RESUMEN
The role of astrocytes in modulating synaptic plasticity is an important question that until recently was not addressed due to limitations of previously existing technology. In the present study, we took an advantage of optogenetics to specifically activate astrocytes in hippocampal slices in order to study effects on synaptic function. Using the AAV-based delivery strategy, we expressed the ionotropic channelrhodopsin-2 (ChR2) or the metabotropic Gq-coupled Opto-a1AR opsins specifically in hippocampal astrocytes to compare different modalities of astrocyte activation. In electrophysiological experiments, we observed a depression of basal field excitatory postsynaptic potentials (fEPSPs) in the CA1 hippocampal layer following light stimulation of astrocytic ChR2. The ChR2-mediated depression increased under simultaneous light and electrical theta-burst stimulation (TBS). Application of the type 2 purinergic receptor antagonist suramin prevented depression of basal synaptic transmission, and switched the ChR2-dependent depression into potentiation. The GABAB receptor antagonist, phaclofen, did not prevent the depression of basal fEPSPs, but switched the ChR2-dependent depression into potentiation comparable to the values for TBS in control slices. In contrast, light stimulation of Opto-a1AR expressed in astrocytes led to an increase in basal fEPSPs, as well as a potentiation of synaptic responses to TBS significantly. A specific blocker of the Gq protein downstream target, the phospholipase C, U73122, completely prevented the effects of Opto-a1AR stimulation on basal fEPSPs or Opto + TBS responses. To understand molecular basis for the observed effects, we performed an analysis of gene expression in these slices using quantitative PCR approach. We observed a significant upregulation of "immediate-early" gene expression in hippocampal slices after light activation of Opto-a1AR-expressing astrocytes alone (cRel, Arc, Fos, JunB, and Egr1) or paired with TBS (cRel, Fos, and Egr1). Activation of ChR2-expressing hippocampal astrocytes was insufficient to affect expression of these genes in our experimental conditions. Thus, we concluded that optostimulation of astrocytes with ChR2 and Opto-a1AR optogenetic tools enables bidirectional modulation of synaptic plasticity and gene expression in hippocampus.
Asunto(s)
Astrocitos , Potenciación a Largo Plazo , Potenciación a Largo Plazo/fisiología , Plasticidad Neuronal , Hipocampo/fisiología , Transmisión Sináptica , Estimulación EléctricaRESUMEN
Proteasomes degrade most intracellular proteins. Several different forms of proteasomes are known. Little is known about the role of specific proteasome forms in the central nervous system (CNS). Inhibitors targeting different proteasome forms are used in clinical practice and were shown to modulate long-term potentiation (LTP) in hippocampal slices of untreated animals. Here, to address the role of non-constitutive proteasomes in hippocampal synaptic plasticity and reveal the consequences of their continuous inhibition, we studied the effect of chronic administration of the non-constitutive proteasome inhibitor ONX-0914 on the LTP induced by two different protocols: tetanic stimulation and theta-burst stimulation (TBS). Both the tetanus- and TBS-evoked potentiation contribute to the different forms of hippocampal-dependent memory and learning. Field-excitatory postsynaptic potentials (fEPSPs) in hippocampal slices from control animals and animals treated with DMSO or ONX-0914 were compared. LTP induced by the TBS was not affected by ONX-0914 administration; however, chronic injections of ONX-0914 led to a decrease in fEPSP slopes after tetanic stimulation. The observed effects correlated with differential expression of genes involved in synaptic plasticity, glutaminergic synapse, and synaptic signaling. Obtained results indicate that non-constitutive proteasomes are likely involved in the tetanus-evoked LTP, but not the LTP occurring after TBS, supporting the relevance and complexity of the role of specific proteasomes in synaptic plasticity, memory, and learning.
Asunto(s)
Potenciación a Largo Plazo , Tétanos , Ratas , Ratones , Animales , Inhibidores de Proteasoma/farmacología , Ratas Sprague-Dawley , Complejo de la Endopetidasa Proteasomal/metabolismo , Tétanos/metabolismo , Hipocampo/metabolismo , Expresión Génica , Glutamatos/metabolismo , Estimulación EléctricaRESUMEN
The search for strategies for strengthening the synaptic efficiency in Aß25-35-treated slices is a challenge for the compensation of amyloidosis-related pathologies. Here, we used the recording of field excitatory postsynaptic potentials (fEPSPs), nitric oxide (NO) imaging, measurements of serine/threonine protein phosphatase (STPP) activity, and the detection of the functional mitochondrial parameters in suspension of brain mitochondria to study the Aß25-35-associated signaling in the hippocampus. Aß25-35 aggregates shifted the kinase-phosphatase balance during the long-term potentiation (LTP) induction in the enhancement of STPP activity. The PP1/PP2A inhibitor, okadaic acid, but not the PP2B blocker, cyclosporin A, prevented Aß25-35-dependent LTP suppression for both simultaneous and delayed enzyme blockade protocols. STPP activity in the Aß25-35-treated slices was upregulated, which is reverted relative to the control values in the presence of PP1/PP2A but not in the presence of the PP2B blocker. A selective inhibitor of stress-induced PP1α, sephin1, but not of the PP2A blocker, cantharidin, is crucial for Aß25-35-mediated LTP suppression prevention. A mitochondrial Na+/Ca2+ exchanger (mNCX) blocker, CGP37157, also attenuated the Aß25-35-induced LTP decline. Aß25-35 aggregates did not change the mitochondrial transmembrane potential or reactive oxygen species (ROS) production but affected the ion transport and Ca2+-dependent swelling of organelles. The staining of hippocampal slices with NO-sensitive fluorescence dye, DAF-FM, showed stimulation of the NO production in the Aß25-35-pretreated slices at the dendrite-containing regions of CA1 and CA3, in the dentate gyrus (DG), and in the CA1/DG somata. NO scavenger, PTIO, or nNOS blockade by selective inhibitor 3Br-7NI partly restored the Aß25-35-induced LTP decline. Thus, hippocampal NO production could be another marker for the impairment of synaptic plasticity in amyloidosis-related states, and kinase-phosphatase balance management could be a promising strategy for the compensation of Aß25-35-driven deteriorations.
Asunto(s)
Amiloidosis , Potenciación a Largo Plazo , Proteínas Amiloidogénicas , Cantaridina , Ciclosporina , Hipocampo/fisiología , Humanos , Potenciación a Largo Plazo/fisiología , Mitocondrias , Óxido Nítrico , Ácido Ocadaico/farmacología , Fosfoproteínas Fosfatasas , Especies Reactivas de Oxígeno , Serina , Intercambiador de Sodio-Calcio , TreoninaRESUMEN
Long-term potentiation (LTP) and long-term depression (LTD) are key forms of synaptic plasticity in the hippocampus. LTP and LTD are believed to underlie the processes occurring during learning and memory. Search of mechanisms responsible for switching from LTP to LTD and vice versa is an important fundamental task. Protein synthesis blockers (PSB) are widely used in models of memory impairment and LTP suppression. Here, we found that blockade of serine/threonine phosphatases 1 (PP1) and 2A (PP2A) with the specific blockers, calyculin A (CalyA) or okadaic acid (OA), and simultaneous blockade of the protein translation by anisomycin or cycloheximide leads to a switch from PSB-impaired LTP to LTD. PP1/PP2A-dependent LTD was extremely sensitive to the intensity of the test stimuli, whose increase restored the field excitatory postsynaptic potentials (fEPSP) to the values corresponding to control LTP in the non-treated slices. PP1/PP2A blockade affected the basal synaptic transmission, increasing the paired-pulse facilitation (PPF) ratio, and restored the PSB-impaired PPF 3 h after tetanus. Prolonged exposure to anisomycin led to the NO synthesis increase (measured using fluorescent dye) both in the dendrites and somata of CA1, CA3, dentate gyrus (DG) hippocampal layers. OA partially prevented the NO production in the CA1 dendrites, as well in the CA3 and DG somas. Direct measurements of changes in serine/threonine phosphatase (STPP) activity revealed importance of the PP1/PP2A-dependent component in the late LTP phase (L-LTP) in anisomycin-treated slices. Thus, serine/threonine phosphatases PP1/PP2A influence both basal synaptic transmission and stimulation-induced synaptic plasticity.
Asunto(s)
Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 2/antagonistas & inhibidores , Inhibidores de la Síntesis de la Proteína/farmacología , Animales , Anisomicina/farmacología , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/fisiología , Cicloheximida/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/fisiología , Masculino , Toxinas Marinas/farmacología , Óxido Nítrico/biosíntesis , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Ratas , Ratas WistarRESUMEN
Protein kinase C is the superfamily of intracellular effector molecules which control crucial cellular functions. Here, we for the first time did the percentage estimation of all known PKC and PKC-related isozymes at the individual cadiomyocyte level. Broad spectrum of PKC transcripts is expressed in the left ventricular myocytes. In addition to the well-known 'heart-specific' PKCα, cardiomyocytes have the high expression levels of PKCN1, PKCδ, PKCD2, PKCε. In general, we detected all PKC isoforms excluding PKCη. In cardiomyocytes PKC activity tonically regulates voltage-gated Ca2+-currents, intracellular Ca2+ level and nitric oxide (NO) production. Imidazoline receptor of the first type (I1R)-mediated induction of the PKC activity positively modulates Ca2+ release through ryanodine receptor (RyR), increasing the Ca2+ leakage in the cytosol. In cardiomyocytes with the Ca2+-overloaded regions of > 9-10 µm size, the local PKC-induced Ca2+ signaling is transformed to global accompanied by spontaneous Ca2+ waves propagation across the entire cell perimeter. Such switching of Ca2+ signaling in cardiac cells can be important for the development of several cardiovascular pathologies and/or myocardial plasticity at the cardiomyocyte level.
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Señalización del Calcio , Miocitos Cardíacos/enzimología , Proteína Quinasa C/metabolismo , Animales , Isoenzimas/metabolismo , Masculino , Ratas , Ratas Wistar , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Dephosphorylation of target proteins at serine/threonine residues is one of the most crucial mechanisms regulating their activity and, consequently, the cellular functions. The role of phosphatases in synaptic plasticity, especially in long-term depression or depotentiation, has been reported. We studied serine/threonine phosphatase activity during the protein synthesis blocker (PSB)-induced impairment of long-term potentiation (LTP). Established protein phosphatase 2B (PP2B, calcineurin) inhibitor cyclosporin A prevented the LTP early phase (E-LTP) decline produced by pretreatment of hippocampal slices with cycloheximide or anisomycin. For the first time, we directly measured serine/threonine phosphatase activity during E-LTP, and its significant increase in PSB-treated slices was demonstrated. Nitric oxide (NO) donor SNAP also heightened phosphatase activity in the same manner as PSB, and simultaneous application of anisomycin + SNAP had no synergistic effect. Direct measurement of the NO production in hippocampal slices by the NO-specific fluorescent probe DAF-FM revealed that PSBs strongly stimulate the NO concentration in all studied brain areas: CA1, CA3, and dentate gyrus (DG). Cyclosporin A fully abolished the PSB-induced NO production in the hippocampus, suggesting a close relationship between nNOS and PP2B activity. Surprisingly, cyclosporin A alone impaired short-term plasticity in CA1 by decreasing paired-pulse facilitation, which suggests bi-directionality of the influences of PP2B in the hippocampus. In conclusion, we proposed a minimal model of signaling events that occur during LTP induction in normal conditions and the PSB-treated slices.
Asunto(s)
Región CA1 Hipocampal/metabolismo , Región CA3 Hipocampal/metabolismo , Calcineurina/genética , Potenciación a Largo Plazo/genética , Potenciales Sinápticos/genética , Animales , Anisomicina/farmacología , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/efectos de los fármacos , Región CA3 Hipocampal/citología , Región CA3 Hipocampal/efectos de los fármacos , Calcineurina/metabolismo , Inhibidores de la Calcineurina/farmacología , Cicloheximida/farmacología , Ciclosporina/farmacología , Giro Dentado/citología , Giro Dentado/efectos de los fármacos , Giro Dentado/metabolismo , Regulación de la Expresión Génica , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Microtomía , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/genética , Óxido Nítrico/química , Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas Wistar , S-Nitroso-N-Acetilpenicilamina/química , S-Nitroso-N-Acetilpenicilamina/farmacología , Potenciales Sinápticos/efectos de los fármacos , Técnicas de Cultivo de TejidosRESUMEN
Spontaneous Ca2+-transient (wave) generation in isolated cardiomyocytes is well established phenomenon which poses a lot of questions about myocardial excitability. Current studies of spontaneous Ca2+-activity in cardiac cells mainly relate to the kinetic characteristics, classification and simulation of Ca2+-events through ryanodine receptor (RyR) activity modeling. Here, for the first time we pay attention to the Ca2+-transients having stationary kinetics for correct estimation of the sarcoplasmic reticulum Ca2+ transport. In cardiomyocytes generating such type of Ca2+-transients, the averaged intracellular calcium ([Ca2+]in) fluorescence practically does not change in time. Stationary Ca2+-transients are observed in different animal models (Wistar, SHR, ground squirrels) revealing a common cardiomyocyte phenomenon. They somewhat depend on external Ca2+ ([Ca2+]ex) because the [Ca2+]ex lowering to 1 µM in the presence of EGTA disrupts Ca2+-wave propagation. At the same time, spontaneous Ca2+-transients do not associated with the forward or reverse mode of Na+/Ca2+ exchanger (NCX), but partially modulated by the L-type Ca2+-channels. Among the sarcoplasmic reticulum targets, RyR and SERCA are crucial for Ca2+-wave generation and sustained self-oscillation activity. Analysis of the spontaneous wave kinetics reveals that both slopes of the rising wave front and the wave front decline are gradually changed during propagation, which well correlates with the RyR and SERCA activity, respectively. On the contrary, in the electrical field-stimulated myocytes, both slope factors are sharply changed corresponding to 'all-or-nothing' rule, which is fundamental principle for action potential in cardiomyocytes. Furthermore, stimulation of single cardiomyocyte using local electrode appears the deterioration in the [Ca2+]in utilization from the cytosol, which limits the time of observation during the protocol. Obtained data suggest that stationary spontaneous Ca2+-transients occurring without actual myocellular excitation represent useful and precise tools for estimation of the sarcoplasmic reticulum Ca2+-transport.
Asunto(s)
Calcio/metabolismo , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Potenciales Evocados , Transporte Iónico , Ratas , Ratas Endogámicas SHR , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.
Asunto(s)
Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas/prevención & control , alfa-Sinucleína/química , Algoritmos , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Productos Biológicos/química , Productos Biológicos/farmacología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Línea Celular Tumoral , Colestanoles/química , Colestanoles/farmacología , Humanos , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Estructura Molecular , Neuroblastoma/metabolismo , Neuroblastoma/patología , Paresia/genética , Paresia/metabolismo , Paresia/prevención & control , Enfermedad de Parkinson/metabolismo , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is an imaging approach that enables analysis of the 3D architecture of cells and tissues at resolutions that are 1-2 orders of magnitude higher than that possible with light microscopy. The slow speeds of data collection and manual segmentation are two critical problems that limit the more extensive use of FIB-SEM technology. Here, we present an easily accessible robust method that enables rapid, large-scale acquisition of data from tissue specimens, combined with an approach for semi-automated data segmentation using the open-source machine learning Weka segmentation software, which dramatically increases the speed of image analysis. We demonstrate the feasibility of these methods through the 3D analysis of human muscle tissue by showing that our process results in an improvement in speed of up to three orders of magnitude as compared to manual approaches for data segmentation. All programs and scripts we use are open source and are immediately available for use by others.
Asunto(s)
Imagenología Tridimensional/métodos , Microscopía Electrónica de Rastreo/métodos , Músculo Esquelético/diagnóstico por imagen , Humanos , Aprendizaje Automático , Programas Informáticos , Factores de TiempoRESUMEN
Imidazoline receptor of the first type (I1R) in addition to the established inhibition of sympathetic neurons may mediate the direct control of myocellular functions. Earlier, we revealed that I1-mediated signaling in the normotensive rat cardiomyocytes suppresses the nitric oxide production by endothelial NO synthase, impairs sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity, and elevates intracellular calcium in the cytosol. Also, I1-agonists counteract ß-adrenoceptor stimulation effects in respect to voltage-gated calcium currents. This study ascertains the I1R signal transduction in the normotensive Wistar and SHR cardiomyocytes. Reduction of Ca2+-currents by rilmenidine, a specific agonist of I1R, ensued from the phosphatidylcholine-specific phospholipase C-mediated activation of protein kinase C. There is a stimulation of serine/threonine phosphatase activity. In SHR cardiomyocytes, both the rilmenidine, and putative endogenous ligand, agmatine, almost twofold less effectively reduced L-type of Ca2+-currents. Average mRNA level of Nischarin, established functional component of I1R, is slightly decreased in SHR, as well as the intracellular Nischarin pool immunolabeled in the cytosol of SHR cardiomyocytes. Disturbance of I1R signal transduction in SHR may aggravate the development of this cardiovascular pathology.
Asunto(s)
Receptores de Imidazolina/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Canales de Calcio Tipo L/metabolismo , Relación Dosis-Respuesta a Droga , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Toxinas Marinas , Oxazoles/farmacología , ARN Mensajero/metabolismo , Ratas Endogámicas SHR , Ratas Wistar , Rilmenidina/farmacologíaRESUMEN
AIMS: to investigate α2-AR subtype distribution and the relationship between receptor amounts and their functionality in normotensive and spontaneously hypertensive rats. METHODS: experiments were performed on left ventricular cardiomyocytes isolated from Wistar rats and SHR (2-2.5 months). Molecular routine tools (RT-PCR, Western blotting, immunocytochemistry) were used for semi-quantitative estimation of α2-AR subtypes. Fluorescence of both the Ca2+-dependent and NO-sensitive probes were used to define functionality of α2-AR, evaluated by changes in the dynamics of spontaneous Ca2+-transients and NO production in cardiomyocytes in response to the α2-AR agonist application. RESULTS: percentage of the three known α2-AR subtypes in Wistar and SHR cardiomyocytes is not principally different. Total amounts of α2A-AR subtype in SHR increases, for both the sarcolemmal and intracellular receptor pools. Total number of α2B-AR is also significantly higher in hypertensive rats with an increase in the sarcolemmal, but not the intracellular immunoreactivity. For α2C-AR subtype, no significant differences between Wistar and SHR were identified, despite the fact that its amounts in cardiomyocytes are somewhat higher than the other two subtypes. Notwithstanding the increased expression of α2-AR subtypes in SHR, α2-AR-agonist guanabenz was ineffective in suppression of spontaneous Ca2+-transients, as well as the lowering of free calcium levels in the cytosol. Guanabenz-induced NO synthesis is well correlated with the Ca2+-loading into sarcoplasmic reticulum and actually decreased in SHR cardiomyocytes. CONCLUSION: data indicate α2-AR dysfunction and ineffectiveness of α2-AR-mediated signaling pathways in this model of cardiovascular pathologies. Results can be used for clinical practice for more effective control of cardiovascular functions in various disease states.
Asunto(s)
Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Animales , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Guanabenzo/farmacología , Masculino , Óxido Nítrico/metabolismo , Ratas Endogámicas SHR , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Intracellular Local Ca releases (LCRs) from sarcoplasmic reticulum (SR) regulate cardiac pacemaker cell function by activation of electrogenic Na/Ca exchanger (NCX) during diastole. Prior studies demonstrated the existence of powerful compensatory mechanisms of LCR regulation via a complex local cross-talk of Ca pump, release and NCX. One major obstacle to study these mechanisms is that LCR exhibit complex Ca release propagation patterns (including merges and separations) that have not been characterized. Here we developed new terminology, classification, and computer algorithms for automatic detection of numerically simulated LCRs and examined LCR regulation by SR Ca pumping rate (Pup) that provides a major contribution to fight-or-flight response. In our simulations the faster SR Ca pumping accelerates action potential-induced Ca transient decay and quickly clears Ca under the cell membrane in diastole, preventing premature releases. Then the SR generates an earlier, more synchronized, and stronger diastolic LCR signal activating an earlier and larger inward NCX current. LCRs at higher Pup exhibit larger amplitudes and faster propagation with more collisions to each other. The LCRs overlap with Ca transient decay, causing an elevation of the average diastolic [Ca] nadir to ~200 nM (at Pup = 24 mM/s). Background Ca (in locations lacking LCRs) quickly decays to resting Ca levels (<100 nM) at high Pup, but remained elevated during slower decay at low Pup. Release propagation is facilitated at higher Pup by a larger LCR amplitude, whereas at low Pup by higher background Ca. While at low Pup LCRs show smaller amplitudes, their larger durations and sizes combined with longer transient decay stabilize integrals of diastolic Ca and NCX current signals. Thus, the local interplay of SR Ca pump and release channels regulates LCRs and Ca transient decay to insure fail-safe pacemaker cell operation within a wide range of rates.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Modelos Cardiovasculares , Miocitos Cardíacos/metabolismo , Nodo Sinoatrial/metabolismo , Animales , Calcio/análisis , Señalización del Calcio/fisiología , Simulación por ComputadorRESUMEN
Sustained cardiac adrenergic stimulation has been implicated in the development of heart failure and ventricular dysrhythmia. Conventionally, α2 adrenoceptors (α2-AR) have been assigned to a sympathetic short-loop feedback aimed at attenuating catecholamine release. We have recently revealed the expression of α2-AR in the sarcolemma of cardiomyocytes and identified the ability of α2-AR signaling to suppress spontaneous Ca2+ transients through nitric oxide (NO) dependent pathways. Herein, patch-clamp measurements and serine/threonine phosphatase assay revealed that, in isolated rat cardiomyocytes, activation of α2-AR suppressed L-type Ca2+ current (ICaL) via stimulation of NO synthesis and protein kinase G- (PKG) dependent activation of phosphatase reactions, counteracting isoproterenol-induced ß-adrenergic activation. Under stimulation with norepinephrine (NE), an agonist of ß- and α-adrenoceptors, the α2-AR antagonist yohimbine substantially elevated ICaL at NE levels >10nM. Concomitantly, yohimbine potentiated triggered intracellular Ca2+ dynamics and contractility of cardiac papillary muscles. Therefore, in addition to the α2-AR-mediated feedback suppression of sympathetic and adrenal catecholamine release, α2-AR in cardiomyocytes can govern a previously unrecognized local cardiomyocyte-delimited stress-reactive signaling pathway. We suggest that such aberrant α2-AR signaling may contribute to the development of cardiomyopathy under sustained sympathetic drive. Indeed, in cardiomyocytes of spontaneously hypertensive rats (SHR), an established model of cardiac hypertrophy, α2-AR signaling was dramatically reduced despite increased α2-AR mRNA levels compared to normal cardiomyocytes. Thus, targeting α2-AR signaling mechanisms in cardiomyocytes may find implications in medical strategies against maladaptive cardiac remodeling associated with chronic sympathoadrenal stimulation.
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Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Sarcolema/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Cardiomegalia/metabolismo , Cardiomegalia/patología , Cardiomegalia/fisiopatología , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Masculino , Contracción Miocárdica , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Óxido Nítrico/metabolismo , Proteína Fosfatasa 2/metabolismo , Ratas , Ratas Endogámicas SHR , Receptores de Neuropéptido Y/agonistas , Receptores de Neuropéptido Y/metabolismo , Sarcolema/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
The fifth-generation mobile networks (5G) will not only enhance mobile broadband services, but also enable connectivity for a massive number of Internet-of-Things devices, such as wireless sensors, meters or actuators. Thus, 5G is expected to achieve a 1000-fold or more increase in capacity over 4G. The use of the millimeter-wave (mmWave) spectrum is a key enabler to allowing 5G to achieve such enhancement in capacity. To fully utilize the mmWave spectrum, 5G is expected to adopt a heterogeneous network (HetNet) architecture, wherein mmWave small cells are overlaid onto a conventional macro-cellular network. In the mmWave-integrated HetNet, splitting of the control plane (CP) and user plane (UP) will allow continuous connectivity and increase the capacity of the mmWave small cells. mmWave communication can be used not only for access linking, but also for wireless backhaul linking, which will facilitate the installation of mmWave small cells. In this study, a proof-of-concept (PoC) was conducted to demonstrate the practicality of a prototype mmWave-integrated HetNet, using mmWave technologies for both backhaul and access.
RESUMEN
Evidence suggests that intracellular Ca(2+) levels and contractility of cardiomyocytes can be modulated by targeting receptors other than already identified adrenergic or non-adrenergic sarcolemmal receptors. This study uncovers the presence in myocardial cells of adrenergic α2 (α2-AR) and imidazoline I1 (I1R) receptors. In isolated left ventricular myocytes generating stationary spontaneous Ca(2+) transients in the absence of triggered action potentials, the prototypic agonist of both receptors agmatine can activate corresponding signaling cascades with opposing outcomes on nitric oxide (NO) synthesis and intracellular Ca(2+) handling. Specifically, activation of α2-AR signaling through PI3 kinase and Akt/protein kinase B stimulates NO production and abolishes Ca(2+) transients, while targeting of I1R signaling via phosphatidylcholine-specific phospholipase C (PC-PLC) and protein kinase C (PKC) suppresses NO synthesis and elevates averaged intracellular Ca(2+). We identified that endothelial NO synthase (eNOS) is a major effector for both signaling cascades. According to the established eNOS transitions between active (Akt-dependent) and inactive (PKC-dependent) conformations, we suggest that balance between α2-AR and I1R signaling pathways sets eNOS activity, which by defining operational states of myocellular sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) can adjust Ca(2+) re-uptake and thereby cardiac inotropy. These results indicate that the conventional catalog of cardiomyocyte sarcolemmal receptors should be expanded by the α2-AR and I1R populations, unveiling previously unrecognized targets for endogenous ligands as well as for existing and potential pharmacological agents in cardiovascular medicine.
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Señalización del Calcio , Receptores de Imidazolina/metabolismo , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Agmatina/farmacología , Animales , Benzofuranos/farmacología , Células Cultivadas , Imidazoles/farmacología , Receptores de Imidazolina/agonistas , Receptores de Imidazolina/antagonistas & inhibidores , Miocitos Cardíacos/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
A number of surface proteins specific to pathogenic strains of Leptospira have been identified. The Lig protein family has shown promise as a marker in typing leptospiral isolates for pathogenesis and as an antigen in vaccines. We used NMR spectroscopy to solve the solution structure of the twelfth immunoglobulin-like (Ig-like) repeat domain from LigB (LigB-12). The fold is similar to that of other bacterial Ig-like domains and comprised mainly of ß-strands that form a ß-sandwich based on a Greek-key folding arrangement. Based on sequence analysis and conservation of structurally important residues, homology models for the other LigB Ig-like domains were generated. The set of LigB models illustrates the electrostatic differences between the domains as well as the possible interactions between neighboring domains. Understanding the structure of the extracellular portion of LigB and related proteins is important for developing diagnostic methods and new therapeutics directed toward leptospirosis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Leptospira/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
The antibiotic squalamine forms a lyotropic liquid crystal at very low concentrations in water (0.3-3.5% w/v), which remains stable over a wide range of temperature (1-40 °C) and pH (4-8). Squalamine is positively charged, and comparison of the alignment of ubiquitin relative to 36 previously reported alignment conditions shows that it differs substantially from most of these, but is closest to liquid crystalline cetyl pyridinium bromide. High precision residual dipolar couplings (RDCs) measured for the backbone (1)H-(15)N, (15)N-(13)C', (1)H(α)-(13)C(α), and (13)C'-(13)C(α) one-bond interactions in the squalamine medium fit well to the static structural model previously derived from NMR data. Inclusion into the structure refinement procedure of these RDCs, together with (1)H-(15)N and (1)H(α)-(13)C(α) RDCs newly measured in Pf1, results in improved agreement between alignment-induced changes in (13)C' chemical shift, (3)JHNHα values, and (13)C(α)-(13)C(ß) RDCs and corresponding values predicted by the structure, thereby validating the high quality of the single-conformer structural model. This result indicates that fitting of a single model to experimental data provides a better description of the average conformation than does averaging over previously reported NMR-derived ensemble representations. The latter can capture dynamic aspects of a protein, thus making the two representations valuable complements to one another.
Asunto(s)
Antibacterianos/química , Cristales Líquidos/química , Ubiquitina/química , Colestanoles/química , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación ProteicaRESUMEN
The effect of plant-based fillers on thermal resistance in highly filled biocomposites based on ethylene-vinyl acetate copolymer (EVA) was studied. Wood flour and microcrystalline cellulose were used as fillers. It was shown that the introduction of microcrystalline cellulose into EVA did not affect the thermal stability of the polymer matrix. In contrast, the introduction of wood flour into EVA led to a significant increase in the thermal stability of the entire biocomposite. Oxidation induction time increased from 0 (pure EVA) to 73 min (EVA + wood flour biocomposites). The low-molecular weight phenolic compounds contained in wood flour are likely able to diffuse into the polymer matrix, exerting a stabilizing effect. The discovered stabilizing effect is a positive development for expanding the possibilities of technological processing of biocomposites, including multiple processing.
RESUMEN
Calmodulin (CaM) is a calcium binding protein that plays numerous roles in Ca-dependent cellular processes, including uptake and release of neurotransmitters in neurons. α-Synuclein (α-syn), one of the most abundant proteins in central nervous system neurons, helps maintain presynaptic vesicles containing neurotransmitters and moderates their Ca-dependent release into the synapse. Ca-Bound CaM interacts with α-syn most strongly at its N-terminus. The N-terminal region of α-syn is important for membrane binding; thus, CaM could modulate membrane association of α-syn in a Ca-dependent manner. In contrast, Ca-free CaM has negligible interaction. The interaction with CaM leads to significant signal broadening in both CaM and α-syn NMR spectra, most likely due to conformational exchange. The broadening is much reduced when binding a peptide consisting of the first 19 residues of α-syn. In neurons, most α-syn is acetylated at the N-terminus, and acetylation leads to a 10-fold increase in binding strength for the α-syn peptide (KD = 35 ± 10 µM). The N-terminally acetylated peptide adopts a helical structure at the N-terminus with the acetyl group contacting the N-terminal domain of CaM and with less ordered helical structure toward the C-terminus of the peptide contacting the CaM C-terminal domain. Comparison with known structures shows that the CaM/α-syn complex most closely resembles Ca-bound CaM in a complex with an IQ motif peptide. However, a search comparing the α-syn peptide sequence with known CaM targets, including IQ motifs, found no homologies; thus, the N-terminal α-syn CaM binding site appears to be a novel CaM target sequence.
Asunto(s)
Calmodulina/química , alfa-Sinucleína/química , Sitios de Unión , Calmodulina/metabolismo , Humanos , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Pliegue de Proteína , alfa-Sinucleína/metabolismoRESUMEN
α-Synuclein (αS) is an intrinsically disordered protein that is water-soluble but also can bind negatively charged lipid membranes while adopting an α-helical conformation. Membrane affinity is increased by post-translational N-terminal acetylation, a common modification in all eukaryotic cells. In the presence of lipid vesicles containing a small fraction of peroxidized lipids, the N-terminal Met residues in αS (Met1 and Met5) rapidly oxidize while reducing the toxic lipid hydroperoxide to a nonreactive lipid hydroxide, whereas C-terminal Met residues remain unaffected. Met oxidation can be probed conveniently and quantitatively by NMR spectroscopy. The results show that oxidation of Met1 reduces the rate of oxidation of Met5 and vice versa as a result of decreased membrane affinity of the partially oxidized protein. The effect of Met oxidation on the αS-membrane affinity extends over large distances, as in the V49M mutant, oxidation of Met1 and Met5 strongly impacts the oxidation rate of Met49 and vice versa. When not bound to membrane, oxidized Met1 and Met5 of αS are excellent substrates for methionine sulfoxide reductase (Msr), thereby providing an efficient vehicle for water-soluble Msr enzymes to protect the membrane against oxidative damage.