RESUMEN
The open-channel block of N-methyl-D-aspartate receptors (NMDARs) and their calcium-dependent desensitization (CDD) represent conventional mechanisms of glutamatergic synapse regulation. In neurotrauma, neurodegeneration, and neuropathic pain the clinical benefits of cure with memantine, ketamine, Mg2+, and some tricyclic antidepressants are often attributed to NMDAR open-channel block, while possible involvement of NMDAR CDD in the therapy is not well established. Here the effects of selective high-affinity sodium-calcium exchanger (NCX) isoform 1 inhibitor, SEA0400, on NMDA-activated whole-cell currents and their block by amitriptyline, desipramine and clomipramine recorded by patch-clamp technique in cortical neurons of primary culture were studied. We demonstrated that in the presence of extracellular Ca2+, 50 nM SEA0400 caused a reversible decrease of the steady-state amplitude of NMDAR currents, whereas loading neurons with BAPTA or the removal of extracellular Ca2+ abolished the effect. The decrease did not exceed 30% of the amplitude and did not depend on membrane voltage. The external Mg2+ block and 50 nM SEA0400 inhibition of currents were additive, suggesting their independent modes of action. In the presence of Ca2+ SEA0400 speeded up the decay of NMDAR currents to the steady state determined by CDD. The measured IC50 value of 27 nM for SEA0400-induced inhibition coincides with that for NCX1. Presumably, SEA0400 effects are induced by an enhancement of NMDAR CDD through the inhibition of Ca2+ extrusion by NCX1. SEA0400, in addition, at nanomolar concentrations could interfere with Ca2+-dependent effect of tricyclic antidepressants. In the presence of 50 nM SEA0400, the IC50s for NMDAR inhibition by amitriptyline and desipramine increased by about 20 folds, as the Ca2+-dependent NMDAR inhibition disappeared. This observation highlights NCX1 involvement in amitriptyline and desipramine effects on NMDARs and unmasks competitive relationships between SEA0400 and these antidepressants. Neither amitriptyline nor desipramine could affect NCX3. The open-channel block of NMDARs by these substances was not affected by SEA0400. In agreement, SEA0400 did not change the IC50 for clomipramine, which acts as a pure NMDAR open-channel blocker. Thus, NCX seems to represent a promising molecular target to treat neurological disorders, because of the ability to modulate NMDARs by decreasing the open probability through the enhancement of their CDD.
RESUMEN
Na/K-ATPase maintains transmembrane ionic gradients and acts as a signal transducer when bound to endogenous cardiotonic steroids. At subnanomolar concentrations, ouabain induces neuroprotection against calcium overload and apoptosis of neurons during excitotoxic stress. Here, the role of lipid rafts in interactions between Na/K-ATPase, sodium-calcium exchanger (NCX), and N-methy-D-aspartate receptors (NMDARs) was investigated. We analyzed 0.5-1-nanometer ouabain's effects on calcium responses and miniature post-synaptic current (mEPSCs) frequencies of cortical neurons during the action of NMDA in rat primary culture and brain slices. In both objects, ouabain attenuated NMDA-evoked calcium responses and prevented an increase in mEPSC frequency, while the cholesterol extraction by methyl-ß-cyclodextrin prevented the effects. The data support the conclusions that (i) ouabain-induced inhibition of NMDA-elicited calcium response involves both pre- and post-synapse, (ii) the presence of astrocytes in the tripartite synapse is not critical for the ouabain effects, which are found to be similar in cell cultures and brain slices, and (iii) ouabain action requires the integrity of cholesterol-rich membrane microdomains in which the colocalization and functional interaction of NMDAR-transferred calcium influx, calcium extrusion by NCX, and Na/K-ATPase modulation of the exchanger occur. This regulation of the molecules by cardiotonic steroids may influence synaptic transmission, prevent excitotoxic neuronal death, and interfere with the pharmacological actions of neurological medicines.
Asunto(s)
Calcio , Ouabaína , Ratas , Animales , Ouabaína/farmacología , Calcio/metabolismo , N-Metilaspartato/farmacología , N-Metilaspartato/metabolismo , Neuronas/metabolismo , Colesterol/metabolismo , Adenosina Trifosfatasas/metabolismoRESUMEN
The facilitated activity of N-methyl-D-aspartate receptors (NMDARs) in the central and peripheral nervous systems promotes neuropathic pain. Amitriptyline (ATL) and desipramine (DES) are tricyclic antidepressants (TCAs) whose anti-NMDAR properties contribute to their analgetic effects. At therapeutic concentrations <1 µM, these medicines inhibit NMDARs by enhancing their calcium-dependent desensitization (CDD). Li+, which suppresses the sodium−calcium exchanger (NCX) and enhances NMDAR CDD, also exhibits analgesia. Here, the effects of different [Li+]s on TCA inhibition of currents through native NMDARs in rat cortical neurons recorded by the patch-clamp technique were investigated. We demonstrated that the therapeutic [Li+]s of 0.5−1 mM cause an increase in ATL and DES IC50s of ~10 folds and ~4 folds, respectively, for the Ca2+-dependent NMDAR inhibition. The Ca2+-resistant component of NMDAR inhibition by TCAs, the open-channel block, was not affected by Li+. In agreement, clomipramine providing exclusively the NMDAR open-channel block is not sensitive to Li+. This Ca2+-dependent interplay between Li+, ATL, and DES could be determined by their competition for the same molecular target. Thus, submillimolar [Li+]s may weaken ATL and DES effects during combined therapy. The data suggest that Li+, ATL, and DES can enhance NMDAR CDD through NCX inhibition. This ability implies a drug−drug or ion−drug interaction when these medicines are used together therapeutically.
Asunto(s)
Amitriptilina , Antidepresivos Tricíclicos , Ratas , Animales , Antidepresivos Tricíclicos/farmacología , Amitriptilina/farmacología , Receptores de N-Metil-D-Aspartato , Litio/farmacología , Calcio/metabolismo , Desipramina/farmacología , Calcio de la DietaRESUMEN
It is known that overexpression of N-methyl-D-aspartate receptors (NMDARs) contributes to central sensitization and development of neuropathic pain. Tricyclic antidepressants (TCAs), amitriptyline (ATL), and desipramine (DES) exhibit analgetic anti-NMDAR activity and are commonly utilized for pain therapy. This property is determined by their ability to enhance the calcium-dependent desensitization (CDD) of NMDARs. Coincidently ethanol and cholesterol, the ubiquitous food supplements, also modulate NMDAR CDD. The convergence of the effects of these compounds on a similar calcium-dependent process allows to assume their interaction on NMDARs. Since there is no information on whether ethanol supplementation and cholesterol deficit interfere with TCA inhibition of NMDARs at a cellular level, here we investigated this issue. Whole-cell NMDA-activated currents were recorded in rat cortical neurons of primary cultures to study how the IC50 values for TCA inhibition of NMDARs are influenced by ethanol and cholesterol extraction from the plasma membrane with methyl-ß-cyclodextrin. Ethanol at 0.03% did not reliably affect the steady-state NMDA-activated currents. At this threshold concentration ethanol, however, increased IC50s for ATL and DES abolishing their calcium-dependent inhibition of NMDARs but did not change IC50 for clomipramine (CLO), which is calcium-independent. Whereas the ethanol effects on ATL-induced NMDAR inhibition reached a maximum at 2 mM external [Ca2+], for DES the maximum was achieved already at 1 mM external [Ca2+], that correlates with the manifestation of the calcium-dependent inhibition of NMDARs by these agents. Cholesterol depletion also increased IC50s for both ATL and DES abolishing the calcium-dependent inhibition of NMDARs. The restitution of cholesterol in the plasma membrane reversed the ATL IC50 back to the low values, by a restoration of calcium-dependence of ATL. These observations are consistent with the explanation that either 0.03% ethanol or cholesterol extraction may interrupt some intermediate step of CDD transduction or augment NMDAR CDD to the maximal level so that ATL and DES could not further enhance CDD. It is likely that anti-NMDAR action of ATL and DES against neuropathic pain could demonstrate peculiarities in therapeutic profiles during cholesterol decline in aging or medical treatments and ethanol supplementations even in quantities that are insufficient to cause the symptoms of intoxication.
RESUMEN
N-methyl-D-aspartate receptors (NMDARs) are an essential target for the analgetic action of tricyclic antidepressants (TCAs). Their therapeutic blood concentrations achieve 0.5-1.5 µM, which, however, are insufficient to cause in vitro the open-channel block known as the only effect of TCAs on NMDARs. Whereas structures of amitriptyline (ATL), desipramine (DES), and clomipramine (CLO) are rather similar these compounds manifest different therapeutic profiles and side effects. To study structure-activity relationships of DES and CLO on NMDARs, we measured IC50s as a function of extracellular calcium ([Ca2+]) and membrane voltage (Vm) of NMDAR currents recorded in cortical neurons. Here two components of TCA action on NMDARs are described, which could be characterized as the Ca2+-dependent inhibition and the open-channel block. DES demonstrated a profound Ca2+-dependent inhibition of NMDARs, while the CLO effect was weak. DES IC50 exhibited an e-fold change with a [Ca2+] shift of 0.59 mM, which is consistent with ATL. The Ca2+ dependence of NMDAR inhibition by DES disappeared in BAPTA loaded neurons, suggesting that Ca2+ acts from the inside. Since CLO differs from DES and ATL by the presence of Cl-atom in the structure, most likely, this is the atom which is responsible for the loss of pronounced [Ca2+] dependence. As for the NMDAR open-channel block, both DES and CLO were about 5-folds more potent than ATL due to their slow rates of dissociation either from open and closed states. DES demonstrated stronger Vm-dependence than CLO, suggesting a deeper location of the DES binding site within the ion pore. Because DES and CLO differ from ATL by the nitrogen-containing tricycle, presumably this moiety of the molecules determines their high-affinity binding with the NMDAR channel, while the aliphatic chain mono-methyl amino-group of DES allows a deep permeation in the channel. Thus, different structure-activity relationships of the Ca2+-dependent inhibition and Vm-dependent open-channel block of NMDARs by DES and CLO suggest that these processes are independent and most likely may represent an action on different molecular targets. The proposed model of TCA action on NMDARs predicts well the experimental values of IC50s at physiological [Ca2+] and within a wide range of Vms.
RESUMEN
Homocysteine (HCY) molecule combines distinct pharmacological properties as an agonist of N-methyl-d-aspartate receptors (NMDARs) and a reducing agent. Whereas NMDAR activation by HCY was elucidated, whether the redox modulation contributes to its action is unclear. Here, using patch-clamp recording and imaging of intracellular Ca2+, we study dithiothreitol (DTT) effects on currents and Ca2+ responses activated by HCY through native NMDARs and recombinant diheteromeric GluN1/2A, GluN1/2B, and GluN1/2C receptors. Within a wide range (1-800 µM) of [HCY]s, the concentration-activation relationships for recombinant NMDARs revealed a biphasicness. The high-affinity component obtained between 1 and 100 µM [HCY]s corresponding to the NMDAR activation was not affected by 1 mM DTT. The low-affinity phase observed at [HCY]s above 200 µM probably originated from thiol-dependent redox modulation of NMDARs. The reduction of NMDAR disulfide bonds by either 1 mM DTT or 1 mM HCY decreased GluN1/2A currents activated by HCY. In contrast, HCY-elicited GluN1/2B currents were enhanced due to the remarkable weakening of GluN1/2B desensitization. In fact, cleaving NMDAR disulfide bonds in neurons reversed the HCY-induced Ca2+ accumulation, making it dependent on GluN2B- rather than GluN2A-containing NMDARs. Thus, estimated concentrations for the HCY redox effects exceed those in the plasma during intermediate hyperhomocysteinemia but may occur during severe hyperhomocysteinemia.
Asunto(s)
Homocisteína/farmacología , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Células Cultivadas , Embrión de Mamíferos , Femenino , Células HEK293 , Humanos , Neuronas/efectos de los fármacos , Neuronas/fisiología , Oxidación-Reducción , Embarazo , Subunidades de Proteína/agonistas , Subunidades de Proteína/genética , Subunidades de Proteína/fisiología , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/genética , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genéticaRESUMEN
Pathological homocysteine (HCY) accumulation in the human plasma, known as hyperhomocysteinemia, exacerbates neurodegenerative diseases because, in the brain, this amino acid acts as a persistent N-methyl-d-aspartate receptor agonist. We studied the effects of 0.1-1 nM ouabain on intracellular Ca2+ signaling, mitochondrial inner membrane voltage (φmit), and cell viability in primary cultures of rat cortical neurons in glutamate and HCY neurotoxic insults. In addition, apoptosis-related protein expression and the involvement of some kinases in ouabain-mediated effects were evaluated. In short insults, HCY was less potent than glutamate as a neurotoxic agent and induced a 20% loss of φmit, whereas glutamate caused a 70% decrease of this value. Subnanomolar ouabain exhibited immediate and postponed neuroprotective effects on neurons. (1) Ouabain rapidly reduced the Ca2+ overload of neurons and loss of φmit evoked by glutamate and HCY that rescued neurons in short insults. (2) In prolonged 24 h excitotoxic insults, ouabain prevented neuronal apoptosis, triggering proteinkinase A and proteinkinase C dependent intracellular neuroprotective cascades for HCY, but not for glutamate. We, therefore, demonstrated here the role of PKC and PKA involving pathways in neuronal survival caused by ouabain in hyperhomocysteinemia, which suggests existence of different appropriate pharmacological treatment for hyperhomocysteinemia and glutamate excitotoxicity.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Neuronas/efectos de los fármacos , Ouabaína/farmacología , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ácido Glutámico/farmacología , Hiperhomocisteinemia/metabolismo , Hiperhomocisteinemia/patología , Transporte Iónico/efectos de los fármacos , Neuronas/citología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Proteína Quinasa C/metabolismo , Ratas WistarRESUMEN
N-methyl-d-aspartate receptor (NMDAR) is an essential target for ethanol action in the central nervous system (CNS). Whereas an alcohol addiction treatment represents a severe medical problem, many aspects of ethanol action at physiologically relevant concentrations on NMDARs are still unclear. Here using the whole-cell patch-clamp recording on cortical neurons in the primary culture, we studied inhibition of NMDAR currents by different ethanol concentrations ([Et]s) and its dependence on extracellular Ca2+. The ethanol action on NMDA-activated currents exhibited a biphasic concentration-inhibition relationship in the presence of extracellular Ca2+. The high-affinity region of the curve was found within the range of [Et]s from 9 mM to 30 mM and was characterized by IC50,H of about 20 mM. The low-affinity region was observed within the range of [Et]s from 85 mM to 200 mM with IC50,L of about 150 mM. In the absence of extracellular Ca2+, the ethanol concentration-inhibition relationship became monophasic, with IC50,L of about 200 mM, since the high-affinity component disappeared. A substitution of Li+ for Na+ in the bathing solution and an extraction of cholesterol from the plasma membrane with methyl-ß-cyclodextrin, which are the treatments that both promote the Ca2+-dependent desensitization (CDD) of NMDARs, abolished the high-affinity Ca2+-dependent component of the NMDAR ethanol inhibition. Besides, this component was not observed when neurons were loaded with BAPTA. These data suggest that most likely, ethanol at low concentrations enhances the NMDAR CDD. In agreement when the dependence of the NMDAR CDD on extracellular Ca2+ was directly measured 22 mM ethanol enhanced the NMDAR CDD since an extracellular Ca2+ concentration that caused 50% of the NMDAR CDD decreased almost 3-folds from 0.81 mM to 0.28 mM, and an extent of the CDD was also more pronounced. The low-affinity component of the NMDAR ethanol inhibition was resistant to the above treatments suggesting CDD-independent direct action on NMDARs. Thus, at a physiologically relevant concentration of extracellular Ca2+ and ethanol that could be reached in the blood during light-mild human alcohol intoxication, ethanol causes an enhancement of the NMDAR CDD, which could be in general accompanied by some disruptions of the CNS excitatory system.
Asunto(s)
Calcio/metabolismo , Etanol/toxicidad , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Espacio Extracelular/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Litio/farmacología , N-Metilaspartato/farmacología , Ratas Wistar , beta-CiclodextrinasRESUMEN
Although the tricyclic antidepressant amitriptyline (ATL) is widely used in the clinic, the mechanism underlying its high therapeutic efficacy against neuropathic pain remains unclear. NMDA receptors (NMDARs) represent a target for ATL and are involved in sensitization of neuropathic pain. Here we describe two actions of ATL on NMDARs: 1) enhancement of Ca2+-dependent desensitization and 2) trapping channel block. Inhibition of NMDARs by ATL was found to be dependent upon external Ca2+ concentration ([Ca2+]) in a voltage-independent manner, with an IC50 of 0.72 µM in 4 mM [Ca2+]. The ATL IC50 value increased exponentially with decreasing [Ca2+], with an e-fold change observed per 0.69 mM decrease in [Ca2+]. Loading neurons with BAPTA abolished Ca2+-dependent inhibition, suggesting that Ca2+ affects NMDARs from the cytosol. Since there is one known Ca2+-dependent process in gating of NMDARs, we conclude that ATL most likely promotes Ca2+-dependent desensitization. We also found ATL to be a trapping open-channel blocker of NMDARs with an IC50 of 220 µM at 0 mV. An e-fold change in ATL IC50 was observed to occur with a voltage shift of 50 mV in 0.25 mM [Ca2+]. Thus, we disclose here a robust dependence of ATL potency on extracellular [Ca2+], and demonstrate that ATL bound in the NMDAR pore can be trapped by closure of the channel.
Asunto(s)
Amitriptilina/farmacología , Antidepresivos Tricíclicos/farmacología , Calcio/metabolismo , Neuronas/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Amitriptilina/uso terapéutico , Animales , Antidepresivos Tricíclicos/uso terapéutico , Células Cultivadas , Corteza Cerebral/citología , Dolor Crónico/complicaciones , Dolor Crónico/dietoterapia , Dolor Crónico/psicología , Citosol/efectos de los fármacos , Citosol/metabolismo , Depresión/tratamiento farmacológico , Depresión/etiología , Depresión/psicología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Femenino , Humanos , Concentración 50 Inhibidora , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Neuralgia/complicaciones , Neuralgia/tratamiento farmacológico , Neuralgia/psicología , Neuronas/citología , Neuronas/metabolismo , Técnicas de Placa-Clamp , Cultivo Primario de Células , Ratas , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Homocysteine (HCY) induced neurotoxicity largely depends on interaction of this endogenous amino acid with glutamate NMDA receptors (NMDARs). This receptor type is composed by GluN1 and different GluN2 (A, B, C or D) subunits. However, the receptor activity of HCY in brain regions which differ in relative contribution of GluN2 subunits was not tested so far. In the current study, we explored the action of HCY on cerebellar neurons which natively express GluN2C and GluN2D subunits of NMDARs and compared this with the action of HCY on cortical neurons which are mainly composed by GluN2A and GluN2B subunits. To validate obtained results, we also studied the responses to HCY in recombinant GluN1/2C and GluN1/2D NMDARs expressed in HEK293T cells. Responses to HCY were compared to membrane currents evoked by glutamate or by the specific agonist NMDA. First, we found that on HEK cells expressing GluN1/2C or GluN1/2D NMDARs, HCY was full agonist producing membrane currents similar in amplitude to currents induced by glutamate. The EC50 values for these particular receptor subtype activation were 80⯵M and 31⯵M, respectively. Then, we found that HCY similarly to NMDA, evoked large slightly desensitizing membrane currents in native NMDARs of cerebellar and cortical neurons. In cortical neurons, the ratio of the respective currents (IHCY/INMDA) was 0.16 and did not significantly change during in vitro maturation. In sharp contrast, in cerebellar neurons, the ratio of currents evoked by HCY and NMDA was dramatically increased from 0.31 to 0.72 from 7 to 21 day in culture. We show that least 75% of HCY-induced currents in cerebellum were mediated by GluN2C- or GluN2D-containing NMDARs. Thus, our data revealed a large population of cerebellar NMDA receptors highly sensitive to HCY which suggest potential vulnerability of this brain region to pathological conditions associated with enhanced levels of this neurotoxic amino acid.
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Cerebelo/citología , Homocisteína/farmacología , Neuronas/metabolismo , Subunidades de Proteína/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Ratas Wistar , Receptores de N-Metil-D-Aspartato/agonistasRESUMEN
BACKGROUND: The plasma membrane Na+/Ca2+-exchanger (NCX) has recently been shown to regulate Ca2+-dependent N-methyl-D-aspartate receptor (NMDAR) desensitization, suggesting a tight interaction of NCXs and NMDARs in lipid nanoclasters or "rafts". To evaluate possible role of this interaction we studied effects of Li+ on NMDA-elicited whole-cell currents and Ca2+ responses of rat cortical neurons in vitro before and after cholesterol extraction by methyl-ß-cyclodextrin (MßCD). RESULTS: Substitution Li+ for Na+ in the external solution caused a concentration-dependent decrease of steady-state NMDAR currents from 440 ± 71 pA to 111 ± 29 pA in 140 mM Na+ and 140 mM Li+, respectively. The Li+ inhibition of NMDAR currents disappeared in the absence of Ca2+ in the external solution (Ca2+-free), suggesting that Li+ enhanced Ca2+-dependent NMDAR desensitization. Whereas the cholesterol extraction with MßCD induced a decrease of NMDAR currents to 136 ± 32 pA in 140 mM Na+ and 46 ± 15 pA in 140 mM Li+, the IC50 values for the Li+ inhibition were similar (about 44 mM Li+) before and after this procedure. In the Ca2+-free Na+ solution the steady-state NMDAR currents after the cholesterol extraction were 47 ± 6% of control values. Apparently this amplitude decrease was not Ca2+-dependent. In the Na+ solution containing 1 mM Ca2+ the Ca2+-dependent NMDAR desensitization was greater when cholesterol was extracted. Obviously, this procedure promoted its development. In agreement, Li+ and KB-R7943, an inhibitor of NCX, both considerably reduced NMDA-activated Ca2+ responses. The cholesterol extraction itself caused a decrease of NMDA-activated Ca2+ responses and, in addition, abolished the effects of Li+ and KB-R7943. The cholesterol loading into the plasma membrane caused a recovery of the KB-R7943 effects. CONCLUSIONS: Taken together our data suggest that NCXs downregulate the Ca2+-dependent NMDAR desensitization. Most likely, this is determined by a tight functional interaction of NCX and NMDAR molecules because of their co-localization in membrane lipid rafts. The destruction of these rafts is accompanied by an enhancement of NMDAR desensitization and a loss of NCX-selective agent effects on NMDARs.
Asunto(s)
Membrana Celular/metabolismo , Colesterol/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Membrana Celular/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Regulación hacia Abajo , Litio/metabolismo , Litio/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , N-Metilaspartato/metabolismo , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Cultivo Primario de Células , Ratas Wistar , Sodio/metabolismo , Tiourea/análogos & derivados , Tiourea/farmacología , beta-Ciclodextrinas/farmacologíaRESUMEN
Transient expression of different NMDA receptors (NMDARs) plays a role in development of the cerebellum. Whether similar processes undergo during neuronal differentiation in culture is not clearly understood. We studied NMDARs in cerebellar neurons in cultures of 7 and 21 days in vitro (DIV) using immunocytochemical and electrophysiological approaches. Whereas at 7 DIV, the vast majority of neurons were immunopositive for GluN2 subunits, further synaptoginesis was accompanied by the time-dependent loss of NMDARs. In contrast to GluN2B- and GluN2C-containing NMDARs, which at 7 DIV exhibited homogenous distribution in extrasynaptic regions, GluN2A-containing receptors were aggregated in spots both in cell bodies and dendrites. Double staining for GluN2A subunits and synaptophysin, a widely used marker for presynaptic terminals, revealed their co-localization in about 75% of dendrite GluN2A fluorescent spots, suggesting postsynaptic origin of GluN2A subunits. In agreement, diheteromeric GluN2A-containing NMDARs contributed to postsynaptic currents recorded in neurons throughout the timescale under study. Diheteromeric GluN2B-containing NMDARs escaped postsynaptic regions during differentiation. Finally, the developmental switch favored the expression of triheteromeric NMDARs assembled of 2 GluN1/1 GluN2B/1 GluN2C or GluN2D subunits in extrasynaptic regions. At 21 DIV, these receptors represented over 60% of the NMDAR population. Thus, cerebellar neurons in primary culture undergo transformations with respect to the expression of di- and triheteromeric NMDARs that should be taken into account when studying cellular aspects of their pharmacology and functions.
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Neurogénesis , Células de Purkinje/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Animales , Células Cultivadas , Potenciales de la Membrana , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Células de Purkinje/citología , Células de Purkinje/fisiología , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/genética , Sinapsis/fisiologíaRESUMEN
Genetic variants of the glutamate activated N-methyl-D-aspartate (NMDA) receptor (NMDAR) subunit GluN2A are associated with the hyperexcitable states manifested by epileptic seizures and interictal discharges in patients with disorders of the epilepsy-aphasia spectrum (EAS). The variants found in sporadic cases and families are of different types and include microdeletions encompassing the corresponding GRIN2A gene as well as nonsense, splice-site and missense GRIN2A defects. They are located at different functional domains of GluN2A and no clear genotype-phenotype correlation has emerged yet. Moreover, GluN2A variants may be associated with phenotypic pleiotropy. Deciphering the consequences of pathogenic GRIN2A variants would surely help in better understanding of the underlying mechanisms. This emphasizes the need for functional studies to unravel the basic functional properties of each specific NMDAR variant. In the present study, we have used patch-clamp recordings to evaluate kinetic changes of mutant NMDARs reconstituted after co-transfection of cultured cells with the appropriate expression vectors. Three previously identified missense variants found in patients or families with disorders of the EAS and situated in the N-terminal domain (p.Ile184Ser) or in the ligand-binding domain (p.Arg518His and p.Ala716Thr) of GluN2A were studied in both the homozygous and heterozygous conditions. Relative surface expression and current amplitude were significantly reduced for NMDARs composed of mutant p.Ile184Ser and p.Arg518His, but not p.Ala716His, as compared with wild-type (WT) NMDARs. Amplitude of whole-cell currents was still drastically decreased when WT and mutant p.Arg518His-GluN2A subunits were co-expressed, suggesting a dominant-negative mechanism. Activation times were significantly decreased in both homozygous and heterozygous conditions for the two p.Ile184Ser and p.Arg518His variants, but not for p.Ala716His. Deactivation also significantly increased for p.Ile184Ser variant in the homozygous but not the heterozygous state while it was increased for p.Arg518His in both states. Our data indicate that p.Ile184Ser and p.Arg518His GluN2A variants both impacted on NMDAR function, albeit differently, whereas p.Ala716His did not significantly influence NMDAR kinetics, hence partly questioning its direct and strong pathogenic role. This study brings new insights into the functional impact that GRIN2A variants might have on NMDAR kinetics, and provides a mechanistic explanation for the neurological manifestations seen in the corresponding human spectrum of disorders.
RESUMEN
Background Blocking the pro-nociceptive action of CGRP is one of the most promising approaches for migraine prophylaxis. The aim of this study was to explore a role for CGRP as a neuroprotective agent for central and peripheral neurons. Methods The viability of isolated rat trigeminal, cortical and cerebellar neurons was tested by fluorescence vital assay. Engagement of Nrf2 target genes was analyzed by qPCR. The neuroprotective efficacy of CGRP in vivo was tested in mice using a permanent cerebral ischemia model. Results CGRP prevented apoptosis induced by the amino acid homocysteine in all three distinct neuronal populations. Using a set of specific kinase inhibitors, we show the role of multi-kinase signaling pathways involving PKA and CaMKII in neuronal survival. Forskolin triggered a very similar signaling cascade, suggesting that cAMP is the main upstream trigger for multi-kinase neuroprotection. The specific CGRP antagonist BIBN4096 reduced cellular viability, lending further support to the proposed neuroprotective function of CGRP. Importantly, CGRP was neuroprotective against permanent ischemia in mice. Conclusion Our data show an unexpected 'positive' role for the endogenous pro-nociceptive migraine mediator CGRP, suggesting more careful examination of migraine prophylaxis strategy based on CGRP antagonism although it should be noted that homocysteine induced apoptosis in primary neuronal cell culture might not necessarily reproduce all the features of cell loss in the living organism.
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Péptido Relacionado con Gen de Calcitonina/uso terapéutico , Cerebelo/enzimología , Corteza Cerebral/enzimología , Trastornos Migrañosos/enzimología , Nocicepción/efectos de los fármacos , Células Receptoras Sensoriales/enzimología , Animales , Péptido Relacionado con Gen de Calcitonina/farmacología , Células Cultivadas , Cerebelo/diagnóstico por imagen , Cerebelo/efectos de los fármacos , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/efectos de los fármacos , Masculino , Trastornos Migrañosos/diagnóstico por imagen , Trastornos Migrañosos/tratamiento farmacológico , Neuronas/efectos de los fármacos , Neuronas/enzimología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Nocicepción/fisiología , Ratas , Ratas Wistar , Células Receptoras Sensoriales/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ganglio del Trigémino/diagnóstico por imagen , Ganglio del Trigémino/efectos de los fármacos , Ganglio del Trigémino/enzimologíaRESUMEN
Intracellular calcium ([Ca2+]i) has been reported to play an important role in autophagy, apoptosis and necrosis, however, a little is known about its impact in senescence. Here we investigated [Ca2+]i contribution to oxidative stress-induced senescence of human endometrium-derived stem cells (hMESCs). In hMESCs sublethal H2O2-treatment resulted in a rapid calcium release from intracellular stores mediated by the activation of PLC/IP3/IP3R pathway. Notably, further senescence development was accompanied by persistently elevated [Ca2+]i levels. In H2O2-treated hMESCs, [Ca2+]i chelation by BAPTA-AM (BAPTA) was sufficient to prevent the expansion of the senescence phenotype, to decrease endogenous reactive oxygen species levels, to avoid G0/G1 cell cycle arrest, and finally to retain proliferation. Particularly, loading with BAPTA attenuated phosphorylation of the main DNA damage response members, including ATM, 53BP1 and H2A.X and reduced activation of the p53/p21/Rb pathway in H2O2-stimulated cells. Next, we revealed that BAPTA induced an early onset of AMPK-dependent autophagy in H2O2-treated cells as confirmed by both the phosphorylation status of AMPK/mTORC1 pathway and the dynamics of the LC3 lipidization. Summarizing the obtained data we can assume that calcium chelation is able to trigger short-term autophagy and to prevent the premature senescence of hMESCs under oxidative stress.
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Autofagia/fisiología , Calcio/metabolismo , Senescencia Celular/fisiología , Estrés Oxidativo/fisiología , Células Madre/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Autofagia/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Endometrio/citología , Endometrio/efectos de los fármacos , Endometrio/metabolismo , Femenino , Humanos , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
Homocysteine (HCY) is an endogenous redox active amino acid, best known as contributor to various neurodegenerative disorders. Although it is known that HCY can activate NMDA receptors (NMDARs), the mechanisms of its action on receptors composed of different NMDA receptor subunits remains almost unknown. In this study, using imaging and patch clamp technique in cultured cortical neurons and heterologous expression in HEK293T cells we tested the agonist activity of HCY on NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors) and GluN1 and GluN2B subunits (GluN1/2B receptors). We demonstrate that the time courses of Ca2+ transients and membrane currents activated by HCY and NMDA in cortical neurons are drastically different. Application of HCY to cortical neurons induced responses, which in contrast to currents induced by NMDA (both in the presence of glycine) considerably decreased to steady state of small amplitude. In contrast to NMDA, HCY-activated currents at steady state were resistant to the selective GluN2B subunit inhibitor ifenprodil. In calcium-free external solution the decrease of NMDA evoked currents was abolished, suggesting the Ca2+-dependent NMDAR desensitization. Under these conditions HCY evoked currents still declined almost to the baseline suggesting Ca2+-independent desensitization. In HEK293T cells HCY activated NMDARs of GluN1/2A and GluN1/2B subunit compositions with EC50s of 9.7 ± 1.8 and 61.8 ± 8.9 µM, respectively. Recombinant GluN1/2A receptors, however, did not desensitize by HCY, whereas GluN1/2B receptors were almost fully desensitized by HCY. Thus, HCY is a high affinity agonist of NMDARs preferring the GluN1/2A subunit composition. Our data suggest that HCY induced native NMDAR currents in neurons are mainly mediated by the "synaptic type" GluN1/2A NMDARs. This implies that in hyperhomocysteinemia, a disorder with enlarged level of HCY in plasma, HCY may persistently contribute to post-synaptic responses mediated by GluN2A-containing NMDA receptors. On the other hand, HCY toxicity may be limited by desensitization typical for HCY-induced activation of GluN2B-containing extrasynaptic receptors. Our findings, therefore, provide an evidence for the physiological relevance of endogenous HCY, which may represent an effective endogenous modulator of the central excitatory neurotransmission.
RESUMEN
To evaluate the possible role of the plasma membrane Na(+)/Ca(2+)-exchanger (NCX) in regulation of N-methyl-d-aspartate (NMDA) receptors (NMDARs), we studied effects of 2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate (KB-R7943; KBR) and lithium (inhibitors of NCX) on NMDA-elicited whole-cell currents using the patch-clamp technique on rat cortical neurons and human embryonic kidney 293T cells expressing recombinant NMDARs. KBR inhibited NMDAR currents in a voltage-independent manner with similar potency for receptors of GluN1/2A and GluN1/2B subunit compositions that excludes open-channel block and GluN2B-selective inhibition. The inhibition by KBR depended on glycine (Gly) concentration. At 30 µM NMDA, the KBR IC50 values were 5.3 ± 0.1 and 41.2 ± 8.8 µM for 1 and 300 µM Gly, respectively. Simultaneous application of NMDA + KBR in the absence of Gly induced robust inward NMDAR currents that peaked and then rapidly decreased. KBR, therefore, is an agonist (EC50 is 1.18 ± 0.16 µM) of the GluN1 subunit coagonist binding sites. The decrease of NMDA-elicited currents in the presence of KBR was abolished in Ca(2+)-free solution and was not observed in the presence of extracellular Ca(2+) on 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded neurons, suggesting that Ca(2+) affects NMDARs from the cytosol. In agreement, the substitution of Li(+) for extracellular Na(+) caused a considerable decrease of NMDAR currents, which was not observed in the absence of extracellular Ca(2+). Most likely, the accumulation of intracellular Ca(2+) is caused by the inhibition of Ca(2+) extrusion via NCX. Thus, KBR and Li(+) provoke Ca(2+)-dependent receptor inactivation due to the disruption of Ca(2+) extrusion by the NCX. The data reveal the role of NCX in regulation of Ca(2+)-dependent inactivation of NMDARs.
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Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Compuestos de Litio/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Tiourea/análogos & derivados , Animales , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Glicina/farmacología , Células HEK293 , Humanos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Tiourea/farmacologíaRESUMEN
Recent studies suggested contribution of homocysteine (HCY) to neurodegenerative disorders and migraine. However, HCY effect in the nociceptive system is essentially unknown. To explore the mechanism of HCY action, we studied short- and long-term effects of this amino acid on rat peripheral and central neurons. HCY induced intracellular Ca²âº transients in cultured trigeminal neurons and satellite glial cells (SGC), which were blocked by the NMDA antagonist AP-5 in neurons, but not in SGCs. In contrast, 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP), the metabotropic mGluR5 (metabotropic glutamate receptor 5 subtype) antagonist, preferentially inhibited Ca²âº transients in SGCs. Prolonged application of HCY induced apoptotic cell death of both kinds of trigeminal cells. The apoptosis was blocked by AP-5 or by the mGluR5 antagonist MTEP. Likewise, in cortical neurons, HCY-induced cell death was inhibited by AP-5 or MTEP. Imaging with 2',7'-dichlorodihydrofluorescein diacetate or mitochondrial dye Rhodamine-123 as well as thiobarbituric acid reactive substances assay did not reveal involvement of oxidative stress in the action of HCY. Thus, elevation of intracellular Ca²âº by HCY in neurons is mediated by NMDA and mGluR5 receptors while SGC are activated through the mGluR5 subtype. Long-term neurotoxic effects in peripheral and central neurons involved both receptor types. Our data suggest glutamatergic mechanisms of HCY-induced sensitization and apoptosis of trigeminal nociceptors.
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Calcio/metabolismo , Corteza Cerebral/efectos de los fármacos , Homocisteína/toxicidad , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neurotoxinas , Receptor del Glutamato Metabotropico 5/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Nervio Trigémino/efectos de los fármacos , Animales , Señalización del Calcio/fisiología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Corteza Cerebral/citología , Relación Dosis-Respuesta a Droga , Indicadores y Reactivos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neuroglía/efectos de los fármacos , Oxidación-Reducción , Cultivo Primario de Células , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Receptor Cross-Talk/fisiología , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Nervio Trigémino/citologíaRESUMEN
Whereas kainate (KA)-induced neurodegeneration has been intensively investigated, the contribution of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in neuronal Ca2+ overload ([Ca2+]i) is still controversial. Using Ca2+ imaging and patch-clamp techniques, we found different types of Ca2+ entry in cultured rat cortical neurons. The presence of Ca2+ in the extracellular solution was required to generate the [Ca2+]i responses to 30 µM N-methyl-d-aspartate (NMDA) or KA. The dynamics of NMDA-induced [Ca2+]i responses were fast, while KA-induced responses developed slower reaching high [Ca2+]i. Ifenprodil, a specific inhibitor of the GluN2B subunit of NMDARs, reduced NMDA-induced [Ca2+]i responses suggesting expression of GluN1/GluN2B receptors. Using IEM-1460, a selective blocker of Ca(2+)-permeable GluA2-subunit lacking AMPARs, we found three neuronal responses to KA: (i) IEM-1460 resistant neurons which are similar to pyramidal neurons expressing Ca(2+)-impermeable GluA2-rich AMPARs; (ii) Neurons exhibiting nearly complete block of both KA-induced currents and [Ca2+]i signals by IEM-1460 may represent interneurons expressing GluA2-lacking AMPARs and (iii) neurons with moderate sensitivity to IEM-1460. Ouabain at 1 nM prevented the neuronal Ca2+ overload induced by KA. The data suggest, that cultured rat cortical neurons maintain functional phenotypes of the adult brain cortex, and demonstrate the key contribution of the Na/K-ATPase in neuroprotection against KA excitotoxicity.
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Calcio/metabolismo , Corteza Cerebral/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Ácido Kaínico/farmacología , Neuronas/metabolismo , Ouabaína/farmacología , Receptores AMPA/metabolismo , Adamantano/análogos & derivados , Adamantano/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Técnicas In Vitro , Ácido Kaínico/efectos adversos , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/patología , Técnicas de Placa-Clamp , Piperidinas/farmacología , Embarazo , Ratas , Ratas WistarRESUMEN
Using a fluorescent viability assay, immunocytochemistry, patch-clamp recordings, and Ca(2+) imaging analysis, we report that ouabain, a specific ligand of the Na(+),K(+)-ATPase cardiac glycoside binding site, can prevent glutamate receptor agonist-induced apoptosis in cultured rat cortical neurons. In our model of excitotoxicity, a 240-min exposure to 30 µM N-methyl-d-aspartate (NMDA) or kainate caused apoptosis in â¼50% of neurons. These effects were accompanied by a significant decrease in the number of neurons that were immunopositive for the antiapoptotic peptide Bcl-2. Apoptotic injury was completely prevented when the agonists were applied together with 0.1 or 1 nM ouabain, resulting in a greater survival of neurons, and the percentage of neurons expressing Bcl-2 remained similar to those obtained without agonist treatments. In addition, subnanomolar concentrations of ouabain prevented the increase of spontaneous excitatory postsynaptic current's frequency and the intracellular Ca(2+) overload induced by excitotoxic insults. Loading neurons with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or inhibition of the plasma membrane Na(+),Ca(2+)-exchanger by 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate (KB-R7943) eliminated ouabain's effects on NMDA- or kainite-evoked enhancement of spontaneous synaptic activity. Our data suggest that during excitotoxic insults ouabain accelerates Ca(2+) extrusion from neurons via the Na(+),Ca(2+) exchanger. Because intracellular Ca(2+) accumulation caused by the activation of glutamate receptors and boosted synaptic activity represents a key factor in triggering neuronal apoptosis, up-regulation of Ca(2+) extrusion abolishes its development. These antiapoptotic effects are independent of Na(+),K(+)-ATPase ion transport function and are initiated by concentrations of ouabain that are within the range of an endogenous analog, suggesting a novel functional role for Na(+),K(+)-ATPase in neuroprotection.