RESUMO
Electrical stimulation (ES) of skeletal muscle partially mimics the benefits of physical activity. However, the stimulation protocols applied clinically to date, often cause unpleasant symptoms and muscle fatigue. Here, we compared the efficiency of a "noisy" stimulus waveform derived from human electromyographic (EMG) muscle patterns, with stereotyped 45 and 1 Hz electrical stimulations applied to mouse myotubes in vitro. Human gastrocnemius medialis electromyograms recorded from volunteers during real locomotor activity were used as a template for a noisy stimulation, called EMGstim. The stimulus-induced electrical activity, intracellular Ca(2+) dynamics and mechanical twitches in the myotubes were assessed using whole-cell perforated patch-clamp, Ca(2+) imaging and optical visualization techniques. EMGstim was more efficient in inducing myotube cell firing, [Ca(2+)]i changes and contractions compared with more conventional electrical stimulation. Its stimulation strength was also much lower than the minimum required to induce contractions via stereotyped stimulation protocols. We conclude that muscle cells in vitro can be more efficiently depolarized using the "noisy" stochastic stimulation pattern, EMGstim, a finding that suggests a way to favor a higher level of electrical activity in a larger number of cells.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Estimulação Elétrica , Eletromiografia , Humanos , CamundongosRESUMO
Fura-2 imaging microscopy was used to study [Ca2+]i in nerve growth factor-differentiated PC12 cells exposed to agonists (bradykinin, carbamylcholine, and ATP) binding to receptors coupled to polyphosphoinositide hydrolysis. With all the treatments employed, the response to an individual agonist was often incomplete, i.e., composed of either release from intracellular stores or influx only. In individual cells the responses were closely similar when only one and the same agonist was employed, and markedly heterogeneous, with considerable variation of the release/influx ratio, when different agonists were delivered in sequence. In a recently isolated PC12 cell clone, heterogeneity of the receptor-induced [Ca2+]i responses was markedly lower than in the overall population, although the release/influx ratio was still variable. We conclude that the large response heterogeneity observed in the overall PC12 cell population is due (a) to the coexistence of multiple clones; and (b) to the variable activation of intracellular transduction mechanisms.
Assuntos
Cálcio/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Trifosfato de Adenosina , Bradicinina , Carbacol , Diferenciação Celular , Linhagem Celular , Membrana Celular/metabolismo , Células Clonais , Fura-2 , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Fatores de Crescimento Neural , Neurônios/metabolismoRESUMO
Spatial and temporal aspects of Ca2+ signaling were investigated in PC12 cells differentiated with nerve growth factor, the well known nerve cell model. Activation of receptors coupled to polyphosphoinositide hydrolysis gave rise in a high proportion of the cells to Ca2+ waves propagating non decrementally and at constant speed (2-4 microns/s at 18 degrees C and approximately 10-fold faster at 37 degrees C) along the neurites. These waves relied entirely on the release of Ca2+ from intracellular stores since they could be generated even when the cells were incubated in Ca(2+)-free medium. In contrast, when the cells were depolarized with high K+ in Ca(2+)-containing medium, increases of cytosolic Ca2+ occurred in the neurites but failed to evolve into waves. Depending on the receptor agonist employed (bradykinin and carbachol versus ATP) the orientation of the waves could be opposite, from the neurite tip to the cell body or vice versa, suggesting different and specific distribution of the responsible surface receptors. Cytosolic Ca2+ imaging results, together with studies of inositol 1,4,5-trisphosphate generation in intact cells and inositol 1,4,5-trisphosphate-induced Ca2+ release from microsomes, revealed the sustaining process of the waves to be discharge of Ca2+ from the inositol 1,4,5-trisphosphate- (and not the ryanodine-) sensitive stores distributed along the neurites. The activation of the cognate receptor appears to result from the coordinate action of the second messenger and Ca2+. Because of their properties and orientation, the waves could participate in the control of not only conventional cell activities, but also excitability and differential processing of inputs, and thus of electrochemical computation in nerve cells.
Assuntos
Cálcio/fisiologia , Condução Nervosa/fisiologia , Neuritos/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/fisiologia , Animais , Bradicinina/farmacologia , Carbacol/farmacologia , Diferenciação Celular , Polaridade Celular/fisiologia , Ativação Enzimática , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Microscopia de Fluorescência , Microscopia de Vídeo , Microssomos/metabolismo , Neuritos/efeitos dos fármacos , Células PC12 , Ratos , Fatores de Tempo , Fosfolipases Tipo C/análiseRESUMO
Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca2+]i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T. M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371-1380). To determine whether stimulation of [Ca2+]i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca2+]i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca2+]i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti-P- and anti-E-selectin mAb, as well as anti-VCAM-1 mAb, induced transient increases in EC [Ca2+]i that were comparable to those induced by 200 microM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca2+]i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLex), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca2+]i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLex and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca2+]i changes in the 5 h-treated EC, whereas the anti-VLA-4 mAb alone was sufficient to inhibit [Ca2+]i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti-PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca2+]i, i.e. , mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.
Assuntos
Moléculas de Adesão Celular/fisiologia , Selectina E/fisiologia , Endotélio Vascular/fisiologia , Selectina-P/fisiologia , Actinas/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Cálcio/metabolismo , Adesão Celular/fisiologia , Moléculas de Adesão Celular/imunologia , Células Cultivadas , Selectina E/imunologia , Fura-2/metabolismo , Histamina/farmacologia , Humanos , Integrina alfa4beta1 , Integrinas/imunologia , Leucócitos , Lipopolissacarídeos/farmacologia , Microscopia de Fluorescência , Músculo Liso Vascular/fisiologia , Oligossacarídeos/imunologia , Selectina-P/imunologia , Receptores de Retorno de Linfócitos/imunologia , Antígeno Sialil Lewis X , Molécula 1 de Adesão de Célula Vascular/imunologia , Molécula 1 de Adesão de Célula Vascular/fisiologiaRESUMO
AIMS: The autocrine activity of the embryonic isoform of the nicotinic acetylcholine receptor is crucial for the correct differentiation and trophism of skeletal muscle cells before innervation. The functional activity of extracellular adenosine and adenosine receptor subtypes expressed in differentiating myotubes is still unknown. In this study, we performed a detailed analysis of the role of adenosine receptor-mediated effects on the autocrine-mediated nicotinic acetylcholine receptor channel openings and the associated spontaneous intracellular calcium 'spikes' generated in differentiating mouse myotubes in vitro. METHODS: Cell-attached patch-clamp recordings and intracellular calcium imaging experiments were performed in contracting myotubes derived from mouse satellite cells. RESULTS: The endogenous extracellular adenosine and the adenosine receptor-mediated activity modulated the properties of the embryonic isoform of the nicotinic acetylcholine receptor in myotubes in vitro, by increasing the mean open time and the open probability of the ion channel, and sustaining nicotinic acetylcholine receptor-driven intracellular [Ca(2+) ]i 'spikes'. The pharmacological characterization of the adenosine receptor-mediated effects suggested a prevalent involvement of the A2B adenosine receptor subtype. CONCLUSION: We propose that the interplay between endogenous adenosine and nicotinic acetylcholine receptors represents a potential novel strategy to improve differentiation/regeneration of skeletal muscle.
Assuntos
Adenosina/metabolismo , Sinalização do Cálcio/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Técnicas de Patch-ClampRESUMO
Correlation between intraspecific phenotypic variability and variation of environmental conditions could reflect adaptation. Different phenotypes may result from differential expression of a genotype in different environments (phenotypic plasticity) or from expression of different genotypes (genetic diversity). Populations of Lacerta vivipara exhibit larger adult body length, lower age at maturity, higher fecundity, and smaller neonatal size in humid habitats compared to dry habitats. We conducted reciprocal transplants of juvenile L. vivipara to test for the genetic or plastic origin of this variation. We captured gravid females from four populations that differed in the relative humidity of their habitats, and during the last 2 to 4 weeks of gestation, we manipulated heat and water availability under laboratory conditions. Juveniles were released into the different populations and families were divided to compare growth rate and survival of half-sibs in two environments. Growth rate and survival were assessed using capture-recapture techniques. Growth rate was plastic in response to postnatal conditions and did not differ between populations of origin. Survival differed between populations of origin, partially because of differences in neonatal body length. The response of juvenile body length and body condition to selection in the different habitats was affected by the population of origin. This result cannot be simply interpreted in terms of adaptation; however, phenotypic plasticity of fecundity or juvenile size most probably resulted in adaptive reproductive strategies. Adaptation to the habitat by means of genetic specialization was not detected. Further investigation is needed to discriminate between genetic and long-term maternal effects.
Assuntos
Adaptação Fisiológica , Lagartos/fisiologia , Fenótipo , Animais , Meio Ambiente , Feminino , Fertilidade , Variação Genética , Lagartos/genética , Longevidade , Masculino , GravidezRESUMO
Lipid rich, soft plaques in the clinic are a common forerunner to occlusive thrombus formation, even with modest arterial stenosis. Animal models of atherosclerosis, obtained by various methods, do not generally allow direct in vivo evaluation of the lesion and, furthermore, cannot be examined more than once. The aim of the study was the generation of a rabbit model of atherosclerosis, with morphological characteristics similar to human lipid-rich, soft atheromatous plaques, and the evaluation of the reliability of intravascular ultrasound (IVUS) technology in the study of the development of atherosclerotic lesions in this model. Briefly, New Zealand white rabbits undergo perivascular electrical injury at both common carotid arteries, together with a 1.5% cholesterol diet for up to 90 days. The lesioned arterial segments show progressive changes, from diffuse cellular mortality, to macrophage infiltration in the media, up to the final migration of macrophages to the neointima, resulting in bulky, eccentric, macrophage and lipid-rich lesions. At IVUS, the produced lesions clearly resemble those described as 'soft plaques' in the clinical setting, with minimal calcification and reduced echo-reflectivity versus the adventitial layer. Quantitative and morphometric analysis of plaques shows a significant correlation between histological and IVUS measurements at each time point. In conclusion, vascular injury in the common carotids of rabbits generates atherosclerotic lipid-rich, soft plaques, that can be properly assessed by the IVUS methodology. The easy accessibility of the arterial lesion allows serial IVUS investigations and the direct evaluation of a number of locally or generally delivered therapeutic agents.
Assuntos
Arteriosclerose/diagnóstico por imagem , Arteriosclerose/patologia , Túnica Íntima/diagnóstico por imagem , Túnica Íntima/patologia , Ultrassonografia de Intervenção , Análise de Variância , Animais , Arteriopatias Oclusivas/diagnóstico por imagem , Arteriopatias Oclusivas/patologia , Colesterol na Dieta , Técnicas de Cultura , Modelos Animais de Doenças , Lipídeos/análise , Masculino , Probabilidade , Coelhos , Valores de Referência , Medição de Risco , Sensibilidade e Especificidade , Fatores de TempoRESUMO
The effect of human myasthenia gravis (MG) sera and complement on isolated adult rat muscle fibres was investigated. Heat-inactivated MG sera reduced the frequency of single acetylcholine receptor (AChR)-channel activity. One of the MG sera tested had a stronger effect on the extrajunctional type of AChRs than on the junctional type. The simultaneous addition of normal human serum (NHS), as source of complement, and MG serum to freshly dissociated muscle fibres caused contraction restricted to the endplate area and progressive depolarization of the muscle membrane, followed by contracture. An MG antibody-dependent complement-mediated damage of the muscle fibres is suggested.
Assuntos
Proteínas do Sistema Complemento/farmacologia , Músculos/efeitos dos fármacos , Miastenia Gravis/sangue , Animais , Técnicas de Cultura , Eletrofisiologia , Humanos , Masculino , Músculos/fisiologia , Ratos , Ratos WistarRESUMO
Various aspects of the rapidly exchanging intracellular Ca2+ stores of neurons and nerve cells are reviewed: their multiplicity, with separate sensitivity to either the second messenger, inositol 1,4,5-trisphosphate, or ryanodine-caffeine (the latter stores are probably activated via Ca(2+)-induced Ca2+ release); their control of the plasma membrane Ca2+ permeability, via the activation of a peculiar type of cation channels; their ability to sustain localized heterogeneities of the [Ca2+]i that could be of physiological key-importance. Finally, the molecular composition of these stores is discussed. They are shown (by high resolution immunocytochemistry and subcellular fractionation) to express: i) a Ca2+ ATPase responsible for the accumulation of the cation; ii) Ca2+ binding protein(s) of low affinity and high capacity to keep Ca2+ stored; and iii) a Ca2+ channel, activated by either one of the mechanisms mentioned above, to release Ca2+ to the cytosol. Results obtained in Purkinje neurons document the heterogeneity of the stores and the strategical distribution of the corresponding organelles (calciosomes; specialized portions of the ER) within the cell body, dendrites and dendritic spines.
Assuntos
Cálcio/metabolismo , Cálcio/fisiologia , Membranas Intracelulares/metabolismo , Neurônios/metabolismo , Animais , Humanos , Cinética , Organelas/metabolismoRESUMO
The action of forskolin (FSK), a stimulator of cAMP-dependent protein kinase (PKA), on nicotinic acetylcholine receptor-(nAChR-) channels was studied on cultured rat muscle fibres. The channel activity was estimated by determining Np, with N, being the number of channels and p, the single channel open probability. In order to elucidate the possible role of PKA in the modulation of nAChRs, FSK (10-50 microM) was added to the bath or to the pipette filling solution in the cell-attached configuration. The first protocol used was to test for indirectly-mediated cytosolic effects, the other, for any direct effects of the drug on nAChR-channels. Using both experimental protocols, no effects on the duration of single-channel openings or conductance were observed, while channel activity was significantly reduced. In particular, FSK 10 microM caused a reduction of Np only when applied to the non-patch membrane. FSK at higher concentrations, produced a more marked decrease of Np when present in the recording pipette. The present work provides evidence that the channel activity of muscle embryonic-type nAChRs can be influenced by a direct action of FSK, and is also significantly reduced by an indirectly-mediated cytosolic mechanism triggered by FSK.
Assuntos
Colforsina/farmacologia , Canais Iônicos/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Animais , Técnicas de Cultura , Embrião de Mamíferos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Ratos , Ratos WistarRESUMO
Neuromuscular junction development is the key process required for successful neuromuscular transmission and functional innervation of skeletal muscle fibres. Various substances can influence these processes, some of which are in common use in clinical practice. In the present study, the effects of the potentially new therapeutic agent agrin were followed, along with the widely used glucocorticoid dexamethasone. The in vitro experimental model used was functional innervation and constitutive interleukin 6 (IL-6) secretion of human muscle cells. Additionally, the selective relaxant binding agent sugammadex and its possible interaction with dexamethasone were followed. Dexamethasone impaired functional innervation while agrin had opposing effects. Furthermore, based on interference with IL-6 secretion, we show potential (chemical) interactions between dexamethasone and sugammadex. The physiological effects of this interaction should be taken into consideration under clinical conditions where these two drugs might be applied simultaneously.
Assuntos
Agrina/farmacologia , Dexametasona/farmacologia , Interleucina-6/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/inervação , gama-Ciclodextrinas/farmacologia , Animais , Interações Medicamentosas , Humanos , Músculo Esquelético/metabolismo , Mioblastos/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Ratos , Ratos Sprague-Dawley , SugammadexRESUMO
Yessotoxins (YTXs) are algal toxins originally included in the diarrheic toxins. After oral intake, YTXs induce only ultra-structural changes (packages of swollen mitochondria) in cardiac cells. The aim of this study was to investigate the possible effects of YTX on the other contractile striated tissue, the skeletal muscle, in vitro and in vivo. In vitro, in skeletal mouse myotubes, YTX (0.01-1.0 microM) influenced cell excitability in a concentration- and time-dependent way. In the in vivo study, transmission electron microscopy analysis did not reveal any ultrastructural alteration of skeletal muscle after acute (1 mg kg(-1)) or repeated (1 and 2mg kg(-1) day(-1), for 7 days) oral administration of YTX to mice. The observation that effects were detected in vitro but not in vivo supports the hypothesis of a low YTX bioavailability to skeletal muscle after oral intake. Therefore, the results seem to exclude a toxic effect in skeletal muscle when YTX is consumed as a food contaminant.
Assuntos
Músculo Esquelético/efeitos dos fármacos , Oxocinas/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Microscopia de Vídeo/métodos , Venenos de Moluscos/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/ultraestrutura , Células Satélites de Músculo Esquelético/efeitos dos fármacosRESUMO
In order to further understand the role of voltage- and ligand-gated ryanodine receptors in the control of intracellular Ca2+ signalling during myogenesis, changes in cytosolic free calcium concentration ([Ca2+]i) were investigated by fura-2 videoimaging in C2C12 mouse myotubes developing in vitro. A synchronous [Ca2+]i increase was observed after depolarisation with high [K+], while the Ca2+ response propagated as a wave following caffeine administration. Application of the two stimuli to the same myotube often revealed the existence of cellular zones that were responsive to depolarisation but not to caffeine. Focal application of high [K+] promoted a [Ca2+]i response detectable only in the cellular areas close to the pipette tip, while focal application of caffeine elicited a [Ca2+]i increase which spread as a Ca2+ wave. Buffering of [Ca2+]i by BAPTA did not affect the pattern of the depolarisation-induced [Ca2+]i transient but abolished the Ca2+ waves elicited by caffeine. When high [K+] and caffeine were applied in sequence, reciprocal inhibition of the [Ca2+]i responses was observed. Our results suggest that the different spatial patterns of [Ca2+]i responses are due to uneven distribution of voltage- and ligand-gated ryanodine receptors within the myotube. These two types of receptor control two functionally distinct Ca2+ pools which are part of a common intracellular compartment. Finally, the two differently operated ryanodine receptor channels appear to be independently activated, so that a mechanism of Ca2+-induced Ca2+ release is not required to sustain the global response in C2C12 myotubes.
Assuntos
Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Cafeína/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Líquido Intracelular/metabolismo , Ativação do Canal Iônico , Ligantes , Camundongos , Desenvolvimento Muscular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/crescimento & desenvolvimento , Potássio/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacosRESUMO
This paper is a short review of the fluorimetric methods used to measure intracellular free Ca++ concetration in living cells. The availability of fluorescent probes has greatly contributed to the understanding of the mechanisms responsible for the cellular homeostasys of this second messenger. Data can be collected from populations of cells by spectrofluorimetry or from small groups or single cells by spectromicroscopy. Finally the fluorescent images can be captured by a high sensitivity camera, digitally processed and convert in Ca++ images of the cell. The technique allows recognition of differences in [Ca++]i transients among adjacent cells in a same field or in different regions of a cell and greatly contributes to the identification of the cellular mechanisms modulating [Ca++]i.
RESUMO
The distribution and biophysical properties of acetylcholine receptors were studied, using morphological and patch-clamp techniques, in adult rat skeletal muscle fibers dissociated by collagenase and maintained in culture. Up to ten hours after dissociation, there were no changes in either the distribution or the biophysical properties of junctional acetylcholine receptors. In long-term culture (5 to 14 days), a new type of acetylcholine receptor was inserted all over the muscle fibers; the channel properties were characterized by a longer open time and a smaller conductance, similar to what has been observed in in vivo denervated muscles. Using autoradiography, we found that during culture an impaired incorporation of new acetylcholine receptors in the former endplates caused a progressive decrease in the density of junctional acetylcholine receptors. This contrasts with muscle fibers denervated in vivo, where the density of receptors does not change after denervation.
Assuntos
Músculos/química , Receptores Colinérgicos/análise , Receptores Colinérgicos/fisiologia , Animais , Autorradiografia , Células Cultivadas , Imuno-Histoquímica , Masculino , Placa Motora/fisiologia , Placa Motora/ultraestrutura , Denervação Muscular , Músculos/citologia , Músculos/ultraestrutura , Ratos , Ratos Wistar , Fatores de TempoRESUMO
This paper is a short review of the fluorimetric methods used to measure intracellular free Ca++ concentration in living cells. The availability of fluorescent probes has greatly contributed to the understanding of the mechanisms responsible for the cellular homeostasis of this second messenger. Data can be collected from populations of cells by spectrofluorimetry or from small groups or single cells by spectromicroscopy. Finally the fluorescent images can be captured by a high sensitivity camera, digitally processed and convert in Ca++ images of the cell. The technique allows recognition of differences in [Ca++]i transients among adjacent cells in a same field or in different regions of a cell and greatly contributes to the identification of the cellular mechanisms modulating [Ca++]i.
Assuntos
Cálcio/metabolismo , Fluorometria/métodos , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Espectrometria de Fluorescência , Gravação em VídeoRESUMO
In PC12 cells, activation of B2 receptors by bradykinin promotes an elevation of cytosolic Ca2+ by two mechanisms: IP3 mediated release of Ca2+ from intracellular stores and stimulation of multiple Ca2+ influx pathways. Since these events can have different kinetics and spatial localizations they can induce diversified effects in different cell areas.
Assuntos
Bradicinina/farmacologia , Cálcio/metabolismo , Receptores de Neurotransmissores/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Cafeína/farmacologia , Carbacol/farmacologia , Ácido Egtázico/farmacologia , Cinética , Fatores de Crescimento Neural/farmacologia , Células PC12 , Receptores da Bradicinina , Receptores de Neurotransmissores/efeitos dos fármacosRESUMO
Fluorescence videomicroscopy was used to monitor changes in the cytosolic free Ca2+ concentration ([Ca2+]i) in the mouse muscle cell line C2Cl2 during in vitro myogenesis. Three different patterns of changes in [Ca2+]i were observed: (i) [Ca2+]i oscillations; (ii) faster Ca2+ events confined to subcellular regions (localized [Ca2+]i spikes) and (iii) [Ca2+]i spikes detectable in the entire myotube (global [Ca2+]i spikes). [Ca2+]i oscillations and localized [Ca2+]i spikes were detectable following the appearance of caffeine-sensitivity in differentiating C2Cl2 cells. Global [Ca2+]i spikes appeared later in the process of myogenesis in cells exhibiting coupling between voltage-operated Ca2+ channels and ryanodine receptors. In contrast to [Ca2+]i oscillations and localized [Ca2+]i spikes, the global events immediately stopped when cells were perfused either with a Ca2+-free solution, or a solution with TTX, TEA and verapamil. To explore further the mechanism of the global [Ca2+]i spikes, membrane currents and fluorescence signals were measured simultaneously. These experiments revealed that global [Ca2+]i spikes were correlated with an inward current. Moreover, while the depletion of the Ca2+ stores blocked [Ca2+]i oscillations and localized [Ca2+]i spikes, it only reduced the amplitude of global [Ca2+]i spikes. It is suggested that, during the earlier stages of the myogenesis, spontaneous and repetitive [Ca2+]i changes may be based on cytosolic oscillatory mechanisms. The coupling between voltage-operated Ca2+ channels and ryanodine receptors seems to be the prerequisite for the appearance of global [Ca2+]i spikes triggered by a membrane oscillatory mechanism, which characterizes the later phases of the myogenic process.
Assuntos
Cálcio/metabolismo , Diferenciação Celular , Músculo Esquelético/fisiologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Apamina/farmacologia , Cafeína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Citosol/metabolismo , Ácido Egtázico/farmacologia , Cinética , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Microscopia Confocal , Microscopia de Vídeo , Músculo Esquelético/citologia , Oscilometria , Cloreto de Potássio/farmacologia , Tetraetilamônio , Compostos de Tetraetilamônio/farmacologia , Tetrodotoxina/farmacologia , Verapamil/farmacologiaRESUMO
PURPOSE: Iomeprol, a new nonionic iodinated compound for intravascular use, is being evaluated as a myelographic contrast agent because of its low neurotoxicity. This study aimed to assess the degree of brain penetration of iomeprol after intrathecal administration. MATERIAL AND METHODS: Brain penetration in dogs was investigated by CT and compared with that of iopamidol, iohexol, and ioversol, currently used as myelographic contrast media (CM). Nervous tissue density was determined in different brain structures by recording Hounsfield values. RESULTS: The experiments revealed that CM diffused from the cisternae into the parenchyma, reaching a maximum at 5-24 h after injection. The density of the examined brain regions was still higher than the preinjection levels 24 h later. No differences in brain penetration were observed among the CM investigated. CONCLUSION: The study has shown that iomeprol penetrates into the brain to the same extent as the most widely used myelographic CM.
Assuntos
Encéfalo/metabolismo , Meios de Contraste/farmacocinética , Iopamidol/análogos & derivados , Animais , Tronco Encefálico/metabolismo , Córtex Cerebral/metabolismo , Meios de Contraste/administração & dosagem , Cães , Feminino , Injeções Espinhais , Iopamidol/administração & dosagem , Iopamidol/farmacocinéticaRESUMO
The effect of interleukin-2 (rIL-2) on nicotinic acetylcholine receptors (nAChR) was examined on cultured muscle fibres isolated from the flexor digitorum brevis muscle (FDB) of the rat and on aneural mouse cultured C2 myotubes. Intracellular measurement of the sensitivity to iontophoretically applied ACh demonstrated that the sensitivity of the extrajunctional nAChRs in cultured fibres showed a transient increase after application of rIL-2 (2,000-3,000 units/ml). Cell-attached patch-clamp experiments on the same fibres proved that rIL-2 (2,000 units/ml) induces a significant increase in the mean open time of the extrajunctional nAChR channel. The other channel parameters were not significantly modified. The same applied also to aneural mouse patch-clamped C2 myotubes exposed to rIL-2 (2,000 units/ml). In freshly dissociated fibres no effects on nAChR channels were observed following rIL-2 application. 125I-rIL-2 binding experiments on either 7-day cultured or freshly dissociated adult muscle fibres showed that a specific binding with a Kd of 2.07 +/- 0.4 nM develops in cultured fibres but fails to occur immediately after dissociation. It is concluded that rIL-2 modulates the duration of extrajunctional nAChR channels in both myotubes and adult muscle cells, and that this effect is probably due to the activation of a second messenger system.