RESUMO
Genetic variability in ten populations of wild-growing ginseng was assessed using AFLP markers with the application of fragment analysis on a genetic analyzer. The variation indices were high in the populations (P = 55.68%; H(S) = 0.1891) and for the species (P = 99.65%; H(S) = 0.2857). Considerable and statistically significant population differentiation was demonstrated (theta = 0.363; Bayesian approach, "full model"; F(ST) = 0.36, AMOVA). The results of AMOVA and Bayesian analysis indicate that 64.46% of variability is found within the populations. Mantel test showed no correlation between the genetic and geographic distances among the populations (r = -0.174; P = 0.817). Hierarchical AMOVA and analysis of genetic relationships based on Euclidean distances (NJ, PCoA, and MST) identified two divergent population groups of ginseng. Low gene flow between these groups (N(m) = 0.4) suggests their demographic independence. In accordance to the concept of evolutionary significant units (ESU), these population groups, in terms of the strategy and tactics for conservation and management of natural resources, should be treated as management units (MUs). The MS tree topology suggests recolonization of southern Sikhote-Alin by ginseng along two directions, from south and west.
Assuntos
Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Variação Genética , Panax/genética , Genética Populacional , Filogeografia , Federação RussaRESUMO
OBJECTIVE: Many risk factors associated with deep infections after primary shoulder arthroplasty remain controversial and have not yet been summarized. As such, the aim of the present study was to quantitatively summarize the risk factors associated with deep infections after primary shoulder arthroplasty. MATERIALS AND METHODS: Computerized and additional manual searches on the Medline, Embase, Chinese National Knowledge Infrastructure (CNKI), and Cochrane central database for potential studies, published from inception to March 2022, were performed. All studies that assessed risk factors for deep infection after primary shoulder arthroplasty were selected without language restrictions. Eligible studies were required to fulfill quality assessment criteria from the Consort statement and to evaluate risk factors for deep infection after primary shoulder arthroplasty. Two reviewers independently extracted the relevant data, with disagreements resolved by consensus. Statistical analyses were performed using Stata version 11.0 (Statacorp LLC, College Station, TX, USA). RESULTS: Seven studies including 493,148 patients who underwent primary shoulder arthroplasty, among whom 1,314 experienced infection (0.3%), were eligible and included in this meta-analysis. Meta-analysis revealed that signiï¬cantly increased risk factors for infection after primary shoulder arthroplasty included male sex (odds ratio [OR] 1.79 [95% confidence interval (CI) 1.23-2.60]), avascular necrosis (OR 2.64 [95% CI 1.61-4.34]), rotator cuff arthropathy (OR 2.14 [95% CI 1.55-2.95]), proximal humerus fracture (OR 2.68 [95% CI 1.93-3.73]), and non-union of humerus fracture (OR 5.32 [95% CI 3.52-8.02]). In contrast, advanced age was associated with a decreased likelihood for development of infection (OR 0.97 [95% CI 0.94-1]). CONCLUSIONS: Surgeons should devote close attention to the above-mentioned medical conditions to reduce deep infection after primary shoulder arthroplasty.
Assuntos
Artroplastia do Ombro , Articulação do Ombro , Artroplastia do Ombro/efeitos adversos , Humanos , Incidência , Masculino , Razão de Chances , Estudos Retrospectivos , Fatores de Risco , Manguito Rotador , Articulação do Ombro/cirurgia , Resultado do TratamentoRESUMO
We have used computer-assisted reconstructions of continuous serial sections to study the cytoplasmic organization of growth cones in vivo. Optic tecta from 6.25-6.5-d-old chicken embryos were quick-frozen and then freeze-substituted in acetone-osmium tetroxide or, for comparison, prepared by conventional fixation. Images of eight freeze-substituted and two conventionally fixed growth cones were reconstructed from aligned serial micrographs. After freeze-substitution, numerous lumenless membrane-bound sacs arrayed in multilamellar stacks appear to replace the abundant smooth endoplasmic reticulum found after chemical fixation. Microtubule fascicles progressively diverge from their typical fascicular organization in the initial segment of the growth cone and are absent in the varicosity and the more distal segment. Mitochondria, in contrast, are concentrated in the proximal segment of the varicosity; multilamellar stacks and endosome-like vacuoles are in the distal segment; and coated pits and vesicles are concentrated near the terminal filopodium, which is the most distal and organelle-poor domain of the growth cone. These observations suggest that dilation and fusion of the lumenless, membrane-bound sacs that occurs during chemical fixation give rise to the network of smooth endoplasmic reticulum. The three-dimensional reconstructions show that the cytoplasmic components of growth cones, including the membrane-bound sacs and multilamellar stacks revealed by freeze substitution, are polarized along the axis of these growth cones, which suggests that they have a role in recycling of membrane during elongation of the growth cone.
Assuntos
Compartimento Celular , Neurônios/ultraestrutura , Organoides/ultraestrutura , Colículos Superiores/ultraestrutura , Animais , Embrião de Galinha , Invaginações Revestidas da Membrana Celular/ultraestrutura , Computadores , Fixadores , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Modelos Anatômicos , Manejo de Espécimes , Colículos Superiores/embriologia , Vacúolos/ultraestruturaRESUMO
Immunocytochemistry has been used to study the distribution of the major 180,000-mol wt protein of coated vesicles in rodent cerebellum. An antibody to the coat protein was prepared in rabbits and characterized by immunodiffusion and immunofixation of polyacrylamide gels. At the light microscope level the protein was primarily localized in punctate profiles surrounding Purkinje cells and within the cerebellar glomeruli. At the electron microscope level the punctate distribution was confined to presynaptic terminals of basket and Golgi II neurons as well as mossy fiber terminals of the glomeruli. This label was heaviest on the lattice coat of coated vesicles but, in addition, label was found within the presynaptic axoplasm and along the cytoplasmic surface of the plasmalemma. Coated vesicles in cell somata were labeled as well as the cytosol around groupings of these vesicles. These data suggest that there may be two forms (or more) of coated vesicle protein in neurons, a lattice form associated with coated vesicles and a soluble form associated with the cytoplasmic matrix.
Assuntos
Encéfalo/metabolismo , Grânulos Citoplasmáticos/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Encéfalo/ultraestrutura , Córtex Cerebelar/ultraestrutura , Clatrina , Feminino , Técnicas Imunológicas , Microscopia Eletrônica , Peso Molecular , Terminações Nervosas/ultraestrutura , Células de Purkinje/ultraestrutura , CoelhosRESUMO
Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by gammac-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Interleucina-2/fisiologia , Proteínas do Leite , Proteínas Tirosina Quinases/fisiologia , Transdução de Sinais , Transativadores/fisiologia , Animais , Linhagem Celular , Ativação Enzimática , Precursores Enzimáticos/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Janus Quinase 3 , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Fosforilação , Receptores de Interleucina-2/fisiologia , Fator de Transcrição STAT5 , Quinase Syk , Proteínas Supressoras de TumorRESUMO
Regulatory effects of extracellular magnesium ions ([Mg2+]o) on intracellular free ionized calcium ([Ca2+]i) were studied in cultured vascular smooth muscle cells (VSMCs) from rat aorta by use of the fluorescent indicator fura-2 and digital imaging microscopy. With normal Mg2+ (1.2 mM)-containing incubation media, [Ca2+]i in VSMCs was 93.6 +/- 7.93 nM with a heterogeneous cellular distribution. Lowering [Mg2+]o to 0 mM or 0.3 mM (the lowest physiological range) resulted in 5.8-fold (579.5 +/- 39.99 nM) and 3.5-fold (348.0 +/- 31.52 nM) increments of [Ca2+]i, respectively, without influencing the cellular distribution of [Ca2+]i. Surprisingly, [Mg2+]o withdrawal induced changes of cell geometry in many VSMCs, i.e., the cells rounded up. However, elevation of [Mg2+]o up to 4.8 mM only induced slight decrements of [Ca2+]i (mean = 72.0 +/- 4.55 nM). The large increment of [Ca2+]i induced by [Mg2+]o withdrawal was totally inhibited when [Ca2+]o was removed. The data suggest that: (1) [Mg2+]o regulates the level of [Ca2+]i in rat aortic smooth muscle cells, and (2) [Mg2+] acts as an important regulatory ion by modulating cell shapes in cultured VSMc and their metabolism to control vascular contractile activities.
Assuntos
Cálcio/fisiologia , Magnésio/fisiologia , Músculo Liso Vascular/fisiologia , Animais , Aorta/citologia , Células Cultivadas , Citoplasma/metabolismo , Técnicas In Vitro , Músculo Liso Vascular/citologia , RatosRESUMO
Primary cultures of rat dorsal root ganglionic (DRG) cells were stained with isoform-specific antibodies against non-muscle myosin II. Antibodies against the brain type myosin (MIIB) stained the peripheries of growth cones and non-neuronal cells. Double staining of the cells with the anti-myosin antibodies and rhodamine-phalloidin or anti-actin antibodies indicated that MIIB co-exists, with F-actin, at the leading edge. Antibodies against platelet myosin stained neither leading edges nor neurites, but stained the cell bodies of neurons and the stress fibers of non-neuronal cells. These results suggest that MIIB functions in the motility of the leading edge of DRG cells.
Assuntos
Gânglios Espinais/química , Miosinas/análise , Neuritos/química , Neurônios/química , Animais , Divisão Celular , Células Cultivadas , Imunofluorescência , Gânglios Espinais/citologia , Neurônios/citologia , RatosRESUMO
Established methods for cryopreservation of living cells were modified for freeze-storage of postnatal retinal ganglion cells from rat. Retinal cell suspensions containing fluorescently labeled ganglion cells were frozen after addition of 8% dimethyl sulfoxide and stored at -80 degrees C for up to 66 days. Viability of identified retinal ganglion cells was assessed by their ability to take up and cleave fluorescein diacetate to fluorescein. No significant difference was found in the number of living retinal ganglion cells when cells obtained from the same dissociation were counted before and after freezing (6.65 +/- 2.37 x 10(4) vs 7.05 +/- 3.67 x 10(4) retinal ganglion cells per ml, respectively; mean +/- S.D., n = 4). In culture following cryopreservation, the cells appeared morphologically normal, and developed neurites and growth cones similar to their freshly dissociated counterparts. Since very little is known about the electrophysiology and membrane properties of neurons after cryopreservation, we used the whole-cell configuration of the patch-clamp technique to study voltage- and ligand-gated conductances in cryopreserved retinal ganglion cells. The cryopreserved retinal ganglion cells studied under current-clamp maintained resting potentials of -60.9 +/- 6.6 mV (n = 10) and upon depolarization fired action potentials. During voltage-clamp in the whole-cell mode, depolarizing voltage steps activated Na(+)-(INa), Ca(2+)-(ICa), and K(+)-currents in all cells tested (n = 122). INa could be reversibly blocked by 1 microM tetrodotoxin added to the external solution. ICa was blocked by external 250 microM Cd2+ or 3 mM Co2+. In some cells, ICa consisted of both a transient and prolonged component. The outward K(+)-current consisted of Ca(2+)-dependent and -independent components. The Ca(2+)-insensitive portion of the K+ outward current was separated into four distinct components based upon pharmacological sensitivity and biophysical properties. In many cells, a rapidly inactivating current similar to the A-type K(+)-current (IA) observed in freshly cultured retinal ganglion cells was isolated by its greater sensitivity to 4-aminopyridine (5 mM) than to tetraethylammonium (20 mM). A tetraethylammonium-sensitive current with a more prolonged time course reminiscent of IK, the delayed rectifier, was also found. When the 4-aminopyridine- and tetraethylammonium-insensitive portions of the outward current were further analysed with voltage protocols, an additional slowly decaying potassium current became apparent. The inhibitory amino acids, GABA (20 microM) and glycine (100 microM), activated chloride-selective currents that were selectively blocked by bicuculline methiodide (10 microM) and strychnine (5 microM), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Canais Iônicos/fisiologia , Células Ganglionares da Retina/metabolismo , Acetilcolina/farmacologia , Aminoácidos/antagonistas & inibidores , Aminoácidos/farmacologia , Animais , Células Cultivadas , Criopreservação , Dimetil Sulfóxido , Eletrodos , Eletrofisiologia , Canais Iônicos/efeitos dos fármacos , Ligantes , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , RatosRESUMO
Interaction of ionized magnesium ([Mg2+]o) and caffeine in regulation of intracellular free calcium concentration ([Ca2+]i) in human aortic endothelial cells was studied using fura-2 and digital imaging microscopy. In 1.2 mM [Mg2+]o, basal [Ca2+]i was 73.7 +/- 22.4 nM, with a heterogeneous distribution within the cells. No significant changes of basal [Ca2+]i were found either when cells were treated with 10 mM caffeine or when [Mg2+]o was lowered from 1.2 mM to 0.3 mM. However, a combined superfusion of the cells with 0.3 mM [Mg2+]o and 10 mM caffeine resulted in a significant elevation of [Ca2+]i to 382.8 +/- 57.1 nM, probably by release of Ca2+ from internal stores, which was attenuated by NiCl2 (1 mM). These results suggest that a Ca(2+)-induced Ca2+ release mechanism is involved in regulation of [Ca2+]i in endothelial cells, which may be either regulated or modulated by Mg2+.
Assuntos
Cafeína/farmacologia , Cálcio/metabolismo , Endotélio Vascular/metabolismo , Magnésio/farmacologia , Aorta/citologia , Aorta/metabolismo , Linhagem Celular , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Fura-2 , HumanosRESUMO
Immunofixation of sodium lauryl sulphate (SDS)-acrylamide gels has been used to study the distribution of the major protein (clathrin) of coated vesicles in various compartments of synaptic terminals. Synaptosomal subcellular fractions were isolated and purified from pig brain homogenates by the procedure of Ueda et al. and lysed in 6 mM Tris-Cl buffer at pH 6.6, 7.8, and 8.1. The synaptosomal particulate and soluble fractions were separated by centrifugation. The synaptic junctional complex (SJC) and postsynaptic density (PSD) fractions were obtained by detergent treatment of the synaptic plasma membrane (SPM). The synaptosomal subcellular fractions and purified coated vesicle (PCV) fractions were subjected to SDS gel electrophoresis (7.5%). The resulting slabs were divided vertically into 4 segments which were stained with Coomassie blue dye, or immunofixed with preimmune and anti-clathrin serum, or affinity labeled with concanavalin A (Con A)-peroxidase. The Comassie blue stained gel indicated the presence of 180,000-molecular weight band in gels of most synaptosomal subcellular fractions. However, immunofixation of an identical gel revealed positive staining of the 180,000-molecular weight protein in PCV, synaptosomal (SF), SPM and synaptoplasmic (SS) fractions only. These findings not only support the contention that a pool of cytosolic-coated vesicle protein is localized at synaptic terminals, they also indicate that clathrin appears highly unlikely to contribute to the structural frameworks of the SJC and PSD of mature synapses.
Assuntos
Química Encefálica , Proteínas de Membrana/análise , Sinaptossomos/análise , Animais , Clatrina , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Dodecilsulfato de Sódio , Frações Subcelulares/análise , SuínosRESUMO
The whole-cell configuration of the patch-clamp technique was used to study nicotinic acetylcholine (ACh) responses in freshly dissociated dorsal root ganglion (DRG) cells from postnatal rat. At negative holding potentials with physiological solutions in the bath and the pipette, ACh (20 microM), nicotine (5 microM) or DMPP (20 microM) activated inward currents in 51% of the cells. Average current density was higher in 1-month-old compared to newborn animals. Nicotinic agonist-induced currents were unaffected by atropine (10 microM) but reversibly blocked by hexamethonium (20 microM). Although labeling with fluorescent alpha-bungarotoxin (BGT) demonstrated the presence of toxin binding sites on DRG cells, DMPP-induced inward currents were unaffected by micromolar BGT. Neuronal bungarotoxin (100 nM), in contrast, led to a largely irreversible block of the nicotinic responses. These results show that postnatal DRG cells express functional nicotinic acetylcholine receptors (nAChR) of a neuronal type.
Assuntos
Gânglios Espinais/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Receptores Nicotínicos/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Bungarotoxinas , Células Cultivadas , Corantes Fluorescentes , Potenciais da Membrana/efeitos dos fármacosRESUMO
Using digital imaging microscopy and fluorescent probes, isolated hippocampal CA1 pyramidal neurons of the guinea-pig were used to examine the roles of [Mg2+]o in regulation of [Ca2+]i and [Mg2+]i. Low extracellular Mg ([Mg2+]o) (0.3 mM) significantly increased [Ca2+]i compared to 1.2 and 4.8 mM [Mg2+]o. In contrast, [Mg2+]i levels remained relatively constant, irrespective of alterations of [Mg2+]o. The sustained rise in [Ca2+]i induced by low [Mg2+]o was reduced 70% by 1 microM verapamil and 42% by 1 mM Ni2+, and completely abolished by 5 mM Ni2+. The data suggest that [Mg2+]o regulates [Ca2+]i in hippocampal neurons, probably by modulating Ca2+ entry via voltage-sensitive Ca2+ channels, which may play important roles in epileptogenesis, memory, learning and brain trauma. Furthermore, the results demonstrate that intracellular Mg2+ concentration does not follow passively the concentration of Mg2+ in the extracellular solution.
Assuntos
Cálcio/metabolismo , Hipocampo/efeitos dos fármacos , Magnésio/farmacologia , Células Piramidais/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Cobaias , Verapamil/farmacologiaRESUMO
Acute exposure of cultured canine cerebral vascular smooth muscle cells to low concentrations of cocaine HCl (10(-9) to (10(-7) M) resulted in significant, rapid (1 min) loss of intracellular free Mg ions ([Mg2+]i); these reductions (12-25%) in [Mg2+]i were reversible upon exposure to normal, Mg(2+)-containing physiological salt solution. These findings help to provide a rational basis for why cocaine can result in cerebrovasospasm and stroke.
Assuntos
Encéfalo/irrigação sanguínea , Cocaína/farmacologia , Magnésio/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Animais , Células Cultivadas , Transtornos Cerebrovasculares/induzido quimicamente , Transtornos Cerebrovasculares/metabolismo , Cães , Hipóxia Encefálica/induzido quimicamente , Ataque Isquêmico Transitório/induzido quimicamente , Espectroscopia de Ressonância Magnética , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , FósforoRESUMO
Chronic exposure of cultured piglet neonatal coronary arterial smooth muscle cells to low concentrations of ethanol (46-115 mg/dl) for 7 days resulted in concentration-dependent elevation in intracellular free Ca2+ ions ([Ca2+i); these rises (22-56%) in [Ca2+]i were not reversible upon short-term exposure to normal, Ca2(+)-containing physiological salt solution. These findings help to provide a rational basis for why ethanol can result in the well-known fetal alcohol syndrome, including cardiac defects and in-utero death.
Assuntos
Cálcio/metabolismo , Vasoespasmo Coronário/prevenção & controle , Vasos Coronários/efeitos dos fármacos , Etanol/farmacologia , Transtornos do Espectro Alcoólico Fetal/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Animais , Células Cultivadas , Vasoespasmo Coronário/induzido quimicamente , Vasoespasmo Coronário/metabolismo , Vasos Coronários/citologia , Vasos Coronários/metabolismo , Hipóxia Fetal/induzido quimicamente , Hipóxia Fetal/metabolismo , Hipóxia Fetal/prevenção & controle , Técnicas In Vitro , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Isquemia Miocárdica/induzido quimicamente , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/prevenção & controle , SuínosRESUMO
Effects of cocaine on intracellular free calcium concentration ([Ca2+]i) in cultured canine cerebral vascular smooth muscle cells were studied using digital imaging microscopy and the calcium molecular fluorescent indicator, fura-2. Acute treatment of cerebral vascular smooth muscle cells with cocaine HCl, from a low concentration of 10(-9) M up to 10(-5) M, induced significant increases of [Ca2+]i. Irrespective of the changes in [Ca2+]i, the subcellular distribution of [Ca2+]i appeared heterogeneous in both normal and cocaine-treated cells. These results suggest that cocaine induces cerebral vasospasm by a rapid elevation of [Ca2+]i in vascular smooth muscle cells; these ionic events could play a crucial role in the pathogenesis of cocaine-induced cerebral ischemia and stroke.
Assuntos
Cálcio/metabolismo , Circulação Cerebrovascular/efeitos dos fármacos , Cocaína/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Animais , Transtornos Cerebrovasculares/etiologia , Cães , Relação Dose-Resposta a Droga , MasculinoRESUMO
Ninety-eight patients admitted to the emergency rooms of three urban hospitals with a diagnosis of either ischemic stroke or hemorrhagic stroke exhibited early and significant deficits in serum ionized Mg2+ (IMg2+), but not total Mg, as measured with a unique Mg2+-sensitive ion-selective electrode. Twenty-five percent of these stroke patients exhibited >65% reductions in the mean serum IMg2+ found in normal healthy human volunteers or patients admitted for minor bruises, cuts or deep lacerations. The stroke patients also demonstrated significant elevation in the serum ionized Ca2+ (ICa2+)/IMg2+ ratio, a sign of increased vascular tone and cerebrovasospasm. Exposure of primary cultured canine cerebral vascular smooth muscle cells to the low concentrations of IMg2+ found in the stroke patients, e.g. 0.30-0.48 mM, resulted in rapid and marked elevations in cytosolic free calcium ions ([Ca2+]i) as measured with the fluorescent probe, fura-2, and digital image analysis. Coincident with the rise in [Ca2+]i, many of the cerebral vascular cells went into spasm. Reintroduction of normal extracellular Mg2+ ion concentrations failed to either lower the [Ca2+]i overload or reverse the rounding-up of the cerebral vascular cells. These results suggest that changes in Mg2+ metabolism play important roles in stroke syndromes and in the etiology of cerebrovasospasm associated with cerebral hemorrhage.
Assuntos
Cálcio/metabolismo , Transtornos Cerebrovasculares/sangue , Magnésio/sangue , Músculo Liso Vascular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Artéria Basilar/metabolismo , Biomarcadores/sangue , Isquemia Encefálica/sangue , Células Cultivadas , Artérias Cerebrais/metabolismo , Hemorragia Cerebral/sangue , Meios de Cultura , Citosol/metabolismo , Cães , Eletroquímica , Feminino , Humanos , Magnésio/farmacologia , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/efeitos dos fármacos , Valores de Referência , Medição de Risco , Sensibilidade e EspecificidadeRESUMO
The acute effects of low concentrations of ethanol on intracellular free magnesium ions ([Mg2+]i) in cultured type-2 astrocytes were studied by digital imaging microscopy using the Mg2+ fluorescent probe, mag-fura-2. In 0-mM ethanol, the basal level of [Mg+]i was 124.7+/-2.56 microM with a heterogeneous distribution within the cells. Treatment of the cells with 10 and 25 mM ethanol (10 min) resulted in rapid concentration-dependent reduction in [Mg2+]i; the greater the concentration of alcohol, the greater the depletion of [Mg2+]i. Exposure of cells to 10 and 25 mM resulted in approximately 27 and 50% reductions in [Mg2+]i, respectively. Reincubation in normal Mg2+-physiological buffer solution restored [Mg2+]i levels. These observations may suggest that acute "binge drinking" of ethanol, which often results in cerebral ischemia and stroke, may do so as a result of depletion of astrocytic [Mg2+]i, possibly producing disruption of the blood-brain barrier.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Deficiência de Magnésio/induzido quimicamente , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Células Cultivadas , Citosol/metabolismo , Relação Dose-Resposta a Droga , Magnésio/metabolismo , Deficiência de Magnésio/metabolismo , Ratos , Ratos Wistar , Acidente Vascular Cerebral/metabolismoRESUMO
The acute effects of ethanol on intracellular free magnesium ions ([Mg2+]i) in cultured canine cerebral vascular smooth muscle cells (VSMCs) were studied by digital imaging microscopy using the Mg2+ fluorescent probe mag-fura-2. In 0 mM ethanol, the basal level of [Mg2+]i was between 500-700 microM with a heterogeneous distribution within the cells; [Mg2+]i was greater in the perinuclear than in the peripheral region. Treatment of the cells with 10, 25, and 100 mM ethanol resulted in rapid (within 30 s) concentration-dependent reduction in [Mg2+]i; the greater the concentration and the greater the duration of acute exposure, the greater the fall in [Mg2+]i. Exposure of cerebral VSMCs to 100 mM ethanol resulted in a 57% reduction in [Mg2+]i (i.e., from 510 +/- 40 to 220 +/- 30 microM). These observations are consistent with the tenet that "binge drinking" of ethanol could result in cerebrovasospasm, ischemia, and rupture of cerebral blood vessels as a consequence of depletion of cerebral VSMC [Mg2+]i. Deficits in [Mg2+]i, O2, and nutrient delivery could account in part for some of the behavioral actions of alcohol.
Assuntos
Circulação Cerebrovascular , Etanol/farmacologia , Membranas Intracelulares/metabolismo , Magnésio/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Células Cultivadas , Transtornos Cerebrovasculares/induzido quimicamente , Cães , Masculino , Músculo Liso Vascular/citologiaRESUMO
Quantitative digital imaging microscopy, confocal laser scanning microscopy (CLSM), and multiple molecular fluorescent probes were utilized to test the hypothesis that cerebral vascular muscle cell nuclear ([Ca(2+)](n)), perinuclear ([Ca(2+)](pn)), and cytoplasmic free calcium ([Ca(2+)](i)) levels are regulated by the concentration of extracellular free magnesium ions ([Mg(2+)](o)). Primary cultured canine cerebral vascular smooth muscle cells were loaded with either fura-2/AM, indo-1/AM, or fluo-3/AM, and the subcellular Ca(2+) responses to stepwise reduction in [Mg(2+)](o) (i.e., from 1.36 to 0.17 mM) were analyzed over time. With normal 1.36 mM [Mg(2+)](o)-containing incubation media, basal mean [Ca(2+)](i) was 89.6+/-15 nM. Lowering [Mg(2+)](o) to 1.07, 0.88, 0.48, and 0.17 mM resulted in rapid (<4 min) increments in [Ca(2+)](i) going to 213+/-43, 368+/-67, 471+/-77, and 642+/-98 nM, respectively; the longer the exposure time (up to 30 min) to lowered [Mg(2+)](o), the higher the [Ca(2+)](i). Restoration of [Mg(2+)](o) to normal caused decreases in [Ca(2+)](i) to 215.9+/-42.3 nM, but only complete removal of [Ca(2+)](o) returned [Ca(2+)](i) to basal levels. Results show that basal [Ca(2+)](pn) (282+/-92 nM) exceeds basal cytoplasmic Ca(2+) (61+/-27.8 nM) and [Ca(2+)](n) (20+/-7.6 nM). However, reduction of normal [Mg(2+)](o) to 0.48 mM resulted in dramatic, rapid rises in all subcellular compartments, where [Ca(2+)](pn) (1503+/-102 nM)>cytoplasmic Ca(2+) (688+/-49 nM) approximately equal to [Ca(2+)](n) (674+/-12 nM). Nuclear Ca(2+) rose dramatically (e.g., 35-40 times basal levels). Both verapamil (1 microM) and Ni(2+) (5 mM) prevented, completely, the rises in Ca(2+) in all compartments, suggesting that Mg(2+)-dependent Ca(2+) accumulation may be dependent on nuclear, endoplasmic reticulum-Golgi, and cytoplasmic L-type voltage membrane-regulated Ca(2+) channels. The normally low [Ca(2+)](n) suggests that Ca(2+) does not transport passively across the nuclear membrane in cerebral vascular smooth muscle cells. These results may help to explain much of the impact of hypomagnesemic states on cerebral-central nervous system pathobiology, and, particularly, alcohol-induced strokes.
Assuntos
Cálcio/metabolismo , Núcleo Celular/efeitos dos fármacos , Magnésio/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Alcoolismo/metabolismo , Animais , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Córtex Cerebral , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Cães , Relação Dose-Resposta a Droga , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Magnésio/fisiologia , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Acidente Vascular Cerebral/metabolismoRESUMO
The influence of chronic treatment of cultured canine cerebral vascular smooth muscle cells, with low concentrations of ethanol, on the intracellular concentrations of free calcium ([Ca2+]i) was studied by use of the fluorescent indicator, fura-2, and digital imaging microscopy. The resting level of [Ca2+]i in the cerebral vascular smooth muscle cells was 89 +/- 3.2 nM. Exposure of these cells to 10 and 25 mM ethanol for 5 days resulted in significant elevation of [Ca2+]i (mean rises to 208 +/- 11.4 and 307 +/- 14.0 nM, respectively), and potentiated the transient rise in [Ca2+]i induced by 10(-7) M PGF2 alpha. However, exposure of these cerebral cells to a high-concentration ethanol (100 mM) resulted in only a slight increase of [Ca2+]i (106 +/- 6.9 nM) and lack of effects on the [Ca2+]i response to PGF2 alpha. Irrespective of the different ethanol treatments, the subcellular distribution of [Ca2+]i was heterogeneous in all the cells tested. Our data suggest that chronic exposure of cerebral vascular smooth muscle cells to ethanol, particularly at low concentrations, results in dramatic increases in [Ca2+]i and the responses of these vascular smooth muscle cells to prostanoids. These results support an hypothesis whereby ethanol induces stroke by causing spasm and rupture of cerebral blood vessels as a consequence of large rises in intracellular Ca2+.