performed for brain-derived neurotrophic factor (BDNF) and cleaved caspase-3 expressions in the prefrontal cortex, striatum, hippocampus, and thalamic nucleus. ω-Agatoxin IVA injected into the right lateral ventricle significantly prolonged the onset of seizures in a dose-dependent manner. In addition, repeated intraperitoneal administrations of ω-agatoxin IVA significantly suppressed the development of kindling and epileptic discharges without altering motor coordination. In addition, ω-agatoxin IVA significantly increased BDNF expressions, and decreased cleaved caspase-3 expressions in the brain when compared to PTZ + saline group. Our current study emphasizes the significance of the inhibition of P/Q type calcium channels by ω-agatoxin IVA, which suppresses the development of epileptogenesis and provides a new potential pathway for epilepsy treatment.
Asunto(s)
Bloqueadores de los Canales de Calcio , Epilepsia , Ratas , Animales , Bloqueadores de los Canales de Calcio/farmacología , omega-Agatoxina IVA , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Caspasa 3/metabolismo , omega-Conotoxina GVIA/metabolismo , omega-Conotoxina GVIA/farmacología , Canales de Calcio Tipo N/metabolismo , Encéfalo/metabolismo , Epilepsia/metabolismo , Convulsiones/metabolismo , Calcio/metabolismoRESUMEN
BACKGROUND: Coordinated calcium influx upon neuronal depolarization activates pathways that phosphorylate CaMKII, ERKs, and the transcription factor CREB and, therefore, expression of pro-survival and neuroprotective genes. Recent evidence indicates that amyloid-ß protein precursor (AßPP) is trafficked to synapses and promotes their formation. At the synapse, AßPP interacts with synaptic proteins involved in vesicle exocytosis and affects calcium channel function. OBJECTIVE: Herein, we examined the role of AßPP in depolarization-induced calcium-mediated signaling using acute cerebral slices from wild-type C57bl/6 mice and AßPP-/- C57bl/6 mice. METHODS: Depolarization of acute cerebral slices from wild-type C57bl/6 and AßPP-/- C57bl/6 mice was used to induce synaptic signaling. Protein levels were examined by western blot and calcium dynamics were assessed using primary neuronal cultures. RESULTS: In the absence of AßPP, decreased pCaMKII and pERKs levels were observed. This decrease was sensitive to the inhibition of N- and P/Q-type Voltage Gated Calcium Channels (N- and P/Q-VGCCs) by ω-conotoxin GVIA and ω-conotoxin MVIIC, respectively, but not to inhibition of L-type VGCCs by nifedipine. However, the absence of AßPP did not result in a statistically significant decrease of pCREB, which is a known substrate of pERKs. Finally, using calcium imaging, we found that down regulation of AßPP in cortical neurons results in a decreased response to depolarization and altered kinetics of calcium response. CONCLUSION: AßPP regulates synaptic activity-mediated neuronal signaling by affecting N- and P/Q-VGCCs.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Nifedipino/farmacología , Sinapsis/efectos de los fármacos , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Sinapsis/metabolismo , omega-Conotoxina GVIA/metabolismo , omega-Conotoxina GVIA/farmacologíaRESUMEN
A set of fluorophenoxyanilides, designed to be simplified analogues of previously reported ω-conotoxin GVIA mimetics, were prepared and tested for N-type calcium channel inhibition in a SH-SY5Y neuroblastoma FLIPR assay. N-type or Cav2.2 channel is a validated target for the treatment of refractory chronic pain. Despite being significantly less complex than the originally designed mimetics, up to a seven-fold improvement in activity was observed.
Asunto(s)
Analgésicos no Narcóticos/farmacología , Anilidas/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/metabolismo , Diseño de Fármacos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Analgésicos no Narcóticos/síntesis química , Analgésicos no Narcóticos/química , Analgésicos no Narcóticos/metabolismo , Anilidas/síntesis química , Anilidas/química , Anilidas/metabolismo , Unión Competitiva , Bloqueadores de los Canales de Calcio/síntesis química , Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio Tipo N/química , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Fluorobencenos/síntesis química , Fluorobencenos/química , Fluorobencenos/metabolismo , Fluorobencenos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Estructura Molecular , Terapia Molecular Dirigida , Proteínas del Tejido Nervioso/metabolismo , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Neuronas/metabolismo , Neurotoxinas/química , Dolor Intratable/tratamiento farmacológico , Dolor Intratable/metabolismo , Relación Estructura-Actividad , omega-Conotoxina GVIA/química , omega-Conotoxina GVIA/metabolismo , omega-Conotoxina GVIA/farmacologíaRESUMEN
SH-SY5Y human neuroblastoma cells provide a useful in vitro model to study the mechanisms underlying neurotransmission and nociception. These cells are derived from human sympathetic neuronal tissue and thus, express a number of the Cav channel subtypes essential for regulation of important physiological functions, such as heart contraction and nociception, including the clinically validated pain target Cav2.2. We have detected mRNA transcripts for a range of endogenous expressed subtypes Cav1.3, Cav2.2 (including two Cav1.3, and three Cav2.2 splice variant isoforms) and Cav3.1 in SH-SY5Y cells; as well as Cav auxiliary subunits α2δ1-3, ß1, ß3, ß4, γ1, γ4-5, and γ7. Both high- and low-voltage activated Cav channels generated calcium signals in SH-SY5Y cells. Pharmacological characterisation using ω-conotoxins CVID and MVIIA revealed significantly (â¼ 10-fold) higher affinity at human versus rat Cav2.2, while GVIA, which interacts with Cav2.2 through a distinct pharmacophore had similar affinity for both species. CVID, GVIA and MVIIA affinity was higher for SH-SY5Y membranes vs whole cells in the binding assays and functional assays, suggesting auxiliary subunits expressed endogenously in native systems can strongly influence Cav2.2 channels pharmacology. These results may have implications for strategies used to identify therapeutic leads at Cav2.2 channels.
Asunto(s)
Canales de Calcio/genética , Canales de Calcio/metabolismo , Expresión Génica , Neuroblastoma/genética , Neuroblastoma/metabolismo , Unión Competitiva , Bloqueadores de los Canales de Calcio/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Unión Proteica , Isoformas de Proteínas , omega-Conotoxina GVIA/metabolismoRESUMEN
Oxytocin (80 ng) injected into the caudal mesencephalic ventral tegmental area (VTA) of male rats induces penile erection. Such an effect occurs together with an increase in nitric oxide (NO) production, as measured by the augmented concentration of NO(2)(-) and NO(3)(-) found in the dialysate obtained from this brain area by means of intracerebral microdialysis. Both effects are abolished by d(CH(2))(5)Tyr(Me)(2)-Orn(8)-vasotocin (1 microg), an oxytocin receptor antagonist, by S-methyl-l-thiocitrulline acetate (20 microg), a neuronal NO synthase inhibitor, or by omega-conotoxin GVIA (50 ng), a N-type Ca(2+) channel blocker, all injected into the VTA 15 min before oxytocin. In contrast, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (40 microg), a guanylate cyclase inhibitor, given into the VTA 15 min before oxytocin, abolishes penile erection, but not the increase in NO production, while haemoglobin (40 microg), a NO scavenger, injected immediately before oxytocin reduces the increase in NO production, but not penile erection. 8-Bromo-cyclic guanosine monophosphate (0.5-10 microg) microinjected into the VTA induces penile erection with an inverted U-shaped dose-response curve; the maximal effective dose being 3 microg. Immunohistochemistry reveals that in the caudal VTA oxytocin-containing axons/fibres (originating from the paraventricular nucleus of the hypothalamus) contact cell bodies of mesolimbic dopaminergic (tyrosine hydroxylase-positive) neurons containing both NO synthase and guanylate cyclase. These results suggest that oxytocin injected into the VTA induces penile erection by activating NO synthase in the cell bodies of mesolimbic dopaminergic neurons. NO in turn activates guanylate cyclase present in these neurons, thereby increasing cyclic GMP concentration.
Asunto(s)
GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , Oxitocina/farmacología , Erección Peniana/efectos de los fármacos , Área Tegmental Ventral/efectos de los fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animales , Conducta Animal/fisiología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/metabolismo , Guanilato Ciclasa/metabolismo , Hemoglobinas/metabolismo , Humanos , Masculino , Microdiálisis , Neurotoxinas/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/metabolismo , Nitritos/metabolismo , Oxitocina/administración & dosificación , Erección Peniana/fisiología , Ratas , Ratas Sprague-Dawley , Tirosina 3-Monooxigenasa/metabolismo , Área Tegmental Ventral/metabolismo , omega-Conotoxina GVIA/metabolismoRESUMEN
We investigated the contribution of L-, N- and P/Q-type Ca(2+) channels to the [Ca(2+)](i) changes, evoked by kainate, in the cell bodies of hippocampal neurons, using a pharmacological approach and Ca(2+) imaging. Selective Ca(2+) channel blockers, namely nitrendipine, omega-Conotoxin GVIA (omega-GVIA) and omega-Agatoxin IVA (omega-AgaIVA) were used. The [Ca(2+)](i) changes evoked by kainate presented a high variability, and were abolished by NBQX, a AMPA/kainate receptor antagonist, but the N-methyl-D-aspartate (NMDA) receptor antagonist, D-AP5, was without effect. Each Ca(2+) channel blocker caused differential inhibitory effects on [Ca(2+)](i) responses evoked by kainate. We grouped the neurons for each blocker in three subpopulations: (1) neurons with responses below 60% of the control; (2) neurons with responses between 60% and 90% of the control, and (3) neurons with responses above 90% of the control. The inhibition caused by nitrendipine was higher than the inhibition caused by omega-GVIA or omega-AgaIVA. Thus, in the presence of nitrendipine, the percentage of cells with responses below 60% of the control was 41%, whereas in the case of omega-GVIA or omega-AgaIVA the values were 9 or 17%, respectively. The results indicate that hippocampal neurons differ in what concerns their L-, N- and P/Q-type Ca(2+) channels activated by stimulation of the AMPA/kainate receptors.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo , Ácido Kaínico/farmacología , Neuronas/efectos de los fármacos , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Células Cultivadas , Antagonistas de Aminoácidos Excitadores/metabolismo , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Neuronas/citología , Neuronas/metabolismo , Nitrendipino/metabolismo , Quinoxalinas/metabolismo , Ratas , Ratas Wistar , omega-Agatoxina IVA/metabolismo , omega-Conotoxina GVIA/metabolismoRESUMEN
Convection-enhanced delivery (CED) permits the homogeneous distribution of therapeutic agents throughout localized regions of the brain parenchyma without causing tissue damage as occurs with bolus injection. Here, we examined whether CED infusion of the N-type calcium channel antagonists omega-conotoxin GVIA (omega-CTX-G) and omega-conotoxin MVIIA (omega-CTX-M) can attenuate kindling measures in fully amygdala-kindled rats. Rats were implanted with a combination infusion cannula-stimulating electrode assembly into the right basolateral amygdala. Fully kindled animals received infusions of vehicle, omega-CTX-G (0.005, 0.05, and 0.5 nmol), omega-CTX-M (0.05, 0.15, and 0.5 nmol), proteolytically inactivated omega-CTX-M (0.5 nmol), or carbamazepine (500 nmol) into the stimulation site. CED of omega-CTX-G and omega-CTX-M over a 20-min period resulted in a dose-dependent increase in the afterdischarge threshold and a decrease in the afterdischarge duration and behavioral seizure score and duration during a period of 20 min to 1 week after the infusion, indicating an inhibitory effect on the triggering and expression of kindled seizures. The protective effects of omega-conotoxins reached a maximum at 48 h postinfusion, and then they gradually resolved over the next 5 days. In contrast, carbamazepine was active at 20 min but not at 24 h after the infusion, whereas CED of vehicle or inactivated omega-CTX-M had no effect. Except for transient tremor in some rats receiving the highest toxin doses, no adverse effects were observed. These results indicate that local CED of high-molecular-weight presynaptic N-type calcium channel blockers can produce long-lasting inhibition of brain excitability and that they may provide prolonged seizure protection in focal seizure disorders.
Asunto(s)
Amígdala del Cerebelo/efectos de los fármacos , Bloqueadores de los Canales de Calcio/administración & dosificación , Excitación Neurológica/efectos de los fármacos , omega-Conotoxina GVIA/administración & dosificación , omega-Conotoxinas/administración & dosificación , Animales , Carbamazepina/farmacología , Relación Dosis-Respuesta a Droga , Inyecciones Intraventriculares , Masculino , Actividad Motora/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , omega-Conotoxina GVIA/metabolismo , omega-Conotoxinas/metabolismoRESUMEN
The magnocellular neurosecretory cells of the hypothalamus (MNCs) regulate water balance by releasing vasopressin (VP) and oxytocin (OT) as a function of plasma osmolality. Release is determined largely by the rate and pattern of MNC firing, but sustained increases in osmolality also produce structural adaptations, such as cellular hypertrophy, that may be necessary for maintaining high levels of neuropeptide release. Since increases in Ca(2+) current could enhance exocytotic secretion, influence MNC firing patterns, and activate gene transcription and translation, we tested whether Ca(2+) currents in MNCs acutely isolated from the supraoptic nucleus (SON) of the hypothalamus are altered by 16-24 h of water deprivation. A comparison of whole-cell patch-clamp recordings demonstrated that dehydration causes a significant increase in the amplitude of current sensitive to the L-type Ca(2+) channel blocker nifedipine (from -56 +/- 6 to -99 +/- 10 pA; P < 0.001) with no apparent change in other components of Ca(2+) current. Post-recording immunocytochemical identification showed that this increase in current occurred in both OT- and VP-releasing MNCs. Radioligand binding studies of tissue from the SON showed there is also an increase in the density of binding sites for an L-type Ca(2+) channel ligand (from 51.5 +/- 4.8 to 68.1 +/- 4.1 fmol (mg protein)(-1); P < 0.05), suggesting that there was an increase in the number of L-type channels on the plasma membrane of the MNCs or some other cell type in the SON. There were no changes in the measured number of binding sites for an N-type Ca(2+) channel ligand. Dehydration was not associated with changes in the levels of mRNA coding for Ca(2+) channel alpha(1) subunits. These data are consistent with the hypothesis that a selective increase of L-type Ca(2+) current may contribute to the adaptation that occurs in the MNCs during dehydration.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Deshidratación/fisiopatología , Neuronas/fisiología , Núcleo Supraóptico/fisiología , Animales , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio Tipo L/biosíntesis , Canales de Calcio Tipo L/genética , Electrofisiología , Inmunohistoquímica , Técnicas In Vitro , Isradipino/metabolismo , Masculino , ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ensayo de Unión Radioligante , Ratas , Ratas Long-Evans , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Núcleo Supraóptico/citología , omega-Conotoxina GVIA/metabolismoRESUMEN
The contribution of voltage-gated calcium channels to excitable cell function depends, critically, upon the mechanisms that control their expression at the cell surface. While co-assembly of the pore forming alpha(1) and auxiliary beta subunits enhances channel surface expression, the levels are still only 30-40% of those seen with the core alpha(1B)/beta(1b)/alpha(2)delta calcium channel complex. To rationalize this observation, it has been suggested that the alpha(2)/delta subunit might stabilize calcium channel expression at the cell surface. To test this notion, we have resolved the effect of the alpha(2)/delta subunit on the rates of binding, internalization and degradation of defined N-type calcium channel surface complexes expressed in HEK293 cells, through pulse-labeling with the selective, cell impermeable, radioligand [(125)I]-omega-CgTx. Through detailed kinetic and sensitivity analysis we show that alpha(1B)/beta(1b)/alpha(2)delta complexes are internalized slowly (k(int) 0.4/h), whereupon, most become degraded (k(deg) 0.02/h). In contrast, alpha(1B)/beta(1b) complexes are internalized more rapidly (k(int) 0.8/h), following which they are either quickly degraded (k(deg) 0.1/h) or are sequestered slowly (k(tra) 0.1/h) to a pool that is metabolically stable within the time-frame of our experiments (24h). In neither case did we find evidence for recycling via the cell surface. Thus, our data argue for a novel mechanism where complexes lacking an alpha(2)/delta subunit are cleared from the cell surface and are rapidly degraded or stored, possibly for further attempts at complexation as new alpha(2)/delta subunits become available. The slower rate of internalization of complexes containing the alpha(2)/delta subunit rationalizes the stabilizing effect this subunit has upon calcium channel surface expression and suggests a mechanism by which alpha(2)delta mutations may cause severe neurological deficits.
Asunto(s)
Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/metabolismo , Canales de Calcio/química , Canales de Calcio/metabolismo , Animales , Canales de Calcio/genética , Canales de Calcio Tipo L , Canales de Calcio Tipo N/genética , Línea Celular , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Modelos Biológicos , Subunidades de Proteína , Ensayo de Unión Radioligante , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , omega-Conotoxina GVIA/metabolismoRESUMEN
1. The Ca(2+)-mediated regulation of interaction between FGF-1 and S100A13 in NG108-15 cells was studied. When the stress by depriving B27 supplement from the culture was given, cellular levels of both proteins were decreased, while their releases were significantly increased within 3 h. These stress-induced changes were all abolished by amlexanox, an anti-allergic drug. 2. These releases were significantly inhibited by the addition of EGTA or BAPTA-AM, cellular or extracellular Ca(2+)-chelating agent, respectively. The addition of omega-conotoxin GVIA, a N-type Ca(2+)-channel blocker caused a complete inhibition of the release, while increased the cytosolic levels of both proteins. However, omega-conotoxin MVIIC, the non-N-type Ca(2+)-channel blocker was ineffective. 3. In NG108-15 cells, which had been transfected with Venus-FGF-1 and CFP-S100A13, the supplement-deprivation stress caused several spike-type fluorescence resonance energy transfer (FRET) signals, suggesting that both proteins showing interaction would be immediately released. These spikes were completely abolished by the addition of omega-conotoxin GVIA. However, the addition of amlexanox caused bell-shaped FRET signals without spikes. 4. Thus, it is suggested that the interaction between FGF-1 and S100A13 responsible for stress-induced non-vesicular release is dependent of Ca(2+)-influx through N-type Ca(2+)-channels.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Exocitosis/fisiología , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Proteínas S100/metabolismo , Vesículas Secretoras/metabolismo , Aminopiridinas/metabolismo , Animales , Antialérgicos/metabolismo , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Línea Celular , Quelantes/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/metabolismo , Factor 1 de Crecimiento de Fibroblastos/genética , Transferencia Resonante de Energía de Fluorescencia , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Estrés Oxidativo , Ratas , Proteínas S100/genética , omega-Conotoxina GVIA/metabolismoRESUMEN
Characteristics for the specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to crude membranes from BHKN101 cells expressing the alpha1B subunits of Cav2.2 channels and from mice brain lacking the alpha1B subunits of Cav2.2 channels, particularly, the effects of CaM and various Ca2+ channel blockers on these specific bindings were investigated. Specific binding of 125I-omega-CTX GVIA to the crude membranes from BHKN101 cells was observed, but not from control BHK6 cells. omega-CTX GVIA, omega-CTX MVIIC and omega-CTX SVIB inhibited the specific binding of 125I-omega-CTX GVIA to crude membranes from BHKN101 cells, and the IC50 values for omega-CTXGVIA, omega-CTX MVIIC and omega-CTX SVIB were 0.07, 8.5 and 1.7 nM, respectively. However, omega-agatoxin IVA and calciseptine at concentrations of 10(-9)-10(-6) M did not inhibit specific binding. Specific binding was also about 80% inhibited by 20 microg protein/ml CaM. The amount of 125I-omega-CTX GVIA (30 pM) specifically bound to membranes from brain of knockout mice lacking alpha1B subunits of Cav2.2 channels was about 30% of that to the crude membranes from brain of wild-type. On the other hand, specific binding of 125I-omega-CTX MVIIC (200 pM) was observed on the crude membranes of both BHKN101 and control BHK6 cells. The specific binding of 125I-omega-CTX MVIIC (200 pM) was not inhibited by omega-CTX GVIA and omega-CTX SVIB, and also omega-Aga IVA and calciseptine at concentrations of 10(-9)-10(-7) M, although specific binding was almost completely dose dependently inhibited by non-radiolabeled omega-CTX MVIIC (IC50 value was about 0.1 nM). 20 microg protein/ml CaM did not inhibit specific binding. Therefore, these results suggest that BHKN101 cells have a typical Cav2.2 channels which are also inhibited by CaM and have not specific binding sites for omega-CTX MVIIC, although omega-CTX MVIIC is a blocker for both Cav2.1 (alpha1A; P/Q-type) and Cav2.2 channels.
Asunto(s)
Encéfalo/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/efectos de los fármacos , Calmodulina/farmacología , omega-Conotoxina GVIA/metabolismo , Animales , Encéfalo/metabolismo , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/metabolismo , Línea Celular , Cricetinae , Radioisótopos de Yodo/metabolismo , Ratones , Ratones Noqueados , Ensayo de Unión Radioligante , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismoRESUMEN
A New Binding Method (NBM) was used to investigate the characteristics of the specific binding of 125I-omega-conotoxin (omega-CTX) GVIA and 125I-omega-CTX MVIIC to Cav2.1 and Cav2.2 channels captured from chick brain membranes by antibodies against B1Nt (a peptide sequence in Car2.1 and Cav2.2 channels). The results for the NBM were as follows. (1) The ED50 values for specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to Cav2.1 and Cav2.2 channels were about 68 and 60 pM, respectively, and very similar to those (87 and 35 pM, respectively) to crude membranes from chick brain. (2) The specific 125I-omega-CTX GVIA (100 pM) binding was inhibited by omega-CTX GVIA (0.5 nM), dynorphine A (Dyn), gentamicin (Gen), neomycin (Neo) and tobramicin (Tob) (100 microM each), but not by omega-agaconotoxin (Aga) IVA, calciseptine, omega-CTX SVIB, omega-CTX MVIIC (0.5 nM each), PN200-110 (PN), diltiazem (Dil) or verapamil (Ver) (100 microM each). Calmodulin (CaM) inhibited the specific binding in a dose-dependent manner (IC50 value of about 100 microg protein/ml). (3) The specific 125I-omega-CTX MVIIC (60 pM) binding was inhibited by omega-CTX MVIIC, omega-CTX GVIA, omega-CTX SVIB (0.5 nM each), Dyn, Neo and Tob (100 microM, each), but not by omega-Aga IVA, calciseptine (0.5 nM each), PN, Dil, Ver (100 microM each) or 100 microg protein/ml CaM. These results suggested that the characteristics of the specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to Cav2.1 and Cav2.2 channels in the NBM were very similar to those to crude membranes from chick brain, although the IC50 values for CaM and free Ca2+ of CaM were about 33- and 5000-fold higher, respectively, than those for the specific binding of 125I-omega-CTX GVIA and 125I-omega-CTX MVIIC to crude membranes.
Asunto(s)
Anticuerpos Bloqueadores , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio Tipo N/metabolismo , omega-Conotoxina GVIA/metabolismo , omega-Conotoxinas/metabolismo , Animales , Especificidad de Anticuerpos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/efectos de los fármacos , Canales de Calcio Tipo N/inmunología , Calmodulina/farmacología , Pollos , Inmunoprecipitación , Técnicas In Vitro , Radioisótopos de Yodo , Cinética , Membranas/efectos de los fármacos , Membranas/metabolismo , omega-Conotoxina GVIA/farmacología , omega-Conotoxinas/farmacologíaRESUMEN
N- and P/Q-type Ca2+ channels are abundant in nerve terminals where they interact with proteins of the release apparatus, including syntaxin 1A and SNAP-25. In previous studies on N- or P/Q-type Ca2+ channels, syntaxin 1A co-expression reduced current amplitudes, increased voltage-dependent inactivation and/or enhanced G-protein inhibition. However, these studies were conducted in Ca2+ channels that exhibited significant voltage-dependent inactivation. We previously reported that N-type current in bovine chromaffin cells exhibits very little voltage-dependent inactivation and we identified the Ca2+ channel subunits involved. This study was undertaken to determine the effect of syntaxin 1A on this weakly inactivating Ca2+ channel. Co-expression of syntaxin 1A with the weakly inactivating bovine N-type Ca2+ channels in Xenopus oocytes did not appear to alter inactivation but dramatically reduced current amplitudes, without changing cell surface expression. To further understand the mechanisms of syntaxin 1A regulation of this weakly inactivating channel, we examined mutants of the alpha1B subunit, beta2a subunit and syntaxin 1A. We determined that the synprint site of alpha1B and the C-terminal third of syntaxin 1A were necessary for the reduced current amplitude. In addition we show that enhanced G-protein-dependent modulation of the Ca2+ current by syntaxin 1A cannot explain the large suppression of Ca2+ current observed. Of most significance, syntaxin 1A increased voltage-dependent inactivation in channels containing mutant beta2a subunits that cannot be palmitoylated. Our data suggest that changes in inactivation can not explain the reduction in current amplitude produced by co-expressing syntaxin and a weakly inactivating Ca2+ channel.
Asunto(s)
Antígenos de Superficie/farmacología , Canales de Calcio Tipo N/efectos de los fármacos , Canales de Calcio Tipo N/metabolismo , Proteínas del Tejido Nervioso/farmacología , Animales , Antígenos de Superficie/química , Antígenos de Superficie/genética , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo N/fisiología , Bovinos , Conductividad Eléctrica , Femenino , Proteínas de Unión al GTP/fisiología , Mutación/fisiología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Isoformas de Proteínas/genética , Estructura Terciaria de Proteína/fisiología , Ratas , Sintaxina 1 , Xenopus , omega-Conotoxina GVIA/metabolismoRESUMEN
To clear the structural specificity of calmodulin (CaM) on the specific 125I-omega-CTX binding to crude membranes from whole chick brain, the following experiments were investigated in this study: (i) the attenuating effect of semisynthetic tetrahydroisoquinoline derivatives on the inhibitory effect of Ca2+/CaM, (ii) the effects of chimeras of yeast and chicken Ca2+/CaM, and (iii) the effects of Ca2+-binding proteins (such as troponin c, S 100 a and b, and annexin I, III-V). The inhibitory effect of Ca2+/CaM was attenuated by isoquinoline derivatives (PX 28, 34, 216, 224, and CPU57) and a CaM antagonist W-7. PX 34, a typical synthesized isoquinoline derivative, showed the attenuating effect in a dose-dependent manner. The ED50 value for the attenuating effect of PX 34 was about 20 microM, which is similar to that of W-7 reported previously. Some chimeric CaMs such as YC 51-53 (which are close to the properties of vertebrate CaM) showed a significant inhibitory effect on the specific 125I-omega-CTX binding, but YC 129 and 130 (which retain the properties of yeast CaM), troponin c, S100 a, b, and annexin I, III-V had no effect on the specific 125I-omega-CTX binding. These results suggest that the characteristic structure containing the EF-hand structure of CaM itself is needed to cause the inhibitory effect on the specific 125I-omega-CTX binding.
Asunto(s)
Calmodulina/farmacología , omega-Conotoxina GVIA/metabolismo , Animales , Pollos , Radioisótopos de Yodo , Ensayo de Unión RadioliganteRESUMEN
In the Lambert-Eaton myasthenic syndrome (LEMS), immunoglobulin G (IgG) autoantibodies to presynaptic voltage-gated calcium channels (VGCCs) at the neuromuscular junction lead to a reduction in nerve-evoked release of neurotransmitter and muscle weakness. We have examined the action of LEMS IgGs on cloned human VGCCs stably expressed in transfected human embryonic kidney (HEK293) cell lines: 10-13 (alpha(1A-2), alpha(2b)delta, beta(4a)) and C2D7 (alpha(1B-1), alpha(2b)delta, beta(1b)). All LEMS IgGs studied showed surface binding to [(125)I]-omega-CTx-MVIIC-labeled VGCCs in the alpha(1A) cell line and two of six IgGs showed surface binding to [(125)I]-omega-CTx-GVIA-labeled VGCCs in the alpha(1B) cell line. We next studied the effect of LEMS IgGs (2 mg/ml) on whole-cell calcium currents in the alpha(1A) and alpha(1B) cell lines. Overnight treatment of alpha(1A) (10-13) cells with LEMS IgGs led to a significant reduction in peak current density without alteration of the current-voltage relationship or the voltage dependence of steady-state inactivation. In contrast, LEMS IgGs did not reduce peak current density in the alpha(1B) cell line. Overall these data demonstrate the specificity of LEMS IgGs for the alpha(1A) cell line and suggest that LEMS IgGs bind to and downregulate VGCCs in this cell line. Although several LEMS IgGs can be shown to bind to the alpha(1B) (C2D7) cell line, no functional effects were seen on this channel.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Canales de Calcio/genética , Clonación Molecular , Inmunoglobulina G/sangre , Inmunoglobulina G/farmacología , Síndrome Miasténico de Lambert-Eaton/sangre , Bario/fisiología , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Línea Celular , Conductividad Eléctrica , Humanos , Técnicas de Placa-Clamp , Pruebas de Precipitina , Temperatura , omega-Conotoxina GVIA/metabolismo , omega-Conotoxinas/metabolismoRESUMEN
The binding of [3H]angiotensin II to AT(1) receptors on Chinese Hamster Ovary cells expressing the human AT(1) receptor (CHO-AT(1) cells) is potently inhibited by venoms of the marine snails Conus geographus and C. betulinus. On the other hand, the binding of the nonpeptide AT(1) receptor-selective antagonist [3H]candesartan is not affected but competition binding curves of angiotensin II and the peptide antagonist [Sar(1),Ile(8)]angiotensin II (sarile) are shifted to the right. These effects resulted from the breakdown of angiotensin II into smaller fragments that do not bind to the AT(1) receptor. In this context, angiotensin-(1-7) is the most prominent fragment and angiotensin-(1-4) and angiotensin-(1-5) are also formed but to a lesser extent. The molecular weight of the involved peptidases exceeds 50 kDa, as determined by gel chromatography and ultrafitration.
Asunto(s)
Angiotensina II/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores de Angiotensina/metabolismo , omega-Conotoxina GVIA/metabolismo , Angiotensina I , Animales , Células CHO , Cricetinae , Humanos , Receptor de Angiotensina Tipo 1 , Receptores de Angiotensina/genética , TritioRESUMEN
In the mammalian central nervous system, transient global ischemia of specific duration causes selective degeneration of CA1 pyramidal neurons in hippocampus. Many of the ischemia-induced pathophysiologic cascades that destroy the neurons are triggered by pre- and postsynaptic calcium entry. Consistent with this, many calcium channel blockers have been shown to be neuroprotective in global models of ischemia. omega-Conotoxin MVIIA, a selective N-type VGCC blocker isolated from the venom of Conus magus, protects CA1 neurons in the rat model of global ischemia, albeit transiently. The mechanism by which this peptide renders neuroprotection is unknown. We performed high-resolution receptor autoradiography with the radiolabeled peptide and observed highest binding in stratum lucidum of CA3 subfield, known to contain inhibitory neurons potentially important in the pathogenesis of delayed neuronal death. This finding suggested that the survival of stratum lucidum inhibitory neurons might be the primary event, leading to CA1 neuroprotection after ischemia. Testing of this hypothesis required the reproduction of its neuroprotective effects in the gerbil model of global ischemia. Surprisingly, we found that omega-MVIIA did not attenuate CA1 hippocampal injury after 5 min of cerebral ischemia in gerbil. Possible reasons are discussed. Lastly, we show that the peptide can be used as a synaptic marker in assessing short and long-term changes that occur in hippocampus after ischemic injury.
Asunto(s)
Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Tipo N/análisis , Hipocampo/irrigación sanguínea , Ataque Isquémico Transitorio/tratamiento farmacológico , Proteínas del Tejido Nervioso/análisis , Neuronas/patología , Fármacos Neuroprotectores/uso terapéutico , omega-Conotoxina GVIA/metabolismo , omega-Conotoxinas/uso terapéutico , Animales , Autorradiografía , Biomarcadores , Calcio/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/efectos de los fármacos , Canales de Calcio Tipo N/metabolismo , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Gerbillinae , Hipocampo/metabolismo , Hipocampo/patología , Interneuronas/efectos de los fármacos , Interneuronas/patología , Transporte Iónico , Ataque Isquémico Transitorio/patología , Masculino , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Ratas , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/prevención & control , Especificidad de la Especie , Factores de Tiempo , omega-Conotoxinas/administración & dosificación , omega-Conotoxinas/farmacologíaRESUMEN
The effect of Ca2+/calmodulin (CaM) on the specific binding of [125I]omega-conotoxin GVIA (125I-omega-CTX) to crude membranes from chick brain was investigated. When we examined the effects of the activation of various endogenous protein kinases on specific [125I]omega-CTX binding to crude membranes, we observed that Ca2+/CaM had an inhibitory effect regardless of whether or not the standard medium contained ATP (0.5 mM). Ca2+/CaM also had an inhibitory effect in a simple binding-assay medium containing HEPES-HCl buffer, BSA, Ca2+ and CaM, and this effect was dependent on the concentration of Ca2+. The effect of Ca2+/CaM was attenuated by the CaM antagonists W-7 and CaM-kinase II fragment (290-309). An experiment with modified ELISA using purified anti omega-CTX antibody indicated that Ca2+/CaM did not affect the direct binding of [125I]omega-CTX and CaM. These results suggest that Ca2+/CaM either directly or indirectly affects specific [125I]omega-CTX binding sites, probably N-type Ca2+ channels in crude membranes from chick whole brain.
Asunto(s)
Encéfalo/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , omega-Conotoxina GVIA/metabolismo , Animales , Canales de Calcio Tipo N/metabolismo , Pollos , Ensayo de Inmunoadsorción Enzimática , Técnicas In Vitro , Radioisótopos de Yodo , MembranasRESUMEN
To understand better the pathophysiological roles of the vagal efferent system in ischemic heart diseases, we examined endogenous acetylcholine (ACh) release in the myocardium in vivo. Acute myocardial ischemia was induced in anesthetized cats by a 60-min occlusion of the left anterior descending coronary artery (LAD). We implanted dialysis probes in the left ventricular free wall and measured the dialysate ACh concentration using liquid chromatography. In the ischemic region, the ACh level increased from 0.68+/-0.12 to 12.3+/-3.3 n M (mean+/-S.E., P<0.01) by LAD occlusion. Bilateral vagotomy did not inhibit ischemia-induced ACh release (20.3+/-6.4 n M). In vagotomized animals, inhibition of the N-type Ca(2+)channel by intravenous administration of omega-conotoxin GVIA (10microg/kg) also failed to suppress ACh release (15.9+/-2.0 n M). However, the inhibition of intracellular Ca(2+)mobilization by local administration of 3,4,5-trimethoxybenzoic acid 8-(dietyl amino)-octyl ester (1 m M) suppressed ACh release (4.4+/-0.8 n M, P<0.05 compared with no pharmacological intervention). In the non-ischemic region, the ACh level increased from 1.9+/-0.4 to 6. 0+/-1.0 n M (P<0.05) by LAD occlusion, which was completely abolished by vagotomy. We concluded that ACh release in the ischemic region was mainly attributed to a local release mechanism, whereas that in the non-ischemic region depended on the presence of intact vagal activity. The local release mechanism would depend on intracellular Ca(2+)mobilization but not on N-type Ca(2+)channel opening.
Asunto(s)
Acetilcolina/metabolismo , Isquemia Miocárdica/metabolismo , Animales , Arteriopatías Oclusivas/metabolismo , Bloqueadores de los Canales de Calcio/administración & dosificación , Bloqueadores de los Canales de Calcio/metabolismo , Gatos , Modelos Animales de Enfermedad , Ácido Gálico/administración & dosificación , Ácido Gálico/análogos & derivados , Ácido Gálico/metabolismo , Norepinefrina/metabolismo , omega-Conotoxina GVIA/administración & dosificación , omega-Conotoxina GVIA/metabolismoRESUMEN
Brief exposure to ethanol inhibits L-type and N-type voltage-gated calcium channels in neural cells. Although chronic ethanol exposure up-regulates the density and function of L-type channels via a protein kinase C (PKC) delta-dependent mechanism, the effect of prolonged ethanol exposure on N-type channels is not known. Using PC12 cells, we found that exposure to 25 to 150 mM ethanol for 0 to 8 days produced a time- and concentration-dependent increase in the density of binding sites for the N-type channel antagonist (125)I-omega-conotoxin GVIA. This was associated with an increase in omega-conotoxin GVIA-sensitive, depolarization-evoked rises in [Ca(2+)](i). Increases in (125)I-omega-conotoxin GVIA binding also were observed in the frontal cortex and the hippocampus, but not in the thalamus of mice exposed to ethanol vapor for 3 days. In PC12 cells, increases in (125)I-omega-conotoxin GVIA binding were blocked by the PKC inhibitor bisindolylmaleimide I and by expression of a selective peptide inhibitor of PKCepsilon. Expression of a selective inhibitor of PKCdelta did not alter ethanol-induced increases in (125)I-omega-conotoxin GVIA binding. These findings indicate that PKCepsilon mediates up-regulation of N-type channels by ethanol. Because N-type channels modulate calcium-dependent neurotransmitter release, these findings suggest a mechanism that may contribute to neuronal hyperexcitability observed during alcohol withdrawal.