RESUMO
Calcium channels derived from sarcoplasmic reticulum of frog skeletal muscle were fused with planar lipid bilayers. Fractional open times displayed two types of calcium dependence: (i) blockable channels showed a bell-shaped calcium dependence with an activation constant of 4.5 microM, a Hill coefficient for activation of 1.46 and a blocking constant of 226 microM, and (ii) non-blockable channels displayed a sigmoidal calcium dependence with an activation constant of 1.1 microM and a Hill coefficient of 1.42; no blocking effect was seen with calcium up to 0.5 mM. These two types of calcium dependence may underlie the coexistence of two different pathways for calcium release in frog skeletal muscle.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Músculos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Anuros , Técnicas In Vitro , Ativação do Canal Iônico , Bicamadas Lipídicas , Músculos/ultraestruturaRESUMO
Endoplasmic reticulum vesicles isolated from rat brain cortex and fused with lipid bilayers displayed ryanodine-sensitive calcium channels, with three cytoplasmic calcium dependences. A: Channels (n=5) stimulated by Ca2+ (K0.5=1.2 microM and nHill=1.9) and not inhibited up to 0.5 mM Ca2+. B: Channels (n=14) cooperatively activated (K0.5=6.9 microM and nHill=1.8), and inhibited by Ca2+ (K0.5=152 microM and nHill=1.8). C: Low Po (<0.1) channels (n=22), non-cooperatively activated and inhibited with the same K0.5=26.3 microM Ca2+. These three types of responses to cytoplasmic [Ca2+] may underlie separate calcium release pathways in neurons of rat brain cortex.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Córtex Cerebral/metabolismo , Retículo Endoplasmático/metabolismo , Rianodina/farmacologia , Animais , Sítios de Ligação , Cálcio/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/ultraestrutura , Eletrofisiologia , Retículo Endoplasmático/efeitos dos fármacos , Fosfatos de Inositol/metabolismo , Bicamadas Lipídicas , Masculino , Fosfolipídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Rianodina/metabolismo , Saxitoxina/metabolismoRESUMO
The postsynaptic density is a highly dynamic structure, which is reorganized in an activity-dependent manner. An animal model for temporal lobe epilepsy, i.e. kainate-induced limbic seizures in rats, was used to study changes in postsynaptic density composition after extensive synaptic activity. Six hours after kainate injection, the protein content of the postsynaptic density fractions from rats that developed strong seizures was increased three-fold compared to saline-treated controls. Immunoblot analysis revealed that the relative amounts of metabotropic glutamate receptor 1alpha, N-ethylmaleimide-sensitive fusion protein, protein kinases C, Fyn and TrkB, as well as the neuronal nitric oxide synthase, were significantly higher in seizure-developing than in control rats. In contrast, the relative contents of the kainate receptor KA2 subunit, beta-actin, alpha-adducin and the membrane-associated guanylate kinase homolog SAP90/PSD-95 were decreased. The relative amounts of additional postsynaptic density proteins, including alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-D-aspartate receptor subunits, calcium/calmodulin-dependent kinase type II, casein kinase 2, tubulin, microtubule-associated protein 2B, the membrane-associated guanylate kinase homolog SAP102, and proline-rich synapse-associated protein 1/cortactin binding protein 1/Shank2 remained essentially unchanged. To assess possible changes in postsynaptic performance, postsynaptic densities were isolated from control and epileptic rats, incorporated into giant liposomes and N-methyl-D-aspartate receptor currents were recorded. A significant reduction in the mean conductance was observed in patches containing postsynaptic densities from animals with high seizure activity. This was due to the presence of reduced conductance levels in each membrane patch compared to control postsynaptic density preparations. From these data, we suggest that intense synaptic activity associated with seizures modifies the composition of postsynaptic densities and has profound consequences on the function of the N-methyl-D-aspartate receptors present in them. This rearrangement may accompany impairment of synaptic plasticity.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Prosencéfalo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsões/metabolismo , Membranas Sinápticas/metabolismo , Animais , Citoesqueleto/metabolismo , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/fisiopatologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Masculino , Proteínas do Tecido Nervoso/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fosforilação , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/fisiopatologia , Ratos , Ratos Wistar , Receptores de Ácido Caínico/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Proteínas Associadas SAP90-PSD95 , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Frações Subcelulares/metabolismo , Membranas Sinápticas/efeitos dos fármacos , Tirosina/metabolismoRESUMO
INTRODUCTION: Cisternal and ventricular blood predisposes to hydrocephalus and cerebral ischemia after high-grade aneurysmal subarachnoid hemorrhage (HGSAH). We studied the role of lamina terminalis fenestration combined with cisternal blood evacuation in HGSAH. PATIENTS/MATERIALS AND METHODS: A clinical, prospective, non-randomized study of a series of HGSAH patients (Modified Fisher>or=3) treated in the acute phase was carried out. The microsurgical treatment included aneurysm clipping, cisternal blood evacuation, and fenestration of the lamina terminalis. A comparable, non-blood-cleansed, endovascular-treated group, was included as a control. Clinical results were evaluated by the Glasgow Outcome Scale (GOS). RESULTS: During a period of 30 months, 95 patients who met the selection criteria were treated by microsurgical procedures and 28 by endovascular procedures. The distribution of GOS scores was superior for the microsurgical group: good results (GOS 4-5) were obtained in 85.3%, with a mortality rate of 5.9%. By contrast, 60.3% of patients in the endovascular group achieved GOS 4-5 scores, and 15.8% died. Good results for the endovascular group correlated inversely with delay of treatment. A permanent ventriculo-peritoneal shunt was necessary in 3.2% and 7.1% of the microsurgical and endovascular groups, respectively. The incidence of cerebral infarct was 3.1% and 14.3% for the microsurgical and endovascular groups, respectively. DISCUSSION: Microsurgical management reduces the usually poor outcome of patients with HGSAH. Lamina terminalis fenestration diminishes the incidence of shunt-dependent hydrocephalus and, combined with extensive cisternal blood cleansing, can lower the incidence of stroke. A procedure for cleansing blood and clots from the cisterns in HGSAH, based on the pathophysiology of vasospasm, is proposed.
Assuntos
Hipotálamo/cirurgia , Trombose Intracraniana/cirurgia , Microcirurgia/métodos , Hemorragia Subaracnóidea/cirurgia , Espaço Subaracnóideo/cirurgia , Ventriculostomia/métodos , Artérias Cerebrais/diagnóstico por imagem , Artérias Cerebrais/patologia , Artérias Cerebrais/fisiopatologia , Feminino , Humanos , Hidrocefalia/etiologia , Hipotálamo/anatomia & histologia , Hipotálamo/fisiologia , Trombose Intracraniana/etiologia , Trombose Intracraniana/fisiopatologia , Masculino , Estudos Prospectivos , Hemorragia Subaracnóidea/diagnóstico por imagem , Hemorragia Subaracnóidea/fisiopatologia , Espaço Subaracnóideo/diagnóstico por imagem , Espaço Subaracnóideo/fisiopatologia , Terceiro Ventrículo/anatomia & histologia , Terceiro Ventrículo/fisiologia , Terceiro Ventrículo/cirurgia , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Vasoespasmo Intracraniano/etiologia , Vasoespasmo Intracraniano/prevenção & controle , Vasoespasmo Intracraniano/cirurgiaRESUMO
El objetivo del tratamiento quirúrgico de los pacientes aquejados de disrrafia espinal es mantener su función neurológica estable y óptima durante toda su vida, sin embargo, existe un riesgo importante implícito en la cirugía. Con el objeto de investigar el impacto del monitoreo electrofisiológico intraoperatorio (MIO) en la evolución postoperatoria de estos pacientes, el cirujano practicó electromiografía libre y estimulada como retroalimentación continua del procedimiento. Las patologías operadas fueron: lipomas del fillum, lipomas del cono medular y re-anclajes de pacientes portadores de mielomeningocele al nacer. En todas las patologías se comparó la evolución post-operatoria con grupos de pacientes operados sin MIO, con el objeto de comparar sus evoluciones. Se observaron diferencias significativas entre los tres grupos de pacientes el uso de MIO evita el deterioro motor y la aparición de vejiga neurogénica en pacientes operados de lipoma del cono, evita la inclusión de raíces nerviosas en la sección del Fillum y evita el deterioro motor y ayuda a mejorar la función vesical de pacientes que se operan por re-anclaje medular por mielomeningocele. Finalmente, fue posible aplicar esta técnica en recién nacidos con mielomeningocele bajo, desarrollándose un protocolo que evita de facto el deterioro motor y debería evitar la inclusión de piel durante la tunelización de la placoda. Los datos mostrados en el presente trabajo permiten afirmar que esta técnica, por primera vez desarrollada en el sistema público chileno, así aplicada mejora muy significativamente los resultados de la cirugía de disrrafia espinal.
Assuntos
Humanos , Recém-Nascido , Disrafismo Espinal/cirurgia , Eletromiografia , Lipoma , Meningomielocele/cirurgia , Monitorização Fisiológica/instrumentação , Período Pós-Operatório , ChileRESUMO
The effect of halothane on calcium channels present in sarcoplasmic reticulum membranes isolated from frog skeletal muscle was studied at the single channel level after fusing the isolated vesicles into planar lipid bilayers. Addition of 91 microM halothane to the cytosolic compartment containing 1 microM free calcium activated the channel by increasing fractional open time from 0.11 to 0.59, without changing the channel conductance. The activation of the channels by halothane was calcium dependent. At resting calcium concentrations in the cytosolic compartment, halothane failed to activate the channel, whereas maximal activation was found at 10 microM calcium. The free energy of halothane binding to the channel decreased from -5.8 kcal/mol at 1 microM calcium to -6.6 kcal/mol at 10 microM calcium. Halothane increased the open time constants and decreased the closed time constants, indicating that it binds to both the open and the closed configurations of the channel.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Cálcio/farmacologia , Halotano/farmacologia , Músculos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Anuros , Condutividade Elétrica , Modelos Biológicos , Tempo de ReaçãoRESUMO
The calcium dependence of ryanodine-sensitive single calcium channels was studied after fusing with planar lipid bilayers sarcoendoplasmic reticulum vesicles isolated from excitable tissues. Native channels from mammalian or amphibian skeletal muscle displayed three different calcium dependencies, cardiac (C), mammalian skeletal (MS), and low fractional open times (low Po), as reported for channels from brain cortex. Native channels from cardiac muscle presented only the MS and C dependencies. Channels with the MS or low Po behaviors showed bell-shaped calcium dependencies, but the latter had fractional open times of <0.1 at all [Ca2+]. Channels with C calcium dependence were activated by [Ca2+] < 10 microM and were not inhibited by increasing cis [Ca2+] up to 0.5 mM. After oxidation with 2,2'-dithiodipyridine or thimerosal, channels with low Po or MS dependencies increased their activity. These channels modified their calcium dependencies sequentially, from low Po to MS and C, or from MS to C. Reduction with glutathione of channels with C dependence (native or oxidized) decreased their fractional open times in 0.5 mM cis [Ca2+], from near unity to 0.1-0.3. These results show that all native channels displayed at least two calcium dependencies regardless of their origin, and that these changed after treatment with redox reagents.
Assuntos
Cálcio/farmacologia , Retículo Endoplasmático/fisiologia , Fibras Musculares de Contração Rápida/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Retículo Sarcoplasmático/fisiologia , Animais , Anuros , Cálcio/metabolismo , Córtex Cerebral/fisiologia , Coração/fisiologia , Cinética , Bicamadas Lipídicas , Mamíferos , Fusão de Membrana , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Músculo Esquelético/fisiologia , Músculo Liso Vascular/fisiologia , Oxirredução , Coelhos , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Compostos de SulfidrilaRESUMO
The effects of redox reagents on the activity of the intracellular calcium release channels (ryanodine receptors) of skeletal and cardiac muscle, or brain cortex neurons, was examined. In lipid bilayer experiments, oxidizing agents (2,2'-dithiodipyridine or thimerosal) modified the calcium dependence of all single channels studied. After controlled oxidation channels became active at sub microM calcium concentrations and were not inhibited by increasing the calcium concentration to 0.5 mM. Subsequent reduction reversed these effects. Channels purified from amphibian skeletal muscle exhibited the same behavior, indicating that the SH groups responsible for modifying the calcium dependence belong to the channel protein. Parallel experiments that measured calcium release through these channels in sarcoplasmic reticulum vesicles showed that following oxidation, the channels were no longer inhibited by sub mM concentrations of Mg2+. It is proposed that channel redox state controls the high affinity sites responsible for calcium activation as well as the low affinity sites involved in Mg2+ inhibition of channel activity. The possible physiological and pathological implications of these results are discussed.
Assuntos
Córtex Cerebral/citologia , Miócitos Cardíacos/metabolismo , Neurônios/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Compostos de Sulfidrila/farmacologia , Animais , Anuros , Oxirredução , Coelhos , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
1. The modulation by Ca2+ of the activation by inositol 1,4,5-trisphosphate (IP3) of Ca2+ channels present in native sarcoplasmic reticulum membranes from frog skeletal muscle was studied after channel incorporation into planar phospholipid bilayers in the presence of Ca2+ or Ba2+ as current carrier species. 2. Channel activity expressed as fractional open time (Po) was low (less than or equal to 0.15) in the presence of varying free Ca2+ concentrations bathing the myoplasmic face of the channel (cis side), and did not increase significantly between 0.01 and 30 microM-Ca2+. 3. Channel activation mediated by IP3 could be elicited from free Ca2+ levels similar to those of resting skeletal muscle (about 0.1 microM) and was found to be strongly regulated by the free Ca2+ concentration present at the myoplasmic moiety of the channel. 4. Channel activation by 10 microM-IP3 depended on the Ca2+ concentration on the cis side. Po reached a maximum between pCa 7.0 and 6.0, but decreased at higher concentrations of free Ca2+. Thus, Ca2+ exerted a modulatory influence on IP3-mediated activation in a concentration range where the channel was insensitive to Ca2+. 5. The results indicate that Ca2+ ions act as modulators of IP3 efficacy to open the channel. This could arise from an interaction of Ca2+ with the channel gating mechanism or with the agonist binding site.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Inositol 1,4,5-Trifosfato/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Animais , Anuros , Bário/farmacologia , Sítios de Ligação , Cálcio/farmacologia , Relação Dose-Resposta a Droga , Técnicas In Vitro , CinéticaRESUMO
Adenosine 5'-cyclic diphosphoribose [cyclic ADP-ribose (cADPR)], a metabolite of NAD+ that promotes Ca2+ release from sea urchin egg homogenates and microsomal fractions, has been proposed to act as an endogenous agonist of Ca2+ release in sea urchin eggs. We describe experiments showing that a microsomal fraction isolated from Tetrapigus nyger sea urchin eggs displayed Ca(2+)-selective single channels with conductances of 155.0 +/- 8.0 pS in asymmetric Cs+ solutions and 47.5 +/- 1.1 pS in asymmetric Ca2+ solutions. These channels were sensitive to stimulation by Ca2+, ATP, and caffeine, but not inositol 1,4,5-trisphosphate, and were inhibited by ruthenium red. The channels were also activated by cADP-ribose in a Ca(2+)-dependent fashion. Calmodulin and Mg2+, but not heparin, modulated channel activity in the presence of cADP-ribose. We propose that these Ca2+ channels constitute the intracellular Ca(2+)-induced Ca2+ release pathway that is activated by cADP-ribose in sea urchin eggs.
Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Cafeína/farmacologia , Canais de Cálcio/metabolismo , Microssomos/metabolismo , Adenosina Difosfato Ribose/farmacologia , Animais , Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Calmodulina/antagonistas & inibidores , Calmodulina/farmacologia , ADP-Ribose Cíclica , Heparina/farmacologia , Técnicas In Vitro , Indicadores e Reagentes/farmacologia , Inositol 1,4,5-Trifosfato/farmacologia , Bicamadas Lipídicas/metabolismo , Magnésio/farmacologia , Microssomos/efeitos dos fármacos , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Rutênio Vermelho/farmacologia , Ouriços-do-Mar , Sulfonamidas/farmacologiaRESUMO
Sarcoplasmic reticulum vesicles isolated from fast-twitch frog skeletal muscle presented two classes of binding sites for ryanodine, one of high affinity (Kd1 = 1.7 nM, Bmax1 = 3.3 pmol per mg) and a second class with lower affinity (Kd2 = 90 nM, Bmax2 = 7.0 pmol per milligram). The calcium channels present in the sarcoplasmic reticulum membranes were studied in vesicles fused into lipid bilayers. Low concentrations of ryanodine (5 to 10 nM) activated a large conductance calcium channel after a short delay (5 to 10 min). The activation, which could be elicited from conditions of high or low fractional open time, was characterized by an increase in channel fractional open time without a change in conductance. The open and closed dwell time distributions were fitted with the sum of two exponentials in the range of 4 to 800 ms. The activating effect of ryanodine was due to an increase of both open time constants and a concomitant decrease in the closed time constants. Under conditions of low fractional open time (less than 0.1), the time spent in long closed periods (greater than 800 ms) between bursts was not affected by ryanodine. Higher concentrations of ryanodine (250 nM) locked the channel in a lower conductance level (approximately 40%) with a fractional open time near unity. These results suggest that the activating effects of nanomolar concentrations of ryanodine may arise from drug binding to high affinity sites. The expression of the lower conductance state obtained with higher concentrations of ryanodine may be associated with the low affinity binding sites observed in frog sarcoplasmic reticulum.
Assuntos
Alcaloides/metabolismo , Canais de Cálcio/fisiologia , Receptores Colinérgicos/metabolismo , Rianodina/metabolismo , Retículo Sarcoplasmático/fisiologia , Animais , Anuros , Canais de Cálcio/efeitos dos fármacos , Condutividade Elétrica , Cinética , Bicamadas Lipídicas , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina , Retículo Sarcoplasmático/efeitos dos fármacosRESUMO
The effects of redox reagents on the activity of the intracellular calcium release channels (ryanodine receptors) of skeletal and cardiac muscle, or brain cortex neurons, was examined. In lipid bilayer experiments, oxidizing agents (2,2'-dithiodipyridine or thimerosal) modified the calcium dependence of all single channels studied. After controlled oxidation channels became active at sub microM calcium concentrations and were not inhibited by increasing the calcium concentration to 0.5 mM. Subsequent reduction reversed these effects. Channels purified from amphibian skeletal muscle exhibited the same behavior, indicating that the SH groups responsible for modifying the calcium dependence belong to the channel protein. Parallel experiments that measured calcium release through these channels in sarcoplasmic reticulum vesicles showed that following oxidation, the channels were no longer inhibited by sub mM concentrations of Mg2+. It is proposed that channel redox state controls the high affinity sites responsible for calcium activation as well as the low affinity sites involved in Mg2+ inhibition of channel activity. The possible physiological and pathological implications of these results are discussed.