RESUMO
To investigate the feasibility and the clinical efficiency of robot navigation combined with wrist arthroscopy in minimally invasive treatment of nondisplaced type Herbert D1 scaphoid fracture. A retrospective analysis was performed on 9 patients who underwent nondisplaced type Herbert D1 scaphoid fracture in Xuzhou Renci Hospital from December 2019 to January 2021. Before the operation and at the last follow-up, grip strength, pinching force, modified wrist Mayo score and visual analogue scale (VAS) of wrist pain were recorded and compared. The average follow-up time was 14.1 months (7.5-24.0 months). All the fractures achieved primary healing after an average of 13.3 weeks (10-18 weeks). The average flexion and dorsal extension activity of the injured wrist was 51.2°±9.4°, 68.0°±7.3°, and the radial and ulnar deviation was 19.3°±6.2°, 45.7°±7.8°, respectively. At the final follow-up, there were statistically significant differences in grip strength, pinch strength, wrist Mayo score and VAS when compared with those before the operation (all P<0.05). The results demonstrated that robot navigation combined with wrist arthroscopy for nondisplaced type Herbert D1 scaphoid fracture is effective and minimally invasive with a short recovery time and satisfactory healing rate.
Assuntos
Fraturas Ósseas , Robótica , Osso Escafoide , Artroscopia , Fixação Interna de Fraturas , Fraturas Ósseas/cirurgia , Humanos , Amplitude de Movimento Articular , Estudos Retrospectivos , Osso Escafoide/cirurgia , Resultado do Tratamento , PunhoRESUMO
Objective: To investigate the incidence and treatment of perioperative anemia in patients with gastrointestinal neoplasms in Hubei Province. Methods: The clinicopathological data of 7 474 patients with gastrointestinal neoplasms in 62 hospitals in 15 cities (state) of Hubei Province in 2019 were collected in the form of network database. There were 4 749 males and 2 725 females. The median age of the patients was 62 years (range: 17 to 96 years). The hemoglobin value of the first time in hospital and the first day after operation was used as the criterion of preoperative anemia and postoperative anemia. Anemia was defined as male hemoglobin <120 g/L and female hemoglobin <110.0 g/L, mild anemia as 90 to normal, moderate anemia as 60 to <90 g/L, severe anemia as <60 g/L. The t test and χ2 test were used for inter-group comparison. Results: The overall incidence of preoperative anemia was 38.60%(2 885/7 474), and the incidences of mild anemia, moderate anemia and severe anemia were 25.09%(1 875/7 474), 11.37%(850/7 474) and 2.14%(160/7 474), respectively. The overall incidence of postoperative anemia was 61.40%(4 589/7 474). The incidence of mild anemia, moderate anemia and severe anemia were 48.73%(3 642/7 474), 12.20%(912/7 474) and 0.47%(35/7 474), respectively. The proportion of preoperative anemia patients receiving treatment was 26.86% (775/2 885), and the proportion of postoperative anemia patients receiving treatment was 14.93% (685/4 589). The proportions of preoperative anemia patients in grade â ¢A, grade â ¢B, and grade â ¡A hospitals receiving treatment were 26.12% (649/2 485), 32.32% (85/263), and 29.93% (41/137), and the proportions of postoperative anemia patients receiving treatment were 14.61% (592/4 052), 22.05% (73/331), and 9.71% (20/206). The proportion of intraoperative blood transfusion (16.74% (483/2 885) vs. 3.05% (140/4 589), χ²=434.555, P<0.01) and the incidence of postoperative complications (17.78% (513/2 885) vs. 14.08% (646/4 589), χ²=18.553, P<0.01) in the preoperative anemia group were higher than those in the non-anemia group, and the postoperative hospital stay in the preoperative anemia group was longer than that in the non-anemia group ((14.1±7.3) days vs. (13.3±6.2) days, t=5.202, P<0.01). Conclusions: The incidence of perioperative anemia in patients with gastrointestinal neoplasms is high. Preoperative anemia can increase the demand for intraoperative blood transfusion and affect the short-term prognosis of patients. At present, the concept of standardized treatment of perioperative anemia among gastrointestinal surgeons in Hubei Province needs to be improved.
Assuntos
Anemia , Neoplasias Gastrointestinais , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia/epidemiologia , Transfusão de Sangue , Feminino , Neoplasias Gastrointestinais/complicações , Neoplasias Gastrointestinais/cirurgia , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
Acid-sensing ion channel 1b (ASIC1b) is a proton-gated Na(+) channel mostly expressed in peripheral sensory neurons. To date, the functional significance of ASIC1b in these cells is unclear due to the lack of a specific inhibitor/blocker. A better understanding of the regulation of ASIC1b may provide a clue for future investigation of its functional importance. One important regulator of acid-sensing ion channels (ASICs) is zinc. In this study, we examined the detailed zinc inhibition of ASIC1b currents and specific amino acid(s) involved in the inhibition. In Chinese hamster ovary (CHO) cells expressing rat ASIC1b subunit, pretreatment with zinc concentration-dependently inhibited the ASIC1b currents triggered by pH dropping from 7.4 to 6.0 with a half-maximum inhibitory concentration of 26 µM. The inhibition of ASIC1b currents by pre-applied zinc was independent of pH, voltage, or extracellular Ca(2+). Further, we showed that the effect of zinc is dependent on the extracellular cysteine, but not histidine residue. Mutating cysteine 149, but not cysteine 58 or cysteine 162, located in the extracellular domain of the ASIC1b subunit abolished the zinc inhibition. These findings suggest that cysteine 149 in the extracellular finger domain of ASIC1b subunit is critical for zinc-mediated inhibition and provide the basis for future mechanistic studies addressing the functional significance of zinc inhibition of ASIC1b.
Assuntos
Cisteína/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Canais de Sódio/metabolismo , Oligoelementos/farmacologia , Zinco/farmacologia , Canais Iônicos Sensíveis a Ácido , Análise de Variância , Animais , Cálcio/farmacologia , Linhagem Celular Transformada , Cricetinae , Cricetulus , Cisteína/genética , Relação Dose-Resposta a Droga , Metanossulfonato de Etila/análogos & derivados , Metanossulfonato de Etila/farmacologia , Líquido Extracelular/metabolismo , Peróxido de Hidrogênio , Potenciais da Membrana/genética , Mutagênese Sítio-Dirigida/métodos , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína/genética , Ratos , Canais de Sódio/genética , TransfecçãoRESUMO
Acid-sensing ion channel 3 (ASIC3) is a proton-gated, voltage-insensitive Na(+) channel that is expressed primarily in peripheral sensory neurons and plays an important role in pain perception, particularly as a pH sensor following cardiac ischemia. We previously reported that ASIC3 currents are not affected by zinc at nanomolar concentrations. In this study, we examined the potential role of micromolar zinc in the regulation of ASIC3. In CHO cells expressing ASIC3, we found that ASIC3 currents triggered by dropping the pH from 7.4 to 6.0 were inhibited by pretreatment with zinc in a concentration-dependent manner; the half-maximum inhibitory concentration of zinc was 61 muM. ASIC currents activated by a relatively small drop in pH from 7.4 to 7.2 or 7.0 were also subject to inhibition by zinc. The inhibition was fast and pH independent, and occurred within a relatively narrow range of zinc concentrations between 30 and 300 muM. Further, increasing extracellular Ca(2+) concentrations from 2 to 10 mM failed to affect inhibition of ASIC3 currents by zinc. Experimentally elevating intracellular zinc levels did not affect the inhibition of ASIC3 currents by equal concentrations of extracellular zinc, and modification of cysteine or histidine residues had no effect on the inhibition of ASIC3 currents by zinc. These collective results suggest that zinc is an important regulator of ASIC3 at physiological concentrations, that zinc inhibits ASIC3 in a pH- and Ca(2+)-independent manner, and that inhibition of ASIC3 currents is dependent upon the interaction of zinc with extracellular domain(s) of ASIC3.
Assuntos
Proteínas do Tecido Nervoso/biossíntese , Canais de Sódio/biossíntese , Zinco/fisiologia , Canais Iônicos Sensíveis a Ácido , Animais , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , Cisteína/metabolismo , Relação Dose-Resposta a Droga , Espaço Extracelular/metabolismo , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas do Tecido Nervoso/antagonistas & inibidores , Técnicas de Patch-Clamp , Ligação Proteica , Ratos , Zinco/farmacologiaRESUMO
Numerous studies have indicated oxidative stress as a key pathological factor in ischemic brain injury. One of the key links between oxidative stress and cell death is excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1), which plays an important role in the ischemic brain damage in male animals. Multiple studies have also suggested that NAD+ depletion mediates PARP-1 cytotoxicity, and NAD+ administration can decrease ischemic brain injury. A number of recent studies have provided novel information regarding the mechanisms underlying the roles of oxidative stress and NAD+-dependent enzymes in ischemic brain injury. Of particular interest, there have been exciting progresses regarding the mechanisms underlying the roles of NADPH oxidase and PARP-1 in cerebral ischemia. For examples, it has been suggested that androgen signaling and binding of PARP-1 onto estrogen receptors could account for the intriguing findings that PARP-1 plays remarkably differential roles in the ischemic brain damage of male and female animals; and some studies have suggested casein kinase 2, copper-zinc superoxide dismutase, and estrogen signaling can modulate the expression and activity of NADPH oxidase. This review summarizes these important current advances, and proposes future perspectives for the studies on the roles of oxidative stress and NAD+ in cerebral ischemia. It is increasingly likely that future studies on NAD- and NADP-dependent enzymes, such as NADPH oxidase, PARP-1, and sirtuins, would expose novel mechanisms underlying the roles of oxidative stress in cerebral ischemia, and suggest new therapeutic strategies for treating the debilitating disease.
Assuntos
Isquemia Encefálica/metabolismo , NAD/metabolismo , Estresse Oxidativo , Animais , Feminino , Humanos , Masculino , NADPH Oxidases/metabolismo , NADPH Oxidases/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/fisiologia , Sirtuínas/metabolismo , Sirtuínas/fisiologiaRESUMO
Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases. They are highly expressed in the striatum, where medium spiny neurons (MSNs) are a major population. Given that the properties of ASICs in MSNs are unknown, in this study, we characterized ASICs in MSNs of the mouse striatum. A rapid drop in extracellular pH induced transient inward currents in all MSNs. The pH value for half-maximal activation was 6.25, close to that obtained in homomeric ASIC1a channels. Based on psalmotoxin 1 and zinc sensitivity, ASIC1a (70.5% of neurons) and heteromeric ASIC1a-2 channels (29.5% of neurons) appeared responsible for the acid-induced currents in MSNs. ASIC currents were diminished in MSNs from ASIC1, but not ASIC2, null mice. Furthermore, a drop in pH induced calcium influx by activating homomeric ASIC1a channels. Activation of ASICs increased the membrane excitability of MSNs and lowering extracellular Ca2+ potentiated ASIC currents. Our data suggest that the homomeric ASIC1a channel represents a majority of the ASIC isoform in MSNs. The potential function of ASICs in the striatum requires further investigation.
Assuntos
Corpo Estriado/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Canais de Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido , Potenciais de Ação/fisiologia , Amilorida/administração & dosagem , Animais , Cálcio/metabolismo , Células Cultivadas , Cloretos/administração & dosagem , Corpo Estriado/citologia , Corpo Estriado/efeitos dos fármacos , Relação Dose-Resposta a Droga , Espaço Extracelular/metabolismo , Concentração de Íons de Hidrogênio , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Peptídeos , Bloqueadores dos Canais de Sódio/administração & dosagem , Canais de Sódio/genética , Venenos de Aranha/administração & dosagem , Compostos de Zinco/administração & dosagemRESUMO
Acidosis is a common feature of brain in acute neurological injury, particularly in ischemia where low pH has been assumed to play an important role in the pathological process. However, the cellular and molecular mechanisms underlying acidosis-induced injury remain unclear. Recent studies have demonstrated that activation of Ca(2+)-permeable acid-sensing ion channels (ASIC1a) is largely responsible for acidosis-mediated, glutamate receptor-independent, neuronal injury. In cultured mouse cortical neurons, lowering extracellular pH to the level commonly seen in ischemic brain activates amiloride-sensitive ASIC currents. In the majority of these neurons, ASICs are permeable to Ca(2+), and an activation of these channels induces increases in the concentration of intracellular Ca(2+) ([Ca(2+)](i)). Activation of ASICs with resultant [Ca(2+)](i) loading induces time-dependent neuronal injury occurring in the presence of the blockers for voltage-gated Ca(2+) channels and the glutamate receptors. This acid-induced injury is, however, inhibited by the blockers of ASICs, and by reducing [Ca(2+)](o). In focal ischemia, intracerebroventricular administration of ASIC1a blockers, or knockout of the ASIC1a gene protects brain from injury and does so more potently than glutamate antagonism. Furthermore, pharmacological blockade of ASICs has up to a 5 h therapeutic time window, far beyond that of glutamate antagonists. Thus, targeting the Ca(2+)-permeable acid-sensing ion channels may prove to be a novel neuroprotective strategy for stroke patients.
Assuntos
Isquemia Encefálica/fisiopatologia , Cálcio/metabolismo , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Canais de Sódio/fisiologia , Canais Iônicos Sensíveis a Ácido , Animais , Isquemia Encefálica/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Canais de Sódio/metabolismoRESUMO
In response to harmful stresses, cells induce programmed cell death (PCD) or apoptosis. Seizures can induce neural damage and activate biochemical pathways associated with PCD. Since seizures trigger intracellular calcium overload, it has been presumed that the intrinsic cell death pathway mediated by mitochondrial dysfunction would modulate cell death following seizures. However, previous work suggests that the extrinsic cell death pathway may initiate the damage program. Here we investigate intrinsic versus extrinsic cell death pathway activation using caspase cleavage as a marker for activation of these pathways in a rat in vitro model of seizures. Hippocampal cells, chronically treated with kynurenic acid, had kynurenic acid withdrawn to induce seizure-like activity for 40 min. Subjecting rat hippocampal cultures to seizures increased cell death and apoptosis-like DNA fragmentation using TUNEL staining. Seizure-induced cell death was blocked by both MK801 (10 microM) and CNQX (40 microM), which suggests multiple glutamate receptors regulate seizure-induced cell death. Cleavage of the initiator caspases, caspase 8 and 12 were increased 4h following seizure, and cleavage of the quintessential executioner caspase, caspase 3 was increased 4h following seizure. In contrast, caspase 9 cleavage only increased 24h following seizure. Using an affinity labeling approach to trap activated caspases in situ, we show that caspase 8 is the apical caspase activated following seizures. Finally, we show that the caspase 8 inhibitor Ac-IETD-CHO was more effective at blocking seizure-induced cell death than the caspase 9 inhibitor Ac-LEHD-CHO. Taken together, our data suggests the extrinsic cell death pathway-associated caspase 8 is activated following seizures in vitro.
Assuntos
Caspases/metabolismo , Morte Celular/fisiologia , Dano ao DNA/fisiologia , Hipocampo/patologia , Neurônios/patologia , Convulsões/patologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Caspase 8 , Caspase 9 , Morte Celular/efeitos dos fármacos , Células Cultivadas , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios , Ácido Cinurênico , L-Lactato Desidrogenase/metabolismo , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamenteRESUMO
Seizure-induced neuronal death may involve engagement of the BCL-2 family of apoptosis-regulating proteins. In the present study we examined the activation of proapoptotic BAD in cultured hippocampal neurons following seizures induced by removal of chronic glutamatergic transmission blockade. Kynurenic acid withdrawal elicited an increase in seizure-like electrical activity, which was inhibited by blockers of AMPA (CNQX) and NMDA (MK801 and AP5) receptor function. However, only NMDA receptor antagonists inhibited calcium entry as assessed by fura-2, and cell death of hippocampal neurons. Seizures increased proteolysis of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of cells. Seizure-like activity induced dephosphorylation of BAD and the disruption of its constitutive interaction with 14-3-3 proteins. In turn, BAD dimerized with antiapoptotic BCL-Xl after seizures. However, the absence of neuroprotective effects of pathway intervention suggests that BAD may perform a reinforcement rather than instigator role in cell death following seizures in vitro.
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Neurônios/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas 14-3-3 , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Fragmentação do DNA/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Ácido Cinurênico/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia , Tacrolimo/farmacologia , Fatores de Tempo , Proteína de Morte Celular Associada a bcl , Proteína bcl-XRESUMO
In human pregnancy, cortisol and PGs are involved in the onset of labor and play an important role in the mechanisms leading to parturition. Recent studies have shown that at term, cortisol increases PG synthesis and decreases PG metabolism in chorion trophoblast (CT) cells. In CT, 11 beta-hydroxysteroid oxidase type 1 (11 beta-HSD1) converts biologically inactive cortisone to cortisol to regulate cortisol availability. In the present study, we have investigated whether 11 beta-HSD1 activity could be influenced by PGs. We have shown that in CT, PGF2alpha rapidly increased 11 beta-HSD1 reductase activity in a dose-dependent manner via the PGF2alpha receptor, localized in the fetal membranes. PGF2alpha stimulated 11 beta-HSD1 activity through increased intracellular calcium mobilization, activation of PKC, and the phosphorylation of the 11 beta-HSD enzyme. We propose that within CT there is a novel feed forward loop by which PGF2alpha acts to promote cortisol production from cortisone through increases in 11beta-HSD1, and this in turn leads to further net PG output for the onset of labor and birth.
Assuntos
Dinoprosta/farmacologia , Hidrocortisona/biossíntese , Hidroxiesteroide Desidrogenases/biossíntese , Trabalho de Parto/fisiologia , 11-beta-Hidroxiesteroide Desidrogenases , Adulto , Cálcio/metabolismo , Células Cultivadas , Retroalimentação/fisiologia , Feminino , Feto/metabolismo , Corantes Fluorescentes , Fura-2 , Humanos , Imuno-Histoquímica , Luteolíticos/farmacologia , Membranas/metabolismo , Testes de Precipitina , Gravidez , Prostaglandinas F Sintéticas/farmacologia , Receptores de Prostaglandina/metabolismo , Estimulação Química , Trofoblastos/metabolismoRESUMO
Concentrations of extracellular calcium ([Ca(2+)](e)) in the CNS decrease substantially during seizure activity. We have demonstrated previously that decreases in [Ca(2+)](e) activate a novel calcium-sensing nonselective cation (csNSC) channel in hippocampal neurons. Activation of csNSC channels is responsible for a sustained membrane depolarization and increased neuronal excitability. Our study has suggested that the csNSC channel is likely involved in generating and maintaining seizure activities. In the present study, the effects of anti-epileptic agent lamotrigine (LTG) on csNSC channels were studied in cultured mouse hippocampal neurons using patch-clamp techniques. At a holding potential of -60 mV, a slow inward current through csNSC channels was activated by a step reduction of [Ca(2+)](e) from 1.5 to 0.2 mM. LTG decreased the amplitude of csNSC currents dose dependently with an IC(50) of 171 +/- 25.8 (SE) microM. The effect of LTG was independent of membrane potential. In the presence of 300 microM LTG, the amplitude of csNSC current was decreased by 31 +/- 3% at -60 mV and 29 +/- 2.9% at +40 mV (P > 0.05). LTG depressed csNSC current without affecting the potency of Ca(2+) block of the current (IC(50) for Ca(2+) block of csNSC currents in the absence of LTG: 145 +/- 18 microM; in the presence of 300 microM LTG: 136 +/- 10 microM. n = 5, P > 0.05). In current-clamp recordings, activation of csNSC channel by reducing the [Ca(2+)](e) caused a sustained membrane depolarization and an increase in the frequency of spontaneous firing of action potentials. LTG (300 microM) significantly inhibited csNSC channel-mediated membrane depolarization and the excitation of neurons. Fura-2 ratiometric Ca(2+) imaging experiment showed that LTG also inhibited the increase in intracellular Ca(2+) concentration induced by csNSC channel activation. The effect of LTG on csNSC channels may partially contribute to its broad spectrum of anti-epileptic actions.
Assuntos
Anticonvulsivantes/farmacologia , Cálcio/metabolismo , Cátions/metabolismo , Hipocampo/metabolismo , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Neurônios/metabolismo , Triazinas/farmacologia , Animais , Cálcio/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Condutividade Elétrica , Eletrofisiologia , Hipocampo/citologia , Membranas Intracelulares/metabolismo , Canais Iônicos/antagonistas & inibidores , Lamotrigina , Camundongos , Concentração OsmolarRESUMO
Positive modulators of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) channels reduce desensitization and alter their gating kinetics. We have discovered a novel compound nitric oxide-mimetic that similarly modulates the AMPA receptor by reducing desensitization. This, designated GT-005, belongs to the organic nitrate family that includes the nitrovasodilator nitroglycerine. In acutely isolated hippocampal neurons, GT-005 enhanced kainate (100 microM)-evoked currents with an EC50 of 1.7+/-0.2 mM and a 176+/-10% maximal increase in the steady-state current response. Similar results were found in cultured hippocampal neurons (EC50 of 1.3+/-0.2 mM and a maximal 83+/-14% increase in the steady-state current response). GT-005 reduced the desensitization of glutamate-evoked currents and slowed the onset of desensitization. This compound also increased the rate of recovery from the desensitized state. With respect to alteration of the excitatory synaptic transmission, GT-005 delayed the decay and increased the frequency of spontaneous miniature excitatory postsynaptic currents (mepsc) recorded in cultured hippocampal neurons.
Assuntos
Neurônios/efeitos dos fármacos , Nitratos/farmacologia , Receptores de AMPA/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Ácido Caínico/farmacologia , Potenciais da Membrana , Camundongos , Neurônios/metabolismo , Técnicas de Patch-Clamp , RatosRESUMO
A point mutation of the GluRdelta2 (A654T) glutamate receptor subunit converts it into a functional channel, and a spontaneous mutation at this site is thought to be responsible for the neurodegeneration of neurons in the Lurcher mouse. This mutation is located in a hydrophobic region of the M3 domain of this subunit, and this alanine is conserved throughout many of the glutamate receptors. We show here that site-directed mutagenesis of the homologous alanine (A636T; GluR1-L(c)) in the GluR1 AMPA receptor subunit alters its channel properties. The apparent potencies of both kainate and glutamate were increased 85- and 2000-fold, respectively. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)was converted from a competitive antagonist into a potent agonist. Our results demonstrate that a single amino acid within or near the putative second transmembrane region of the GluR1 subunit is critical for the binding/gating properties of this AMPA receptor.
Assuntos
6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Ácido Glutâmico/farmacologia , Ácido Caínico/farmacologia , Mutação Puntual , Receptores de AMPA/genética , Sequência de Aminoácidos , Animais , Condutividade Elétrica , Potenciais Evocados , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Canais Iônicos/agonistas , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/genética , Camundongos , Camundongos Mutantes , Modelos Moleculares , Dados de Sequência Molecular , Receptores de AMPA/agonistas , Receptores de AMPA/antagonistas & inibidores , Homologia de Sequência de AminoácidosRESUMO
Transient changes in the intracellular concentration of Ca2+ provide a major signal for the regulation of many ion channels and enzymes in central neurones. In contrast, changes in extracellular Ca2+ are thought to play little or no signaling role. However, concentrations of extracellular calcium in the central nervous system do change dramatically during intense physiological and pathological stimulation, and recent studies have identified a number of membrane proteins that can sense and respond to changes in extracellular Ca2+. These include the recently cloned Ca(2+)-sensing receptor, hemi-gap-junction channels, and a potential Ca(2+)-sensing cation channel. Lowering extracellular Ca2+ strongly depolarizes and excites cultured hippocampal neurones. The excitation can be detected with decreases from physiological concentrations of as little as 100 microM. The depolarization results from activation of a nonselective cation current, which is sensitive to block by divalent and polyvalent cations. In outside-out patches, lowering Ca2+ induces a single-channel current with a conductance of 36 pS. Activation of this cation channel, in response to decreases in extracellular Ca2+, likely plays a key role in a positive feedback system of excessive neuronal depolarization, which accompanies intense excitatory activity in the hippocampus.
Assuntos
Cálcio/metabolismo , Neurônios/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Humanos , Receptores de Detecção de Cálcio , XenopusRESUMO
Growth factor receptors provide a major mechanism for the activation of the nonreceptor tyrosine kinase c-Src, and this kinase in turn up-regulates the activity of N-methyl-D-aspartate (NMDA) receptors in CA1 hippocampal neurons (1). Unexpectedly, applications of platelet-derived growth factor (PDGF)-BB to cultured and isolated CA1 hippocampal neurons depressed NMDA-evoked currents. The PDGF-induced depression was blocked by a PDGF-selective tyrosine kinase inhibitor, by a selective inhibitor of phospholipase C-gamma, and by blocking the intracellular release of Ca(2+). Inhibitors of cAMP-dependent protein kinase (PKA) also eliminated the PDGF-induced depression, whereas a phosphodiesterase inhibitor enhanced it. The NMDA receptor-mediated component of excitatory synaptic currents was also inhibited by PDGF, and this inhibition was prevented by co-application of a PKA inhibitor. Src inhibitors also prevented this depression. In recordings from inside-out patches, the catalytic fragment of PKA did not itself alter NMDA single channel activity, but it blocked the up-regulation of these channels by a Src activator peptide. Thus, PDGF receptors depress NMDA channels through a Ca(2+)- and PKA-dependent inhibition of their modulation by c-Src.
Assuntos
Hipocampo/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Células Piramidais/fisiologia , Receptores do Fator de Crescimento Derivado de Plaquetas/fisiologia , Transmissão Sináptica/fisiologia , Animais , Becaplermina , Cálcio/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Potenciais Evocados/efeitos dos fármacos , Heparina/farmacologia , Homeostase , Técnicas In Vitro , Isoenzimas/metabolismo , Camundongos , N-Metilaspartato/farmacologia , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/farmacologia , Fosfolipase C gama , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-sis , Piridinas/farmacologia , Pirrolidinonas/farmacologia , Ratos , Ratos Wistar , Transmissão Sináptica/efeitos dos fármacos , Fosfolipases Tipo C/metabolismoRESUMO
The protein-tyrosine kinase Src is known to potentiate the function of NMDA receptors, which is necessary for the induction of long-term potentiation in the hippocampus. With recombinant receptors composed of NR1-1a/NR2A or NR1-1a/2B subunits, Src reduces voltage-independent inhibition by the divalent cation Zn2+. Thereby the function of recombinant NMDA receptors is potentiated by Src only when the Zn2+ level is sufficient to cause tonic inhibition. Here we investigated whether the Src-induced potentiation of NMDA receptor function in neurons is caused by reducing voltage-independent Zn2+ inhibition. Whereas chelating extracellular Zn2+ blocked the Src-induced potentiation of NR1-1a/2A receptors, we found that Zn2+ chelation did not affect the potentiation of NMDA receptor (NMDAR) currents by Src applied into hippocampal CA1 or CA3 neurons. Moreover, Src did not alter the Zn2+ concentration-inhibition relationship for NMDAR currents in CA1 or CA3 neurons. Also, chelating extracellular Zn2+ did not prevent the upregulation of NMDA single-channel activity by endogenous Src in membrane patches from spinal dorsal horn neurons. Taking these results together we conclude that Src-induced potentiation of NMDAR currents is not mediated by reducing Zn2+ inhibition in hippocampal and dorsal horn neurons.
Assuntos
Hipocampo/fisiologia , Potenciais da Membrana/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Medula Espinal/citologia , Medula Espinal/fisiologia , Zinco/farmacologia , Quinases da Família src/fisiologia , Animais , Eletrofisiologia , Hipocampo/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Medula Espinal/efeitos dos fármacosRESUMO
The N-methyl-D-aspartate (NMDA) receptor contributes to synaptic plasticity in the central nervous system and is both serine-threonine and tyrosine phosphorylated. In CA1 pyramidal neurons of the hippocampus, activators of protein kinase C (PKC) as well as the G-protein-coupled receptor ligands muscarine and lysophosphatidic acid enhanced NMDA-evoked currents. Unexpectedly, this effect was blocked by inhibitors of tyrosine kinases, including a Src required sequence and an antibody selective for Src itself. In neurons from mice lacking c-Src, PKC-dependent upregulation was absent. Thus, G-protein-coupled receptors can regulate NMDA receptor function indirectly through a PKC-dependent activation of the non-receptor tyrosine kinase (Src) signaling cascade.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Células Piramidais/efeitos dos fármacos , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G , Receptores Muscarínicos/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Transdução de Sinais/fisiologia , Alcaloides , Sequência de Aminoácidos , Animais , Benzofenantridinas , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Isoflavonas/farmacologia , Lisofosfolipídeos/farmacologia , Camundongos , Camundongos Knockout , Microinjeções , Dados de Sequência Molecular , Muscarina/farmacologia , Plasticidade Neuronal , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fenantridinas/farmacologia , Fenóis/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Proteínas Proto-Oncogênicas pp60(c-src)/deficiência , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Células Piramidais/fisiologia , Ratos , Ratos Wistar , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Ácidos Lisofosfatídicos , Receptores Muscarínicos/efeitos dos fármacos , Salicilatos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/química , Acetato de Tetradecanoilforbol/farmacologia , Xenopus laevis , meta-AminobenzoatosRESUMO
The ability of the constitutively active fragment of protein kinase C (PKM) to modulate N-methyl-D-aspartate (NMDA)-activated currents in cultured mouse hippocampal neurons and acutely isolated CA1 hippocampal neurons from postnatal rats was studied using patch-clamp techniques. The responses of two heterodimeric combinations of recombinant NMDA receptors (NR1a/NR2A and NR1a/NR2B) expressed in human embryonic kidney 293 cells were also examined. Intracellular applications of PKM potentiated NMDA-evoked currents in cultured and isolated CA1 hippocampal neurons. This potentiation was observed in the absence or presence of extracellular Ca2+ and was prevented by the coapplication of the inhibitory peptide protein kinase inhibitor(19-36). Furthermore, the PKM-induced potentiation was not a consequence of a reduction in the sensitivity of the currents to voltage-dependent blockade by extracellular Mg2+. We also found different sensitivities of the responses of recombinant NMDA receptors to the intracellular application of PKM. Some potentiation was observed with the NR1a/NR2A subunits, but none was observed with the NR1a/NR2B combination. Applications of PKM to inside-out patches taken from cultured neurons increased the probability of channel opening without changing single-channel current amplitudes or channel open times. Thus, the activation of protein kinase C is associated with potentiation of NMDA receptor function in hippocampal neurons largely through an increase in the probability of channel opening.
Assuntos
Hipocampo/metabolismo , Proteína Quinase C/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Potenciais Evocados , Hipocampo/embriologia , Hipocampo/crescimento & desenvolvimento , Humanos , Magnésio/metabolismo , Camundongos , N-Metilaspartato , Neurônios/metabolismo , Peptídeos/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Células Piramidais/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/químicaRESUMO
1. The effects of neomycin on NMDA-evoked currents in isolated CA1 hippocampal pyramidal neurones were investigated and single channel activity was examined in outside-out patches taken from cultured hippocampal neurones. The effects of neomycin on two combinations of NMDA receptor subunits (NR1a-NR2A and NR1a-NR2B) expressed in human embryonic kidney (HEK293) cells were also studied. 2. Neomycin (0. 01-1 mM) caused a potentiation of NMDA-activated currents in all neurones examined. No evidence of a voltage-dependent depression was observed in whole-cell recordings. 3. In outside-out patch recordings relatively low concentrations (30 and 100 microM) of neomycin caused a voltage-dependent reduction in single channel current amplitude as well as a large increase in the frequency of channel opening. 4. In saturating concentrations of glycine, neomycin enhanced NMDA-activated currents and this glycine-independent enhancement was confirmed using recombinant NR1a-NR2B receptors. Neomycin substantially increased the potency of glycine for the receptor by reducing the rate of dissociation of glycine from the receptor. Neomycin demonstrated a glycine-dependent enhancement of currents mediated by the NR1a-NR2A combination of subunits but a paradoxical depression was observed in saturating concentrations of glycine. 5. Neomycin increased the rate of deactivation of glutamate-activated currents consistent with neomycin causing a reduction in the affinity of the receptor for agonist. 6. These results indicate that neomycin has multiple and complex effects on NMDA receptors.