RESUMEN
Agonists of the Gq/11-activated G-protein-coupled receptors (GPCRs) combined with strong membrane depolarization (high KCl) induce a synergistic amplification of transmitter release. The molecular basis for the synergy is unknown. Here, we investigated this potentiated transmitter release (PTR) phenomenon at the single cell level by monitoring catecholamine (CA) release in chromaffin cells using amperometry. We found that the 60 mM KCl (K60)-triggered release in bovine chromaffin cells synergizes with bradykinin (BK) or histamine (Hist) to potentiate CA release. PTR was independent of Ca2+ influx through voltage-gated calcium channels (VGCC), but required Ca2+ at the extracellular medium and was abolished by inhibitors of phospholipase Cß (PLCß). Theâ¼four-fold PTR induced in mouse chromaffin cells by BK and K60, was not observed in chromaffin cells prepared from TRPC1 KO mice and was restored by expressing hTRPC1. The synergy between strong voltage perturbation (K60) in the presence of VGCC blockers, and the activation of the Gq/11-PLCß signal-transduction cascade generates unique and fundamental amplified signaling machinery. The concerted activation of two independent cellular pathways could reinforce physiological signals that impinge on regulation of secretory events during repeated sequence of high-frequency excitation, hyperpolarization, and relief of inhibition such as long-term potentiation (LTP), that lead to neuronal synaptic plasticity.
Asunto(s)
Catecolaminas/metabolismo , Células Cromafines/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Receptores Acoplados a Proteínas G/fisiología , Canales Catiónicos TRPC/fisiología , Animales , Bradiquinina/farmacología , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Bovinos , Membrana Celular/fisiología , Células Cultivadas , Activación Enzimática , Histamina/farmacología , Activación del Canal Iónico , Potenciales de la Membrana , Ratones , Ratones Noqueados , Nifedipino/farmacología , Fosfolipasa C beta/fisiología , Receptor de Bradiquinina B2/fisiología , Receptores Histamínicos H1/fisiología , Transducción de Señal , Especificidad de la Especie , Canales Catiónicos TRPC/genéticaRESUMEN
Objetivo: Evaluar en conductos curvos, utilizando la técnica de fuerzas balanceadas, la extensión de paredes instrumentadas (sin predeterminar) y libres de restos orgánicos, producida por limas de Flex-R, Onyx-R y Nitiflex. Materiales y Métodos: Se seleccionaron 60 molares inferiores. Se fotomicrografiaron cortes histológicos del tercio apical de cada espécimen y sobre las fotos, en hojas transparentes, se realizó un trazado en diferentes colores, de las zonas a evaluar. Estas imágenes fueron escaneadas y medidas. Los datos fueron estudiados con test no paramétrico de Kruskall-Wallis. Resultados: Los resultados no mostraron diferencias estadísticas significativas entre los grupos, en términos de porcentaje de pared del conducto con restos orgánicos (Kruskall-Wallis p=0,35) y porcentaje de pared del conducto sin restos orgánicos (Kruskall-Wallis p=0,374). Los resultados no mostraron diferencias estadísticas significativas entre los grupos, respecto al porcentaje de pared del conducto conformada (Kruskall-Wallis p=0.44) y porcentaje de pared del conducto no conformada (Kruskall-Wallis p=0,176). Los resultados no mostraron diferencias significativas entre las limas respecto a la instrumentación y limpieza de los conductos. Además, se evaluó la forma final de los conductos según el trazado de dos ejes. Los datos fueron analizados con un test Chi-Cuadrado (p= 0,88). No se encontró diferencia significativa entre los grupos. Los conductos mostraron un contorno redondeado u ovalado en la mayor parte de los casos. Conclusión: Ninguno de los instrumentos fue capaz de lograr una limpieza completa del conducto ni una instrumentación total de las paredes. En la mayoría de los casos, los instrumentos, produjeron preparaciones de sección redonda o ligeramente oval, conductos clínicamente deseables para la obturación (AU)
Objetive: To compare incurved root Canals using balanced forcé techniques, the amount of planed canal wall (áreas with predentin) and without organic debris, during preparation with Flex-R, Onyx-R y Nitiflex files. Study Design: Sixty mandibular molars were chosen. The areas to be evaluated were outlined in different colors on tracings over microphotographs form histological sections of each specimen. These images scanned and measured. Statistical analysis was perfomed employing the non-parametric Kruskall-Wallis test. Results: The test showed no statistically significant differences between groups in terms of the percentage of canal wall area with organic debris (KruskallWallis p=0,35) or percentage of canal wall area with no organic debris (Kruskall-Wallis p=0.374). The test showed no statistically significant differneces between groups in terms of the percentage of planed canal wall area (Kruskall-Wallis p=0.44) and non-planed canal wall area (Kruskall-Wallis p=0.176). The results showed no statistically significant differences were found between quality preparation, shape and the different instruments. The final canal shape was evaluated in terms of their two axes, (p=0,88) by Chi-square test. None of the instruments were capable of complete cleansing canal´s system or planning walls. Canals exhibited mosthly round or oval contours. Conclusion: None of the instruments were capable of complete cleansing canal´s system or planning walls. In most cases, we obtained round or slightly oval preparations that are clinically adequate for obturation (AU)
Asunto(s)
Humanos , Preparación del Conducto Radicular/instrumentación , Cavidad Pulpar/anomalías , Instrumentos Dentales , Cavidad Pulpar/ultraestructura , Tratamiento del Conducto Radicular/instrumentación , Capa de Barro Dentinario , Dentina/ultraestructuraRESUMEN
Objetivo: El propósito de este trabajo fue evaluar el espesor remanente de dentina /cemento en conductos mesio vestibulares de molares inferiores instrumentados con limas Flexo-File sistemas rotatorios Profile, ProTaper y RaCe. Material y Métodos: Se seleccionaron 75 molares inferiores, con angulaciones en su conducto mesio-vestibular entre 15º a 45º. Las raíces fueron incluidas en resina transparente utilizando como llave un dispositivo plástico. Las piezas fueron distribuidas de acuerdo a su angulación en cinco grupos: Grupo 1: Técnica escalonada con limas Flexo-File. Grupo 2: Sistema Profile, Grupo 3: Sistema ProTaper, Grupo 4: Sistema RaCe y Grupo 5: Testigo. Luego de la Instrumentación la raíces fueron seccionadas horizontalmente a nivel de la furcación, en el punto donde se inicia la curva y a 3 mm. Del ápice. Se midió en centésima de milímetro el menor espesor posoperatorio de cada raíz en mesial y distal en los tres niveles de corte. Resultados y conclusiones: El análisis estadístico mediante el test ANOVA no mostró diferencias significativas en los espesores dejados por las distintas técnicas a nivel cervical, medio y apical (AU)
Objective: The purpose of this investigation was to evaluate the dentin/cementum remanent thickness in lower molar mesio-vestiular root Canals instrumented with Flexo-files, Profile, Protaper and RaCe rotatory systems. Materials and Methods: Seventy five mandibular molars were selected, with 15º to 45º angles in their mesio-vetibular canals. The roots were included in transparent resin using a plastic device. The specimens were distributed according to their angles in five groups: Group 1: Step-back technique with Flexo-Files, Group 2: Profile system; Group 3: Pro Taper system; Group 4: Race system and Group 5: control group. After instrumentation the roots were horizontally sectioned at the bifurcation level, where the curvature begins and at 3 mm from the apex. The least post operatory thickness of each mesial and distal root in the three cutting levels were measured. Results and Conclusions: The statistical analysis using the ANOVA test did not show significant thickness differences resulting form the different techniques at cervical, medium or apical leves (AU)
Asunto(s)
Humanos , Tratamiento del Conducto Radicular/métodos , Dentina/anatomía & histología , Cemento Dental/anatomía & histología , Rotación , Preparación del Conducto Radicular/instrumentación , Instrumentos DentalesRESUMEN
The inactivation of voltage-gated L-type Ca(2+) channels (Ca(V)1) regulates Ca(2+) entry and controls intracellular Ca(2+) levels that are essential for cellular activity. The molecular entities implicated in L-channel (Ca(V)1.2) inactivation are not fully identified. Here we show for the first time the functional impact of one of the two highly conserved clusters of six negatively charged glutamates and aspartate (802-807; poly ED motif) at the II-III loop of the alpha 1 subunits of rabbit of Ca(v)1.2, alpha(1)1.2 and alpha(1)1.2 DeltaN60-Delta1733) on voltage-dependent inactivation. Mutation of the poly ED motif to alanine or glutamine/asparagine greatly enhanced voltage-dependent inactivation, shifting the voltage dependence to negative potentials by >50 mV and conferring a neuronal like inactivation kinetics onto Ca(V)1.2. The large shift in the midpoint of inactivation of the steady-state inactivation kinetics was observed also in Ca(2+) or Ba(2+) and was not altered by the beta2A subunit. Missing from the fast inactivating neuronal P/Q (Ca(V)2.1)-, N (Ca(V)2.2)- or R (Ca(V)2.3)-type channels and modulating Ca(V)1.2 inactivation kinetics, the poly ED motif is likely to be a specific L-type Ca(2+) channels inactivating domain. Our results fit a model in which the poly ED either by itself or as part of a larger inactivating motif acts as Ca(V)1.2 specific built-in "stopper." In this model, Ca(V)1 accomplishes a large Ca(2+) influx during depolarization, possibly by the poly ED hindering occlusion at the pore. Furthermore, the selective designed poly ED perhaps clarifies major inactivation differences between L- and non-L-type calcium channels.
Asunto(s)
Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/fisiología , Activación del Canal Iónico/fisiología , Alanina/genética , Alanina/metabolismo , Animales , Bario/metabolismo , Calcio/metabolismo , Clonación Molecular/métodos , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Microinyecciones/métodos , Modelos Moleculares , Conformación Molecular , Mutación/fisiología , Oocitos , Técnicas de Placa-Clamp/métodos , Conejos , Estroncio/metabolismo , XenopusRESUMEN
El objetivo del presente trabajo fue comparar, el espesor residual en conductos curvos instrumentados con técnicas manual y mecanizada. Se seleccionaron 60 molares inferiores, con angulaciones en su conducto mesio-vestibular de 15° a 45°. Siguiendo el procedimiento de Bramante modificado, las piezas fueron incluidas en resina transparente utilizando como llave un dispositivo plástico. Las raíces fueron seccionadas horizontalmente a nivel de la furcación, en el punto donde se inicia la curva y a 3 mm del ápice. Se midió y registró en centésima de milímetro el menor espesor preoperatorio de cada raíz en mesial y distal en los tres niveles de corte. Las piezas fueron reposicionadas en el dispositivo plástico e instrumentadas. Luego los segmentos fueron desmontados y medido el menor espesor posoperatorio radicular. Los datos fueron analizados con un test de comparación de proporciones». Se encontraron diferencias estadísticamente significativas, con menor proporción de espesores de riesgo en las técnicas manuales
The purpose of present work was to compare the residual canal thickness in curved canals ínstrumentated with manual and mechanícal techniques. Sixty recently removed human mandibular molars with mesio-bucal curved root canals ranging 15° to 45°, were selected. The teeth were enclosed in transparent resin using a plastic model system with Bramantes modification procedure. The root canals were cross-sectioned at the furcation level, at the point where the root canal curvature begins and at 3 mm from the apex. It was measured and recorded in hundredth of millimetet, the pre operative least thickness mesially and distaliy of the root canals at the three levels of the cross-sections. The teeth were replaced in the plastic model and instrumentated. After been instrumentated, the segment were removed again, and the pos operatíve least thickness root measured. The data were anaiyzed statistically with t test. There were significant dífferences in the residual thickness comparingmanual or mechanical techniques, with the thinnest proportíon risk thickness in manual technique
Asunto(s)
Humanos , Preparación del Conducto Radicular/métodos , Cementos Dentales , Materiales de Obturación del Conducto Radicular , Dentina/lesionesRESUMEN
El objetivo del presente trabajo fue evaluar, utilizando la técnica de fuerzas balanceadas, la efectividad de corte de limas de acero inoxidable (Flex-R) y de níquel- titanio(Onyx-R y Nitiflex). Se seleccionaron 36 molares inferiores, las piezas dentarias, antes y después de ser instrumentados, fueron secadas con papel absorbente, jeringa de aire, colocados en estufa y posteriormente pesados en balanza de precisión. La eficacia de corte se midió por la diferencia entre el peso pre y el postoperatorio. Los datos fueron estudiados estadísticamente con Análisis de Varianza de un Factor. La relación entre eficacia de corte y angulación de los conductos fue evaluada según el "t" test. Los resultados mostraron que la diferencia de los pesos entre los grupos fue estadísticamente significativa, las limas de Nitiflex fueron las más efectivas. La eficacia de corte de los instrumentos no se vió afectada por la angulación de los conductos
The aim of tbe present study was to evaluate tbe cutting efficiency of stainless steel (Flex-R) and nickel-titanium(Onyx-R y Nitiflex) files used with balanced force tecbnique. Thirty-six lower molars were collected. Before and after teeth were instrumentated, root canals were dried on absorbent paper and with air to eliminate residual humidity, dried in a stove and then weighed on a precision balance. Cutting efficiency was measured in term of the difference in weight before and after instrumentation. The date were statistically analyzed by one factor Analysis of Variance. The relation between cutting efficiency and canal curvature was assessed employing Student´s "t" test- The results showed a statistically significant difference in weight variation between groups and revealed tbat tbe Nitiflex files weretbe most effective. 1be cutting efficiency of the instruments did not depend on canal curvature
Asunto(s)
Humanos , Instrumentos Dentales , Tratamiento del Conducto Radicular/instrumentación , Preparación del Conducto Radicular/instrumentación , Fuerza CompresivaRESUMEN
It is well established that syntaxin 1A, synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptotagmin either alone or in combination, modulate the kinetic properties of voltage-gated Ca(2+) channels Ca(v)1.2 (Lc-channel) Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type). The interaction interface was found to reside at the cytosolic II-III domain of the alpha1 subunit of the channels. In this study, we demonstrated a functional coupling of human neuronal Ca(v)2.3 (R-type channel) with syntaxin 1A, SNAP-25 and synaptotagmin in BAPTA injected Xenopus oocytes. The kinetic properties of Ca(v)2.3 assembled with syntaxin 1A, SNAP-25 or synaptotagmin individually differed from Ca(v)2.3 associated with binary complexes syntaxin 1A/SNAP-25, syntaxin 1A/synaptotagmin or SNAP-25/synaptotagmin. Co-expression of Ca(v)2.3 with syntaxin 1A, SNAP-25 and synaptotagmin together, produced a channel with distinctive kinetic properties analogous to excitosome multiprotein complex generated by Ca(v)1.2 and Ca(v)2.2. Exchanging the current-carrying ions altered the kinetics of channel/synaptic proteins interaction, indicating a tight crosstalk formed between the permeation pathway of Ca(v)2.3 and the fusion apparatus during membrane depolarization. This putative coupling could predict how the release site might be organized to allow a rapid communication between the channel and the release machinery. In vivo confocal imaging of oocytes revealed GFP-synaptotagmin at the plasma membrane when the channel was present, as opposed to random distribution in its absence, consistent with Ca(2+)-independent molecular link of synaptotagmin and the channel. Synaptotagmin was detected at the membrane also in oocytes co-expressing the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Both imaging studies and protein-protein interactions in Xenopus oocytes show that channel linkage to synaptotagmin precedes Ca(2+) influx. Altogether, the R-type channel appears to associate with synaptic proteins to generate a multiprotein excitosome complex prior to Ca(2+)-entry. We propose that the distinct kinetics of the Ca(2+)-channel acquired by the close association with the vesicle and the t-SNAREs within the excitosome complex may be essential for depolarization evoked transmitter release.
Asunto(s)
Antígenos de Superficie/metabolismo , Canales de Calcio Tipo R/metabolismo , Proteínas de Unión al Calcio/metabolismo , Membrana Celular/metabolismo , Ácido Egtácico/análogos & derivados , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Oocitos/citología , Animales , Antígenos de Superficie/farmacología , Bario/metabolismo , Calcio/metabolismo , Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/farmacología , Proteínas de Transporte de Catión/metabolismo , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Estimulación Eléctrica/métodos , Técnica del Anticuerpo Fluorescente/métodos , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Indicadores y Reactivos/farmacología , Glicoproteínas de Membrana/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/farmacología , Proteínas del Tejido Nervioso/farmacología , Técnicas de Placa-Clamp/métodos , Proteína 25 Asociada a Sinaptosomas , Sinaptotagminas , Sintaxina 1 , XenopusRESUMEN
The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.
Asunto(s)
Linfocitos B/metabolismo , Canales de Calcio/metabolismo , Exocitosis , Insulina/metabolismo , Páncreas/metabolismo , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Electrofisiología , Ratones , Ratones Noqueados , Microscopía Fluorescente , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Syntaxin 1A has a pronounced inhibitory effect on the activation kinetics and current amplitude of voltage-gated Ca(2+) channels. This study explores the molecular basis of syntaxin interaction with N- and Lc-type Ca(2+) channels by way of functional assays of channel gating in a Xenopus oocytes expression system. A chimera of syntaxin 1A and syntaxin 2 in which the transmembrane domain of syntaxin 2 replaced the transmembrane of syntaxin 1A (Sx1-2), significantly reduced the rate of activation of N- and Lc-channels. This shows a similar effect to that demonstrated by syntaxin 1A, though the current was not inhibited. The major sequence differences at the transmembrane of the syntaxin isoforms are that the two highly conserved cysteines Cys 271 and Cys 272 in syntaxin 1A correspond to the valines Val 272 and Val 273 in syntaxin 2 transmembrane. Mutating either cysteines in Sx1-1 (syntaxin 1A) to valines, did not affect modulation of the channel while a double mutant C271/272V was unable to regulate inward current. Transfer of these two cysteines to the transmembrane of syntaxin 2 by mutating Val 272 and Val 273 to Cys 272 and Cys 273 led to channel inhibition. When cleaved by botulinum toxin, the syntaxin 1A fragments, amino acids 1-253 and 254-288, which includes the transmembrane domain, were both unable to inhibit current amplitude but retained the ability to modify the activation kinetics of the channel. A full-length syntaxin 1A and the integrity of the two cysteines within the transmembrane are crucial for coordinating Ca(2+) entry through the N- and Lc-channels. These results suggest that upon membrane depolarization, the voltage-gated N- and Lc-type Ca(2+)-channels signal the exocytotic machinery by interacting with syntaxin 1A at the transmembrane and the cytosolic domains. Cleavage with botulinum toxin disrupts the coupling of the N- and Lc-type channels with syntaxin 1A and abolishes exocytosis, supporting the hypothesis that these channels actively participate in Ca(2+) regulated secretion.
Asunto(s)
Antígenos de Superficie/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/metabolismo , Membrana Celular/metabolismo , Potenciales de la Membrana/genética , Mutación/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos/genética , Animales , Antígenos de Superficie/genética , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo N/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Cisteína/genética , Cisteína/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , ARN Complementario/farmacología , Proteínas Recombinantes de Fusión/genética , Sintaxina 1 , Xenopus laevisRESUMEN
The secretion of neurotransmitters is a rapid Ca(2+)-regulated process that brings about vesicle fusion with the plasma membrane. This rapid process (< 100 microseconds) involves multiple proteins located at the plasma and vesicular membranes. Because of their homology to proteins participating in constitutive secretion and protein trafficking, they have been characterized extensively. The sequential events that lead these proteins to vesicle docking and fusion are still unclear. We will review recent studies that demonstrate the operative role played by voltage-sensitive Ca(2+) channels and discuss the relevance for the process of evoked transmitter release. The regulation of Ca(2+) influx by syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and synaptotagmin, and the reciprocity of these proteins in controlling the kinetic properties of the channel will be discussed. Calcium channel and synaptic proteins expressed in Xenopus oocytes demonstrate a strong functional interaction, which could be pertinent to the mechanism of secretion. First, the voltage-sensitive Ca(2+) channels are negatively modulated by syntaxin: this inhibition is reversed by synaptotagmin. Second, the modulation of N-type Ca(2+) channel activation kinetics strongly suggests that the vesicle could be docked at the plasma membrane through direct interaction with synaptotagmin. Finally, these interactions provide evidence for the assembly of the voltage-sensitive Ca(2+) channel with syntaxin 1A, SNAP-25 and synaptotagmin into an excitosome complex: a putative fusion complex with a potential role in the final stages of secretion. Studies suggest that cross-talk between the synaptic proteins and the channel in a tightly organized complex may enable a rapid secretory response to an incoming signal such as membrane depolarization.
Asunto(s)
Canales de Calcio/fisiología , Exocitosis/fisiología , Neurotransmisores/fisiología , Sinapsis/fisiología , Proteínas de Transporte Vesicular , Animales , Calcio/fisiología , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Oocitos/fisiología , Proteínas Recombinantes/metabolismo , Proteínas SNARE , Xenopus laevisRESUMEN
Previously it demonstrated that in the absence of Ca2+ entry, evoked secretion occurs neither by membrane depolarization, induction of [Ca2+]i rise, nor by both combined (Ashery, U., Weiss, C., Sela, D., Spira, M. E., and Atlas, D. (1993). Receptors Channels 1:217-220.). These studies designate Ca2+ entry as opposed to [Ca2+]i rise, essential for exocytosis. It led us to propose that the channel acts as the Ca+ sensor and modulates secretion through a physical and functional contact with the synaptic proteins. This view was supported by protein-protein interactions reconstituted in the Xenopus oocytes expression system and release experiments in pancreatic cells (Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G., and Rorsman, P. (2001). Biophys. J; Wiser, O., Bennett, M. K., and Atlas, D. (1996). EMBO J 15:4100-4110; Wiser, O., Trus, M.. Hernandez, A., Renström, E., Barg, S., Rorsman. P., and Atlas, D. (1999). Proc. Natl. Acad. Sci. U.S.A. 96:248-253). The kinetics of Ca(v)1.2 (Lc-type) and Ca(v)2.2 (N-type) Ca2+ channels were modified in oocytes injected with cRNA encoding syntaxin 1A and SNAP-25. Conserved cysteines (Cys271, Cys272) within the syntaxin 1A transmembrane domain are essential. Synaptotagmin 1, a vesicle-associated protein, accelerated the activation kinetics indicating Ca(v)2.2 coupling to the vesicle. The unique modifications of Ca(v)1.2 and Ca(v)2.2 kinetics by syntaxin 1A, SNAP-25, and synaptotagmin combined implied excitosome formation, a primed fusion complex of the channel with synaptic proteins. The Ca(v)1.2 cytosolic domain Lc(753-893), acted as a dominant negative modulator, competitively inhibiting insulin release of channel-associated vesicles (CAV), the readily releasable pool of vesicles (RRP) in islet cells. A molecular mechanism is offered to explain fast secretion of vesicles tethered to SNAREs-associated Ca2+ channel. The tight arrangement facilitates the propagation of conformational changes induced during depolarization and Ca2+-binding at the channel, to the SNAREs to trigger secretion. The results imply a rapid Ca2+-dependent CAV (RRP) release, initiated by the binding of Ca2+ to the channel, upstream to intracellular Ca2+ sensor thus establishing the Ca2+ channel as the Ca2+ sensor of neurotransmitter release.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Proteínas de Unión al Calcio , Sistema Nervioso Central/metabolismo , Exocitosis/fisiología , Neuronas/metabolismo , Neurotransmisores/metabolismo , Transmisión Sináptica/fisiología , Animales , Antígenos de Superficie/metabolismo , Canales de Calcio Tipo L/metabolismo , Femenino , Sustancias Macromoleculares , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Neurológicos , Proteínas del Tejido Nervioso/metabolismo , Oocitos , Embarazo , Estructura Terciaria de Proteína/fisiología , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Proteína 25 Asociada a Sinaptosomas , Sinaptotagmina I , Sinaptotagminas , Sintaxina 1 , Xenopus laevisRESUMEN
The regioselective dibenzylphosphorylation of 2 followed by catalytic reduction in the presence of N-methyl-D-glucamine afforded 2-(S)-(1-(R)-(3, 5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoro)phenyl-4-(5-(2- phosphoryl-3-oxo-4H,-1,2,4-triazolo)methylmorpholine, bis(N-methyl-D-glucamine) salt, 11. Incubation of 11 in rat, dog, and human plasma and in human hepatic subcellular fractions in vitro indicated that conversion to 2 would be expected to occur in vivo most readily in humans during hepatic circulation. Conversion of 11 to 2 occurred rapidly in vivo in the rat and dog with the levels of 11 being undetectable within 5 min after 1 and 8 mg/kg doses iv in the rat and within 15 min after 0.5, 2, and 32 mg/kg doses iv in the dog. Compound 11 has a 10-fold lower affinity for the human NK-1 receptor as compared to 2, but it is functionally equivalent to 2 in preclinical models of NK-1-mediated inflammation in the guinea pig and cisplatin-induced emesis in the ferret, indicating that 11 acts as a prodrug of 2. Based in part on these data, 11 was identified as a novel, water-soluble prodrug of the clinical candidate 2 suitable for intravenous administration in humans.
Asunto(s)
Acetales/síntesis química , Acetales/metabolismo , Antiinflamatorios no Esteroideos/síntesis química , Antieméticos/síntesis química , Morfolinas/síntesis química , Morfolinas/metabolismo , Antagonistas del Receptor de Neuroquinina-1 , Profármacos/síntesis química , Acetales/química , Acetales/farmacología , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Antieméticos/química , Antieméticos/metabolismo , Antieméticos/farmacología , Antineoplásicos , Aprepitant , Cisplatino , Perros , Evaluación Preclínica de Medicamentos , Hurones , Cobayas , Humanos , Morfolinas/química , Morfolinas/farmacología , Profármacos/química , Profármacos/metabolismo , Profármacos/farmacología , Ratas , Solubilidad , Estereoisomerismo , Relación Estructura-Actividad , Vómitos/inducido químicamente , Vómitos/tratamiento farmacológico , AguaRESUMEN
A water-soluble phosphoramidate prodrug (L-758,298, compound I) of the potent and selective human Substance P receptor antagonist L-754, 030 (compound II) is under development as an i.v. drug for treatment of emesis, migraine, and chronic pain. Compound I undergoes hydrolysis readily to II under acidic conditions. In the studies reported herein, we investigated the stability of I in blood and hepatic subcellular fractions from rats, dogs, and humans as well as the conversion of I to II in rats and dogs after i.v. dosing. Compound I was converted to II rapidly in rat blood but was stable in dog and human blood. However, the conversion was rapid in liver microsomes prepared from dogs and humans. As expected from the results of in vitro studies, the in vivo conversion of I to II was rapid after i.v. dosing of I to rats and dogs. The relative extent of exposure of II after i.v. dosing of I was estimated by comparing the dose-adjusted area under the plasma concentration versus time curve values of II after i.v. dosing of I with those after i.v. dosing of II. In rats, the extent of exposure was estimated to be approximately 90 and approximately 100% at 1 and 8 mg/kg, respectively; in dogs, that was approximately 59% at 0.5 mg/kg. A nonproportional increase in the area under the concentration versus time curve value of II with dose was observed after i.v. administration of I in dogs from 0.5 to 32 mg/kg, suggesting that the elimination of II might have been saturated at higher doses.
Asunto(s)
Acetales/farmacocinética , Analgésicos no Narcóticos/farmacocinética , Antieméticos/farmacocinética , Morfolinas/farmacocinética , Antagonistas del Receptor de Neuroquinina-1 , Profármacos/farmacocinética , Acetales/administración & dosificación , Analgésicos no Narcóticos/administración & dosificación , Analgésicos no Narcóticos/sangre , Animales , Antieméticos/administración & dosificación , Antieméticos/sangre , Aprepitant , Área Bajo la Curva , Perros , Humanos , Inyecciones Intravenosas , Hígado/metabolismo , Masculino , Morfolinas/administración & dosificación , Morfolinas/sangre , Profármacos/administración & dosificación , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/metabolismoRESUMEN
Although N- and P-type Ca2+ channels predominant in fast-secreting systems, Lc-type Ca2+ channels (C-class) can play a similar role in certain secretory cells and synapses. For example, in retinal bipolar cells, Ca2+ entry through the Lc channels triggers ultrafast exocytosis, and in pancreatic beta-cells, evoked secretion is highly sensitive to Ca2+. These findings suggest that a rapidly release pool of vesicles colocalizes with the Ca2+ channels to allow high Ca2+ concentration and a tight coupling of the Lc channels at the release site. In binding studies, we show that the Lc channel is physically associated with synaptotagmin (p65) and the soluble N-ethylmaleimide-sensitive attachment proteins receptors: syntaxin and synaptosomal-associated protein of 25 kDa. Soluble N-ethylmaleimide-sensitive attachent proteins receptors coexpressed in Xenopus oocytes along with the Lc channel modify the kinetic properties of the channel. The modulatory action of syntaxin can be overcome by coexpressing p65, where at a certain ratio of p65/syntaxin, the channel regains its unaltered kinetic parameters. The cytosolic region of the channel, Lc753-893, separating repeats II-III of its alpha1C subunit, interacts with p65 and "pulls" down native p65 from rat brain membranes. Lc753-893 injected into single insulin-secreting beta-cell, inhibits secretion in response to channel opening, but not in response to photolysis of caged Ca2+, nor does it affect Ca2+ current. These results suggest that Lc753-893 competes with the endogenous channel for the synaptic proteins and disrupts the spatial coupling with the secretory apparatus. The molecular organization of the Lc channel and the secretory machinery into a multiprotein complex (named excitosome) appears to be essential for an effective depolarization evoked exocytosis.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio , Exocitosis , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Canales de Calcio/genética , Señalización del Calcio , Ácido Egtácico/análogos & derivados , Ácido Egtácico/metabolismo , Conductividad Eléctrica , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Fotólisis , Proteínas Qa-SNARE , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteína 25 Asociada a Sinaptosomas , Sinaptosomas , Sinaptotagmina I , SinaptotagminasRESUMEN
OBJECTIVE: To examine the presence of anti-L-type calcium channel antibodies in the serum of ALS patients. BACKGROUND: Autoimmunity has been hypothesized as one of the mechanisms underlying the pathogenesis of sporadic ALS. Previous studies reported that sera from patients with sporadic ALS contain antibodies against voltage-gated calcium channels (L-type and P-type), but others do not support these findings. METHODS: Regulated secretion of tritiated dopamine ([3H]DA) in PC12 cells is mediated exclusively by calcium entry through L-type calcium channels. To examine whether purified ALS immunoglobulin G (IgG) inhibits [3H]DA release by interfering with calcium entry through L-type calcium channels, evoked release in PC12 cells was determined in the presence of ALS IgG. This functional assay provides a sensitive way to examine L-type calcium channel interaction with IgG from ALS patients. RESULTS: A significant inhibition of depolarization-evoked [3H]DA release (32+/-4%) was observed by purified IgG from ALS patients compared with control subjects (11+/-2%; p < 0.01). Significant inhibition by IgG occurred in 79% (15/19) of the ALS patients compared with only 29% (5/17) in the control group (p < 0.01). The level of calcium channel inhibition by ALS IgG correlated positively with disease duration (r = 0.68; p < 0.01) and correlated negatively with age (r = -0.48; p < 0.05). CONCLUSIONS: These results confirm the presence of antibodies against the L-type calcium channel in the majority of sera from ALS patients, supporting their role in the pathogenesis of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/inmunología , Autoanticuerpos/farmacología , Canales de Calcio/inmunología , Dopamina/metabolismo , Inmunoglobulina G/farmacología , Animales , Autoanticuerpos/sangre , Canales de Calcio/metabolismo , Canales de Calcio Tipo L , Humanos , Inmunoglobulina G/sangre , Potenciales de la Membrana/inmunología , Persona de Mediana Edad , Proteínas Musculares/inmunología , Proteínas Musculares/metabolismo , Células PC12 , Ratas , TritioRESUMEN
1. Ivermectin was extensively metabolized by human liver microsomes to at least 10 metabolites. The structure of many of them (mostly hydroxylated and demethylated) was determined by 1H-NMR and LC/MS. 2. To determine which human cytochrome P450 isoform(s) is responsible for the metabolism of ivermectin, chemical inhibitors including sulphaphenazole, quinidine, furafylline, troleandomycin (TAO) and diethyldithiocarbamate (DDC) were used to evaluate their effect on ivermectin metabolism. TAO, a specific inhibitor of cytochrome P4503A4, was the most potent inhibitor, inhibiting the total metabolism as well as formation of each metabolite. Metabolism was also inhibited by an anti-human cytochrome 3A4 antibody by 90%. 3. When ivermectin was incubated with microsomes from cells expressing CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 or 3A4 at 4 mg/ml protein concentrations, metabolic activity was only detected with the microsomes containing CYP3A4. The metabolic profile from cDNA-expressed CYP3A4 microsomes was qualitatively similar to that from human liver microsomes. 4. Thus, cytochrome P4503A4 is the predominant isoform responsible for the metabolism of ivermectin by human liver microsomes.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Ivermectina/metabolismo , Microsomas Hepáticos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP3A , Humanos , Proteínas Recombinantes/metabolismoRESUMEN
Expression of the N-type voltage sensitive calcium channel in Xenopus oocytes along with syntaxin and p65 showed that the syntaxin-modified N-type channel properties, were fully reversed by p65. The inward current was restored to a significantly higher amplitude when all three proteins were present, suggesting that the channel interacts with syntaxin, p65 and SNAP-25 in a quaternary complex. Further support to a multicomplex formation between the channel and the synaptic proteins was drawn from the steady-state voltage inactivation profiles. A physical interaction of the N-type calcium channel with the vesicular protein synaptotagmin (p65) was demonstrated biochemically, using recombinant fusion proteins. The interaction is confined to a cytosolic channel domain that separates segments II and III amino acids 710-1090 of the N-type channel (N-Loop710-1090). In vitro binding of recombinant N-Loop710-1090 to p65 (amino acids 96-421) involves the two C2 domains of p65, C2A domain [amino acids 96-265; p65(1-3)] and C2B domain [amino acids 248-421; p65(3-5)]. While the binding of C2A and C2B domains was calcium independent, C2B domain binding to the N-Loop was inositol-hexaphosphate (IP6)-sensitive. The N-Loop710-1090 binding to p65 was competed by syntaxin and SNAP-25, which are synaptic plasma membrane proteins. These combined functional and biochemical approaches provide evidence for a complex formation between the N-type channel and the exocytotic machinery which by generating fusion-competent vesicles may function to regulate the process of synaptic secretion.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Unión al Calcio , Activación del Canal Iónico , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Unión Competitiva , Calcio/metabolismo , Ácido Fítico/metabolismo , Unión Proteica , Proteínas Qa-SNARE , Proteínas Recombinantes/metabolismo , Proteína 25 Asociada a Sinaptosomas , Sinaptotagmina I , Sinaptotagminas , Xenopus laevisRESUMEN
The disposition of L-694,458, a potent monocyclic beta-lactam inhibitor of human leukocyte elastase, was studied in male Sprague-Dawley rats and rhesus monkeys. After iv dosing, L-694,458 exhibited similar pharmacokinetic parameters in rats and rhesus monkeys. The mean values for its plasma clearance, terminal half-life, and volume of distribution at steady state were 27 ml/min/kg, 1.8 hr, and 4.0 liters/kg in rats and 34 ml/min/kg, 2.3 hr, and 5 liters/kg in rhesus monkeys. The bioavailability of a 10 mg/kg oral dose was higher in rats (65%) than in rhesus monkeys (39%). In both species, concentrations of L-694,458 in plasma increased more than proportionally when the oral dose was increased from 10 mg/kg to 40 mg/kg. In monkeys a protracted plasma concentration-time profile was observed at 40 mg/kg, characterized by a delayed T(max) (8-24 hr) and a long terminal half-life (6 hr). [3H]L-694,458 was well absorbed after oral dosing to rats at 10 mg/kg, as indicated by the high recovery of radioactivity in bile (83%) and urine (6%) of bile duct-cannulated rats. Only approximately 5% or less of the radioactivity in bile, urine, and feces was a result of intact L-694,458, indicating that the compound was being eliminated by metabolism, followed by excretion of the metabolites in feces, via bile. Demethylenation of the methylenedioxyphenyl group resulting in the catechol was the primary metabolic pathway in human and rhesus monkey liver microsomes. In rat liver microsomes, the major metabolite was the N-oxide of the methyl-substituted piperazine nitrogen. In rats dosed iv and orally with [3H]L-694,458, concentrations of radioactivity were highest in the lung (the primary target tissue), adrenals, and liver. L-694,458 was unstable in rat blood and plasma, degrading via a pathway believed to be catalyzed by B-esterases and to involve cleavage of the beta-lactam ring and loss of the methylpiperazine phenoxy group. In vitro studies indicated that in human liver, L-694,458 was metabolized by CYP3A and 2C isozymes, and in both monkey and human liver microsomes the compound acted as an inhibitor of testosterone 6beta-hydroxylation.
Asunto(s)
Azetidinas/farmacocinética , Inhibidores Enzimáticos/farmacocinética , Elastasa de Leucocito/antagonistas & inhibidores , Piperazinas/farmacocinética , Administración Oral , Animales , Azetidinas/administración & dosificación , Azetidinas/sangre , Disponibilidad Biológica , Sistema Enzimático del Citocromo P-450/metabolismo , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/sangre , Humanos , Isoenzimas/metabolismo , Macaca mulatta , Masculino , Piperazinas/administración & dosificación , Piperazinas/sangre , Ratas , Ratas Sprague-Dawley , Testosterona/metabolismoRESUMEN
The in vitro and in vivo metabolism of N-[1(R)-(1,3-benzodioxol-5-yl)butyl]-3,3-diethyl-2(S)-[4-[(4-methy l-1-piperazinyl)carbonyl]phenoxy]-4-oxo-1-azetidinecarboxamide (L-694,458) was studied in male Sprague-Dawley rats and rhesus monkeys. Analysis by LC-MS/MS and NMR revealed that the major metabolite generated in incubations with rat liver microsomes resulted from N-oxidation of the piperazine group, while the major metabolite generated in monkey liver microsomes was the catechol that resulted from O-dealkylation of the methylenedioxyphenyl group. Other metabolites observed in these incubations include the piperazine N-desmethyl, several monohydroxylated derivatives of the parent compound, and three products that resulted from cleavage of the beta-lactam ring. Incubations of parent compound with rat hepatocytes in culture generated two major metabolites that resulted from cleavage of the piperazine ring with the loss of an ethylene group from one side of the ring; one of these metabolites retained the piperazine N-methyl group, while the other did not. The metabolite profiles in vivo were similar to those observed in vitro, but they were much more complex owing to secondary and, in some cases, tertiary biotransformations of many of the primary metabolites. Bile obtained from orally dosed rats contained more than 40 parent-related components, and many of these metabolites had arisen from piperazine ring cleavage.
Asunto(s)
Azetidinas/química , Cromatografía Liquida/métodos , Elastasa de Leucocito/antagonistas & inhibidores , Espectrometría de Masas/métodos , Piperazinas/química , Administración Oral , Animales , Azetidinas/administración & dosificación , Azetidinas/farmacología , Células Cultivadas , Humanos , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Macaca mulatta , Masculino , Piperazinas/administración & dosificación , Piperazinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The voltage sensitive N-type calcium channel interacts functionally and biochemically with synaptotagmin (p65). N-type channel interaction with p65 is demonstrated in the Xenopus oocyte expression system, where p65 alters the steady state voltage inactivation of the N-channel, and fully restores the syntaxin-modified current amplitude and inactivation kinetics in a calcium dependent manner. In agreement with the functional results, GST-p65 fusion protein binds to a cytosolic region, amino acids 710-1090 of the N-type channel (N-loop(710-1090)). The results of the combined approach provide a functional and biochemical basis for proposing that p65 interaction with the N-type channel brings p65 into a close association with a syntaxin-coupled channel. In turn, calcium entry through the liberated channel initiates fusion of the primed vesicles with the cell membrane at a short distance from the site of calcium entry.