RESUMEN
The goal of this work was to investigate the role of t-tubule (TT) remodeling in abnormal Ca2+ cycling in ventricular myocytes of failing dog hearts. Heart failure (HF) was induced using rapid right ventricular pacing. Extensive changes in echocardiographic parameters, including left and right ventricular dilation and systolic dysfunction, diastolic dysfunction, elevated left ventricular filling pressures, and abnormal cardiac mechanics, indicated that severe HF developed. TT loss was extensive when measured as the density of total cell volume, derived from three-dimensional confocal image analysis, and significantly increased the distances in the cell interior to closest cell membrane. Changes in Ca2+ transients indicated increases in heterogeneity of Ca2+ release along the cell length. When critical properties of Ca2+ release variability were plotted as a function of TT organization, there was a complex, nonlinear relationship between impaired calcium release and decreasing TT organization below a certain threshold of TT organization leading to increased sensitivity in Ca2+ release below a TT density threshold of 1.5%. The loss of TTs was also associated with a greater incidence of triggered Ca2+ waves during rapid pacing. Finally, virtually all of these observations were replicated by acute detubulation by formamide treatment, indicating an important role of TT remodeling in impaired Ca2+ cycling. We conclude that TT remodeling itself is a major contributor to abnormal Ca2+ cycling in HF, reducing myocardial performance. The loss of TTs is also responsible for a greater incidence of triggered Ca2+ waves that may play a role in ventricular arrhythmias arising in HF.NEW & NOTEWORTHY Three-dimensional analysis of t-tubule density showed t-tubule disruption throughout the whole myocyte in failing dog ventricle. A double-linear relationship between Ca2+ release and t-tubule density displays a steeper slope at t-tubule densities below a threshold value (â¼1.5%) above which there is little effect on Ca2+ release (T-tubule reserve). T-tubule loss increases incidence of triggered Ca2+ waves. Chemically induced t-tubule disruption suggests that t-tubule loss alone is a critical component of abnormal Ca2+ cycling in heart failure.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Estimulación Cardíaca Artificial , Modelos Animales de Enfermedad , Perros , Femenino , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Masculino , Miocitos Cardíacos/patología , Función Ventricular Izquierda , Función Ventricular Derecha , Presión VentricularRESUMEN
The sodium channel NaX (encoded by the SCN7A gene) was originally identified in the heart and skeletal muscle and is structurally similar to the other voltage-gated sodium channels but does not appear to be voltage gated. Although NaX is expressed at high levels in cardiac and skeletal muscle, little information exists on the function of NaX in these tissues. Transcriptional profiling of ion channels in the heart in a subset of patients with Brugada syndrome revealed an inverse relationship between the expression of NaX and NaV 1.5 suggesting that, in cardiac myocytes, the expression of these channels may be linked. We propose that NaX plays a role in excitation-contraction coupling based on our experimental observations. Here we show that in cardiac myocytes, NaX is expressed in a striated pattern on the sarcolemma in regions corresponding to the sarcomeric M-line. Knocking down NaX expression decreased NaV 1.5 mRNA and protein and reduced the inward sodium current (INa+ ) following cell depolarization. When the expression of NaV 1.5 was knocked down, ~85% of the INa+ was reduced consistent with the observations that NaV 1.5 is the main voltage-gated sodium channel in cardiac muscle and that NaX likely does not directly participate in mediating the INa+ following depolarization. Silencing NaV 1.5 expression led to significant upregulation of NaX mRNA. Similar to NaV 1.5, NaX protein levels were rapidly downregulated when the intracellular [Ca2+ ] was increased either by CaCl2 or caffeine. These data suggest that a relationship exists between NaX and NaV 1.5 and that NaX may play a role in excitation-contraction coupling.
Asunto(s)
Miocitos Cardíacos/metabolismo , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Síndrome de Brugada/genética , Calcio/metabolismo , Células Cultivadas , Perros , Técnicas de Silenciamiento del Gen , Humanos , Contracción Miocárdica/fisiología , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Ratas , Sarcómeros/metabolismo , Canales de Sodio Activados por Voltaje/genéticaRESUMEN
Mossy fiber synapses act as the critical mediators of highly dynamic communication between hippocampal granule cells in the dentate gyrus and CA3 pyramidal neurons. Excitatory synaptic strength at mossy fiber to CA3 pyramidal cell synapses is potentiated rapidly and reversibly by brief trains of low-frequency stimulation of mossy fiber axons. We show that slight modifications to the pattern of stimulation convert this short-term potentiation into prolonged synaptic strengthening lasting tens of minutes in rodent hippocampal slices. This low-frequency potentiation of mossy fiber EPSCs requires postsynaptic mGlu1 receptors for induction but is expressed presynaptically as an increased release probability and therefore impacts both AMPA and NMDA components of the mossy fiber EPSC. A nonconventional signaling pathway initiated by mGlu1 receptors contributes to induction of plasticity, because EPSC potentiation was prevented by a tyrosine kinase inhibitor and only partially reduced by guanosine 5'-O-(2-thiodiphosphate). A slowly reversible state of enhanced synaptic efficacy could serve as a mechanism for altering the integrative properties of this synapse within a relatively broad temporal window.
Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Fibras Musgosas del Hipocampo/fisiología , Plasticidad Neuronal/fisiología , Receptores de Glutamato Metabotrópico/fisiología , Transducción de Señal/fisiología , Animales , Potenciales Postsinápticos Excitadores/genética , Femenino , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Fibras Musgosas del Hipocampo/metabolismo , Plasticidad Neuronal/genética , Técnicas de Cultivo de Órganos , Terminales Presinápticos/metabolismo , Terminales Presinápticos/fisiología , Receptores de Glutamato Metabotrópico/deficiencia , Receptores de Glutamato Metabotrópico/genética , Transducción de Señal/genéticaRESUMEN
BIN1 (amphyphysin-II) is a structural protein involved in T-tubule (TT) formation and phosphatidylinositol-4,5-bisphosphate (PIP2) is responsible for localization of BIN1 to sarcolemma. The goal of this study was to determine if PIP2-mediated targeting of BIN1 to sarcolemma is compromised during the development of heart failure (HF) and is responsible for TT remodeling. Immunohistochemistry showed co-localization of BIN1, Cav1.2, PIP2, and phospholipase-Cß1 (PLCß1) in TTs in normal rat and human ventricular myocytes. PIP2 levels were reduced in spontaneously hypertensive rats during HF progression compared to age-matched controls. A PIP Strip assay of two native mouse cardiac-specific isoforms of BIN1 including the longest (cardiac BIN1 #4) and shortest (cardiac BIN1 #1) isoforms as well human skeletal BIN1 showed that all bound PIP2. In addition, overexpression of all three BIN1 isoforms caused tubule formation in HL-1 cells. A triple-lysine motif in a short loop segment between two helices was mutated and replaced by negative charges which abolished tubule formation, suggesting a possible location for PIP2 interaction aside from known consensus binding sites. Pharmacological PIP2 depletion in rat ventricular myocytes caused TT loss and was associated with changes in Ca2+ release typically found in myocytes during HF, including a higher variability in release along the cell length and a slowing in rise time, time to peak, and decay time in treated myocytes. These results demonstrate that depletion of PIP2 can lead to TT disruption and suggest that PIP2 interaction with cardiac BIN1 is required for TT maintenance and function.
RESUMEN
BACKGROUND: We have identified a novel form of abnormal Ca2+ wave activity in normal and failing dog atrial myocytes which occurs during the action potential (AP) and is absent during diastole. The goal of this study was to determine if triggered Ca2+ waves affect cellular electrophysiological properties. METHODS: Simultaneous recordings of intracellular Ca2+ and APs allowed measurements of maximum diastolic potential and AP duration during triggered calcium waves (TCWs) in isolated dog atrial myocytes. Computer simulations then explored electrophysiological behavior arising from TCWs at the tissue scale. RESULTS: At 3.3 to 5 Hz, TCWs occurred during the AP and often outlasted several AP cycles. Maximum diastolic potential was reduced, and AP duration was significantly prolonged during TCWs. All electrophysiological responses to TCWs were abolished by SEA0400 and ORM10103, indicating that Na-Ca exchange current caused depolarization. The time constant of recovery from inactivation of Ca2+ current was 40 to 70 ms in atrial myocytes (depending on holding potential) so this current could be responsible for AP activation during depolarization induced by TCWs. Modeling studies demonstrated that the characteristic properties of TCWs are potentially arrhythmogenic by promoting both conduction block and reentry arising from the depolarization induced by TCWs. CONCLUSIONS: Triggered Ca2+ waves activate inward NCX and dramatically reduce atrial maximum diastolic potential and prolong AP duration, establishing the substrate for reentry which could contribute to the initiation and maintenance of atrial arrhythmias.
Asunto(s)
Potenciales de Acción , Arritmias Cardíacas/metabolismo , Señalización del Calcio , Frecuencia Cardíaca , Miocitos Cardíacos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Arritmias Cardíacas/fisiopatología , Simulación por Computador , Diástole , Perros , Femenino , Masculino , Modelos Cardiovasculares , Factores de TiempoRESUMEN
Kainate receptors (KARs) are neuronal proteins that exhibit a highly polarized distribution in the mammalian CNS. Assembly, intracellular trafficking, and synaptic targeting of KARs and other ionotropic glutamate receptors are processes controlled, in part, by various determinants within the constituent subunit proteins themselves. Here, we demonstrate that the linker region between the M3 and S2 domains, which in current structural models is thought to transduce ligand-binding energy into channel opening, additionally has an essential role in receptor biogenesis. Our results show that this gating-associated domain is engaged at two distinct critical stages of KAR biogenesis: first, during the transition from dimeric to tetrameric assembly states and, second, at a postassembly trafficking checkpoint within the endoplasmic reticulum. Alteration of a basic residue, arginine 663, altered the desensitization properties of the GluR6 kainate receptor in response to glutamate application, and these changes were weakly correlated with intracellular retention of the mutant receptors. Elimination of the positive charge also significantly attenuated oligomerization and stability of the intracellular subunit protein. Furthermore, charge swapping with an adjacent residue, glutamate 662, normalized the receptor physiological behavior and reversed the deficits in assembly and degradation, but only partially restored plasma membrane expression of the receptors. These results reveal a new role for this linker domain in glutamate receptor biogenesis and contribute to understanding the cellular controls of receptor assembly and trafficking, which will be important for relating receptor stoichiometry to their neuronal targeting and function.
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Hipocampo/fisiología , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/fisiología , Receptores de Ácido Kaínico/biosíntesis , Animales , Membrana Celular/fisiología , Mutación , Neuronas/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Ácido Kaínico/genética , Receptor de Ácido Kaínico GluK2RESUMEN
BACKGROUND: The peculiarities of transverse tubule (T-tubule) morphology and distribution in the atrium-and how they contribute to excitation-contraction coupling-are just beginning to be understood. OBJECTIVES: The objectives of this study were to determine T-tubule density in the intact, live right and left atria in a large animal and to determine intraregional differences in T-tubule organization within each atrium. METHODS: Using confocal microscopy, T-tubules were imaged in both atria in intact, Langendorf-perfused normal dog hearts loaded with di-4-ANEPPS. T-tubules were imaged in large populations of myocytes from the endocardial surface of each atrium. Computerized data analysis was performed using a new MatLab (Mathworks, Natick, MA) routine, AutoTT. RESULTS: There was a large percentage of myocytes that had no T-tubules in both atria with a higher percentage in the right atrium (25.1%) than in the left atrium (12.5%) (P < .02). The density of transverse and longitudinal T-tubule elements was low in cells that did contain T-tubules, but there were no significant differences in density between the left atrial appendage, the pulmonary vein-posterior left atrium, the right atrial appendage, and the right atrial free wall. In contrast, there were significant differences in sarcomere spacing and cell width between different regions of the atria. CONCLUSION: There is a sparse T-tubule network in atrial myocytes throughout both dog atria, with significant numbers of myocytes in both atria-the right atrium more so than the left atrium-having no T-tubules at all. These regional differences in T-tubule distribution, along with differences in cell width and sarcomere spacing, may have implications for the emergence of substrate for atrial fibrillation.
Asunto(s)
Acoplamiento Excitación-Contracción/fisiología , Atrios Cardíacos , Miocitos Cardíacos/ultraestructura , Animales , Perros , Procesamiento Automatizado de Datos , Atrios Cardíacos/patología , Atrios Cardíacos/ultraestructura , Microscopía Confocal/métodos , Proyectos de Investigación , Sarcómeros/fisiologíaRESUMEN
A highly organized transverse-tubule (TT) system is essential to normal Ca2+ cycling and cardiac function. We explored the relationship between the progressive disruption of TTs and resulting Ca2+ cycling during the development of heart failure (HF). Confocal imaging was used to measure Ca2+ transients and 2-D z-stack images in left ventricular epicardial myocytes of intact hearts from spontaneously hypertensive rats (SHR) and Wistar-Kyoto control rats. TT organization was measured as the organizational index (OI) derived from a fast Fourier transform of TT organization. We found little decrease in the synchrony of Ca2+ release with TT loss until TT remodeling was severe, suggesting a TT "reserve" characterized by a wide range of TT remodeling with little effect on synchrony of release but beyond which variability in release shows an accelerating sensitivity to TT loss. To explain this observation, we applied a computational model of spatially distributed Ca2+ signaling units to investigate the relationship between OI and excitation-contraction coupling. Our model showed that release heterogeneity exhibits a nonlinear relationship on both the spatial distribution of release units and the separation between L-type Ca2+ channels and ryanodine receptors. Our results demonstrate a unique relationship between the synchrony of Ca2+ release and TT organization in myocytes of intact rat ventricle that may contribute to both the compensated and decompensated phases of heart failure.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Insuficiencia Cardíaca/fisiopatología , Ventrículos Cardíacos/fisiopatología , Miocitos Cardíacos/metabolismo , Animales , Progresión de la Enfermedad , Insuficiencia Cardíaca/metabolismo , Ventrículos Cardíacos/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
AIMS: Abnormal intracellular Ca2+ cycling contributes to triggered activity and arrhythmias in the heart. We investigated the properties and underlying mechanisms for systolic triggered Ca2+ waves in left atria from normal and failing dog hearts. METHODS AND RESULTS: Intracellular Ca2+ cycling was studied using confocal microscopy during rapid pacing of atrial myocytes (36 °C) isolated from normal and failing canine hearts (ventricular tachypacing model). In normal atrial myocytes (NAMs), Ca2+ waves developed during rapid pacing at rates ≥ 3.3 Hz and immediately disappeared upon cessation of pacing despite high sarcoplasmic reticulum (SR) load. In heart failure atrial myocytes (HFAMs), triggered Ca2+ waves (TCWs) developed at a higher incidence at slower rates. Because of their timing, TCW development relies upon action potential (AP)-evoked Ca2+ entry. The distribution of Ca2+ wave latencies indicated two populations of waves, with early events representing TCWs and late events representing conventional spontaneous Ca2+ waves. Latency analysis also demonstrated that TCWs arise after junctional Ca2+ release has occurred and spread to non-junctional (cell core) SR. TCWs also occurred in intact dog atrium and in myocytes from humans and pigs. ß-adrenergic stimulation increased Ca2+ release and abolished TCWs in NAMs but was ineffective in HFAMs making this a potentially effective adaptive mechanism in normals but potentially arrhythmogenic in HF. Block of Ca-calmodulin kinase II also abolished TCWs, suggesting a role in TCW formation. Pharmacological manoeuvres that increased Ca2+ release suppressed TCWs as did interventions that decreased Ca2+ release but these also severely reduced excitation-contraction coupling. CONCLUSION: TCWs develop during the atrial AP and thus could affect AP duration, producing repolarization gradients and creating a substrate for reentry, particularly in HF where they develop at slower rates and a higher incidence. TCWs may represent a mechanism for the initiation of atrial fibrillation particularly in HF.
Asunto(s)
Fibrilación Atrial/metabolismo , Señalización del Calcio , Calcio/metabolismo , Atrios Cardíacos/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , Potenciales de Acción , Animales , Antiarrítmicos/farmacología , Fibrilación Atrial/fisiopatología , Fibrilación Atrial/prevención & control , Señalización del Calcio/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Estimulación Cardíaca Artificial , Modelos Animales de Enfermedad , Perros , Acoplamiento Excitación-Contracción , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/fisiopatología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/fisiopatología , Frecuencia Cardíaca , Humanos , Contracción Miocárdica , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Sus scrofa , Factores de TiempoRESUMEN
Several drugs are in clinical use for symptomatic treatment of Alzheimer's disease patients. Since Alzheimer's disease is known to be associated with down-regulation of the cholinergic and N-methyl-D-aspartate (NMDA) systems, most of these drugs inhibit acetylcholinesterase, potentiate the activity of nicotinic acetylcholine receptors (nAChRs), or modulate NMDA receptors. Galantamine is an anticholinesterase and allosterically potentiates the activity of the nicotinic receptors. We have recently found that galantamine potentiates the activity of NMDA receptors as well. Memantine is unique in that it inhibits the NMDA receptors. We have developed a hypothesis that combining galantamine and memantine will be more effective for improving the patient's conditions than monotherapy with either drug. Patch clamp and intracellular Ca(2+) imaging experiments using rat cortical and hippocampal neurons clearly provided the in vitro bases for our hypothesis. Memantine blocked the extrasynaptic NMDA receptor 100 times more potently than the synaptic NMDA receptor at negative membrane potentials and the block of both types of NMDA receptors was attenuated with depolarization. However, galantamine potentiation of the NMDA receptors was not voltage dependent. Thus, co-application of memantine with galantamine prevented the galantamine potentiation and the activation of extrasynaptic NMDA receptors, but membrane depolarization revealed the galantamine potentiation. Therefore, cell death is expected to be prevented by memantine near the resting potential while the NMDA-mediated synaptic transmission, which is down-regulated in the patients, is maintained and potentiated by galantamine. These results provide in vitro bases for the beneficial actions of galantamine and memantine combinations.
Asunto(s)
Inhibidores de la Colinesterasa/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Galantamina/farmacología , Memantina/farmacología , Fármacos Neuroprotectores/farmacología , Agonistas Nicotínicos/farmacología , Receptores de N-Metil-D-Aspartato/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/administración & dosificación , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Potenciales de Acción/efectos de los fármacos , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Bicuculina/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Células Cultivadas/efectos de los fármacos , Corteza Cerebral/citología , Corteza Cerebral/embriología , Inhibidores de la Colinesterasa/administración & dosificación , Cuerpo Estriado/citología , Cuerpo Estriado/embriología , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Galantamina/administración & dosificación , Glicina/farmacología , Técnicas In Vitro , Concentración 50 Inhibidora , Memantina/administración & dosificación , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/fisiología , Fármacos Neuroprotectores/administración & dosificación , Agonistas Nicotínicos/administración & dosificación , Técnicas de Placa-Clamp , Perfusión/instrumentación , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/fisiología , Estricnina/farmacología , Transmisión Sináptica/efectos de los fármacos , Irrigación Terapéutica/instrumentación , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/antagonistas & inhibidoresRESUMEN
The desensitization of alpha-bungarotoxin-insensitive native neuronal nicotinic receptors was studied in rat cortical cell cultures using the patch clamp technique. Thirty-minute perfusions of nicotine reduced currents evoked by short test pulses of 300 microM acetylcholine over a range of 3 to 300 nM, with an IC50 of 51 nM. The time course of desensitization onset was fit by a biexponential function consisting of a fast time constant of about 1 min and a slower component of 6-10 min. The desensitization recovery process was also biexponential and was dominated by a slow time constant of 12-20 min, as well as a minor component of about 1 min. The intracellular dialysis of either the protein kinase C activator phorbol-12-myristate-13 acetate or the phosphatase inhibitor cyclosporin A accelerated the desensitization recovery rate by 2-fold. The data imply that endogenous cortical nicotinic receptor channels may enter one of two desensitization states. The first state (D1) is characterized by rapid entry and recovery, whereas transitions into and out of the second state (D2) occur at slower rates. The D2 receptor state may arise by a sequential transition from the D1 conformation. Protein kinase C activation or phosphatase 2B inhibition may favor the D1 receptor state over that of D2 to promote faster overall rates of desensitization recovery.
Asunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Proteína Quinasa C/metabolismo , Receptores Nicotínicos/fisiología , Acetilcolina/farmacología , Animales , Células Cultivadas , Corteza Cerebral/citología , Ciclosporina/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/fisiología , Nicotina/farmacología , Técnicas de Placa-Clamp , Ésteres del Forbol/farmacología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
While a variety of hypotheses have been proposed for the cause of Alzheimer's disease, our knowledge is far from complete to explain the disease making it difficult to develop the methods for treatment. In the brain of Alzheimer's patients, both neuronal nicotinic acetylcholine (nACh) receptors and NMDA receptors are known to be down-regulated. Thus four anticholinesterases have been developed and approved for the treatment in the U.S.A. However, these are not ideal drugs considering their side effects and limited effectiveness. Nefiracetam is being developed for the treatment of Alzheimer's and other patients with dementia, and has unique actions in potentiating the activity of both nACh and NMDA receptors as demonstrated by in vitro patch clamp experiments using rat cortical neurons in primary culture. Nefiracetam potentiated alpha4beta2-like ACh- and NMDA-induced currents at nanomolar concentrations forming bell-shaped dose-response curves with the maximum potentiation occurring at 1 and 10 nM, respectively. Nefiracetam potentiated nACh receptor currents via G(s) proteins, but not G(i)/G(o) proteins, PKA or PKC. Nefiracetam potentiation of NMDA currents occurred via interactions with the glycine binding site of the NMDA receptor. The nefiracetam potentiation of both nACh and NMDA receptors in a potent and efficacious manner is deemed responsible for its cognitive enhancing action.
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Aconitina/análogos & derivados , Enfermedad de Alzheimer/tratamiento farmacológico , Química Encefálica/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/agonistas , Receptores Nicotínicos/efectos de los fármacos , Acetilcolina/farmacología , Aconitina/farmacología , Animales , Bungarotoxinas/farmacología , Dihidro-beta-Eritroidina/farmacología , Femenino , Proteínas de Unión al GTP/metabolismo , Hexametonio/farmacología , Mecamilamina/farmacología , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Nootrópicos/farmacología , Técnicas de Placa-Clamp , Embarazo , Proteínas Quinasas/metabolismo , Pirrolidinonas/farmacología , RatasRESUMEN
Tetramethylrhodamine methyl ester (TMRM) is a fluorescent dye used to study mitochondrial function in living cells. Previously, we reported that TMRM effectively labeled mitochondria of neurons deep within mouse brain slices. Use of micromolar concentration of dye, which was required to get sufficient staining for two-photon imaging, resulted in typical fluctuations of TMRM. With prolonged exposure, we recorded additional responses in some neurons that included slow oscillations and propagating waves of fluorescence. (Note: We use the terms "fluctuation" to refer to a change in the fluorescent state of an individual mitochondrion, "oscillation" to refer to a localized change in fluorescence in the cytosol, and "wave" to refer to a change in cytosolic fluorescence that propagated within a cell. Use of these terms does not imply any underlying periodicity.) In this report we describe similar results using cultured rat hippocampal neurons. Prolonged exposure of cultures to 2.5 µM TMRM produced a spontaneous increase in fluorescence in some neurons, but not glial cells, after 45-60 minutes that was followed by slow oscillations, waves, and eventually apoptosis. Spontaneous increases in fluorescence were insensitive to high concentrations of FCCP (100 µM) and thapsigargin (10 µM) indicating that they originated, at least in part, from regions outside of mitochondria. The oscillations did not correlate with changes in intracellular Ca(2+), but did correlate with differences in fluorescence lifetime of the dye. Fluorescence lifetime and one-photon ratiometric imaging of TMRM suggested that the spontaneous increase and subsequent oscillations were due to movement of dye between quenched (hydrophobic) and unquenched (hydrophilic) compartments. We propose that these movements may be correlates of intracellular events involved in early stages of apoptosis.
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Hipocampo/citología , Imagenología Tridimensional/métodos , Mitocondrias/metabolismo , Neuronas/metabolismo , Rodaminas/farmacología , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Células Cultivadas , Femenino , Fluorescencia , Ratones , Mitocondrias/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Tapsigargina/farmacología , Factores de TiempoRESUMEN
Proton channels are gated by voltage and pH gradients, and play an important role in the microglial production of pro-inflammatory cytokines, which are known to be suppressed by antidepressants. In the present study we tested the hypothesis that cytokine inhibition by antidepressants is due to an inhibitory action on proton currents by comparing their effects on tumor necrosis factor-α production with the effects on the proton currents in BV2 murine microglial cells. Imipramine, amitriptyline, desipramine and fluoxetine potently and reversibly inhibited proton currents at micromolar concentrations at an intracellular/extracellular pH gradient of 5.5/7.3. Raising extracellular pH to 8.3 sped up the rate and enhanced the extent of block whereas raising intracellular pH to 6.3 reduced the blocking potency of imipramine. These results support a mechanism where the uncharged drug form penetrates the cell membrane, and the charged form blocks the proton channel from the internal side of membrane. This mode of action was corroborated by an experiment with imipraminium, a permanently charged quaternary derivative, which showed far less block compared to imipramine. The lipopolysaccharide-induced release of tumor necrosis factor-α was inhibited by imipramine at concentrations comparable to those inhibiting the proton current. These results support the hypothesis that tumor necrosis factor-α inhibition by imipramine is related to its inhibitory effects on proton channels.
Asunto(s)
Antidepresivos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Microglía/efectos de los fármacos , Protones , Factor de Necrosis Tumoral alfa/metabolismo , Análisis de Varianza , Animales , Biofisica , Línea Celular Transformada , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Concentración de Iones de Hidrógeno , Lipopolisacáridos/farmacología , Potenciales de la Membrana/fisiología , RatonesRESUMEN
BACKGROUND: The gamma-aminobutyric acid-A (GABA(A)) receptor and glutamate receptors are among the most important target sites for the behavioral effects of ethanol. However, data in the literature concerning the ethanol modulation of the GABA(A) and glutamate receptors have been controversial. The activity of the neuronal nicotinic acetylcholine (ACh) receptors (nAChRs) has recently been reported to be potently augmented by ethanol. The activation of nAChRs is also known to cause the release of various neurotransmitters including GABA and glutamate. Thus, ethanol potentiation of nAChRs is expected to stimulate the GABAergic and glutamatergic systems. METHODS: Whole-cell patch clamp experiments were performed using rat cortical neurons in primary culture to record spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and spontaneous miniature excitatory postsynaptic currents (mEPSCs). RESULTS: Two types of neurons were distinguished: bipolar neurons possessed alpha4beta2 nAChRs generating a steady current in response to 30 nM ACh, and multipolar neurons that did not generate a current by ACh application. Acetylcholine greatly increased the frequency of mEPSCs and mIPSCs in bipolar neurons but not in multipolar neurons. The amplitude of neither type of neuron was affected by ACh. Ethanol at 10 to 100 mM suppressed the amplitude of mEPSCs while augmenting the amplitude of mIPSCs in both bipolar and multipolar neurons, indicating the direct action on the respective receptors. In bipolar neurons, ACh plus 100 mM ethanol greatly increased the frequency of mIPSCs beyond the levels achieved by ACh alone, while no such increases were observed in multipolar neurons. CONCLUSIONS: It is concluded that ethanol stimulation of nAChRs modulates the activity of both glutamate and GABA receptors in rat cortical bipolar neurons.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Corteza Cerebral/fisiología , Etanol/farmacología , Neuronas/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Polaridad Celular/efectos de los fármacos , Corteza Cerebral/citología , Interpretación Estadística de Datos , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Técnicas de Placa-Clamp , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
Nicotinic acetylcholine receptors and N-methyl-D-aspartate (NMDA) receptors are known to be down-regulated in the brain of Alzheimer's disease patients. We have previously demonstrated that the nootropic drug nefiracetam potentiates the activity of both nicotinic acetylcholine and NMDA receptors and that nefiracetam modulates the glycine binding site of the NMDA receptor. Because the NMDA receptor is also modulated by Mg2+ and protein kinases, we studied their roles in nefiracetam action on the NMDA receptor by the whole-cell patch-clamp technique and immunoblotting analysis using rat cortical or hippocampal neurons in primary culture. The nefiracetam potentiation of NMDA currents was inhibited by the protein kinase C (PKC) inhibitor chelerythrine, but not by the protein kinase A (PKA) inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89). In immunoblotting analysis, nefiracetam treatment increased the PKCalpha activity with a bell-shaped dose-response relationship peaking at 10 nM, thereby increasing phosphorylation of PKC substrate and NMDA receptor. Such an increase in PKCalpha-mediated phosphorylation was prevented by chelerythine. Nefiracetam treatment did not affect the PKA activity. Analysis of the current-voltage relationships revealed that nefiracetam at 10 nM largely eliminated voltage-dependent Mg2+ block and that this action of nefiracetam was sensitive to PKC inhibition. It was concluded that nefiracetam potentiated NMDA currents not by acting as a partial agonist but by interacting with PKC, allosterically enhancing glycine binding, and attenuating voltage-dependent Mg2+ block.
Asunto(s)
Magnesio/farmacología , Proteína Quinasa C/metabolismo , Pirrolidinonas/farmacología , Receptores de N-Metil-D-Aspartato/agonistas , Regulación Alostérica , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electroquímica , Glicina/metabolismo , N-Metilaspartato , Neuronas/citología , Técnicas de Placa-Clamp , Fosforilación/efectos de los fármacos , Proteína Quinasa C/efectos de los fármacos , Ratas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
The effects of ethanol on the GABA(A) receptors, which are regarded as one of the most important target sites of ethanol, are very controversial, ranging from potentiation to no effect. The delta subunit-containing GABA(A) receptors expressed in Xenopus oocytes were recently reported to be potently augmented by ethanol. We performed patch-clamp experiments using the cerebellar granule cells and mammalian cells expressing recombinant GABA(A) receptors. In granule cells, the sensitivity to GABA increased from 7 to 11 days in vitro. Furosemide, an antagonist of alpha6-containing GABA(A) receptors, inhibited GABA-induced currents more potently at 11 to 14 days than at 7 days. Ethanol at 30 mM had either no effect or an inhibitory effect on currents induced by low concentrations of GABA in granule cells. On alpha4beta2delta, alpha6beta2delta, or alpha6beta3deltaGABA(A) receptors expressed in Chinese hamster ovary cells, ethanol at 10, 30, and 100 mM had either no effect or an inhibitory effect on GABA currents. Ethanol inhibition of GABA(A) receptor was observed in all of the subunit combinations examined. In contrast, the perforated patch-clamp method to record the GABA currents revealed ethanol effects on the alpha6beta2delta subunits ranging from slight potentiation to slight inhibition. Ethanol seems to exert a dual action on the GABA(A) receptors and the potentiating action may depend on intracellular milieu. Thus, the differences between the GABA(A) receptors expressed in mammalian host cells and those in Xenopus oocytes in the response to ethanol might be due to changes in intracellular components under patch-clamp conditions.
Asunto(s)
Cerebelo/efectos de los fármacos , Etanol/farmacología , Receptores de GABA-A/efectos de los fármacos , Animales , Azidas/farmacología , Benzodiazepinas/farmacología , Células CHO , Células Cultivadas , Cricetinae , Furosemida/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/fisiología , Proteínas Recombinantes/efectos de los fármacos , Xenopus , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Nicotinic acetylcholine receptors and N-methyl-D-aspartate (NMDA) receptors are known to be down-regulated in the brain of patients with Alzheimer's disease. It was previously shown that the nootropic drugs nefiracetam and galantamine potentiate the activity of both nicotinic and NMDA receptors. We hypothesized that donepezil, a nootropic with a potent anticholinesterase activity, might also affect the NMDA system. NMDA-induced currents were recorded from rat cortical neurons in primary culture using the whole-cell patch-clamp technique at a holding potential of -70 mV in Mg2+-free solutions. In multipolar neurons, NMDA currents were decreased by bath and U-tube applications of 1 to 10 microM donepezil but were increased by 30 to 100 microM donepezil. Donepezil suppression occurred in a manner independent of NMDA concentrations ranging from 3 to 1000 microM. The donepezil suppression of NMDA currents was prevented by inhibition of protein kinase C (PKC) but unaffected by protein kinase A (PKA) and G proteins. In bipolar neurons, however, NMDA currents were potently augmented by bath and U-tube applications of 0.01 to 100 microM donepezil. Donepezil potentiation occurred at high NMDA concentrations that evoked the saturating responses and in a manner independent of NMDA concentrations ranging from 3 to 1000 microM. The potentiation of NMDA currents by donepezil was decreased by inhibition of PKC and abolished by modulation of G proteins but not by PKA inhibition. It was concluded that donepezil at low therapeutic concentrations (0.01-1 microM) potentiated the activity of the NMDA system and that this action together with cholinesterase inhibition would contribute to the improvement of learning, memory, and cognition in patients with Alzheimer's disease.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Indanos/farmacología , Neuronas/efectos de los fármacos , Nootrópicos/farmacología , Piperidinas/farmacología , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Acetilcolina/farmacología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Donepezilo , Relación Dosis-Respuesta a Droga , Femenino , Proteínas de Unión al GTP/fisiología , Embarazo , Proteína Quinasa C/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
General anesthetics are thought to act on both excitatory and inhibitory neuronal pathways at both post- and presynaptic sites. However, the literature in these regards is somewhat controversial. The aim of the present study was to reassess the relative importance of the various anesthetic actions using a common preparation. Rat cortical neurons in primary culture were used to record spontaneous miniature postsynaptic currents by the whole-cell patch-clamp technique. Halothane at clinically relevant concentrations prolonged the decay phase of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in the presence of tetrodotoxin and at higher concentrations decreased the frequency of mIPSCs. The mIPSC amplitudes underwent little change. Spontaneous action potential-dependent IPSCs recorded in the absence of tetrodotoxin were similarly affected by halothane. Halothane also decreased the frequency of spontaneous miniature non-N-methyl-D-aspartate (NMDA) excitatory postsynaptic currents (mEPSCs) as well as spontaneous action potential-dependent NMDA EPSCs and non-NMDA EPSCs without affecting their decay phase. The halothane effect on mIPSC and mEPSC frequency was dependent on the external calcium concentration. In contrast to halothane, the only effect of propofol was the prolongation of the decay phase of mIPSCs and IPSCs. The prolongation of mIPSCs and IPSCs by halothane and propofol coupled with the ineffectiveness on mEPSCs and EPSCs suggests a selective postsynaptic modulation of GABA(A) receptors. The additional calcium-dependent inhibition of mIPSC and mEPSC frequency by halothane (but not propofol) suggests a more general mechanism by this anesthetic on presynaptic transmitter release.
Asunto(s)
Anestésicos por Inhalación/farmacología , Anestésicos Intravenosos/farmacología , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Halotano/farmacología , Neuronas/efectos de los fármacos , Propofol/farmacología , Transmisión Sináptica/efectos de los fármacos , Anestésicos Locales/farmacología , Animales , Corteza Cerebral/efectos de los fármacos , Electrofisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Técnicas de Placa-Clamp , Embarazo , Terminales Presinápticos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Canales de Sodio/metabolismo , Tetrodotoxina/farmacologíaRESUMEN
No strategies for curing Alzheimer's disease have been developed yet as we do not know the exact cause of the disease. The only therapy that is available for patients is symptomatic treatment. Since Alzheimer's disease is associated with downregulation of the cholinergic system in the brain, its stimulation is expected to improve the patients' cognition, learning, and memory. Four anticholinesterases have been approved in the U.S.A. for the treatment of Alzheimer's disease patients. However, because of the inhibition of cholinesterases, these drugs have side effects and their effectiveness does not last long. Thus new approaches are needed. One approach is to stimulate directly nicotinic acetylcholine (nACh) receptors in the brain, and another is to stimulate NMDA receptors which are also known to be downregulated in Alzheimer's patients. Nefiracetam has been shown to potentiate ACh currents in the alpha4beta2 receptor of rat cortical neurons with a bell-shaped dose-response relationship and the maximum effect at 1 nM. This effect was exerted via G(s) proteins. The alpha7 receptor was almost unaffected by nefiracetam. Nefiracetam also potentiated NMDA currents with the maximum effect at 10 nM via interaction with the glycine-binding site of the receptor. Galantamine had a moderate potentiating effect on the alpha4beta2 receptor and potentiated NMDA currents with the maximum effect at 1 microM. However, galantamine did not interact with the glycine-binding site. Donepezil, a potent anticholinesterase, also potentiated NMDA currents at 1-10000 nM. In conclusion, these three drugs potentiate the activity not only of the cholinergic system but also of the NMDA system, thereby stimulating the downregulated nACh receptors and NMDA receptors to improve patients' learning, cognition, and memory.