RESUMEN
The purpose of this study is to identify factors in the sera of highly sensitized (HS) patients (pts) that inhibit T-cell alloresponses. An in vitro assay was used to measure HLA class I and class II-like antiidiotypic antibodies (anti-ids). The stimulation index (SI) was used to measure PBL and T-cell responses to alloantigens. All HS sera (32 pts) and the IgG fraction inhibited PBL and CD4(+) T-cell responses to alloantigens. The SI with HS IgG was 7.9 +/- 1.7 as compared to 31.5 +/- 5.9 with normal IgG (p = 0.0003). In a subset of pts who were transiently sensitized, the SI was 6.6 +/- 1.0 with a high panel reactive antibody (PRA), but when their PRA was zero, the SI was 17.8 +/- 1.3 (p = 0.0000001). Anti-ids were found in 100% of 17 pts with a high PRA. The T-cell inhibitory factors reduced CD4(+) T-cell responses of HS pts to alloantigens in the presence of autologous anti-ids, were MHC restricted and were inactivated by in vitro generated antibodies to HLA class II-like anti-ids. The HLA class II-like anti-id IgG molecules bind to the TCR of CD4(+) T cells and may impair their ability to help in the downregulating antibody response to anti-ids.
Asunto(s)
Anticuerpos Antiidiotipos/fisiología , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Sueros Inmunes/fisiología , Inmunización , Idiotipos de Inmunoglobulinas/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Anticuerpos Antiidiotipos/sangre , Linfocitos B/inmunología , Células Cultivadas , Femenino , Humanos , Inmunoglobulina G/sangre , Masculino , Linfocitos T/metabolismoRESUMEN
AIM: In patients with stable angina pectoris both morphological and functional information about the coronary artery tree should be present before revascularization therapy is performed. High accuracy was shown for spiral computed tomography (MDCT) angiography acquired with a 64-slice CT scanner compared to invasive coronary angiography (ICA) in detecting "obstructive" coronary artery disease (CAD). Gated myocardial SPECT (MPI) is an established method for the noninvasive assessment of functional significance of coronary stenoses. Aim of the study was to evaluate the combination of 64-slice CT angiography plus MPI in comparison to ICA plus MPI in the detection of hemodynamically relevant coronary artery stenoses in a clinical setting. PATIENTS, METHODS: 30 patients (63 +/- 10.8 years, 23 men) with stable angina (21 with suspected, 9 with known CAD) were investigated. MPI, 64-slice CT angiography and ICA were performed, reversible and fixed perfusion defects were allocated to determining lesions separately for MDCT angiography and ICA. The combination of MDCT angiography plus MPI was compared to the results of ICA plus MPI. RESULTS: Sensitivity, specificity, negative and positive predictive value for the combination of MDCT angiography plus MPI was 85%, 97%, 98% and 79%, respectively, on a vessel-based and 93%, 87%, 93% and 88%, respectively, on a patient-based level. 19 coronary arteries with stenoses > or =50% in both ICA and MDCT angiography showed no ischemia in MPI. CONCLUSION: The combination of 64-slice CT angiography and gated myocardial SPECT enabled a comprehensive non-invasive view of the anatomical and functional status of the coronary artery tree.
Asunto(s)
Angina de Pecho/diagnóstico por imagen , Angiografía Coronaria/métodos , Estenosis Coronaria/diagnóstico por imagen , Imagen de Acumulación Sanguínea de Compuerta/métodos , Anciano , Enfermedad Coronaria/diagnóstico por imagen , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Tomografía Computarizada de Emisión de Fotón Único , Tomografía Computarizada por Rayos XRESUMEN
This literature review shows that the Nd:YAG laser and the Er:YAG laser can be beneficially used in periodontology for subgingival curettage and gingivectomy. Studies demonstrated that subgingival laser treatment can result in significant reduction of the initial levels of periodontal pathogens. It is beneficial to perform laser curettage under water irrigation prior to scaling in order to destroy of the structure of the calculus and its attachment to the root surface. During laser curettage sufficient haemostasis is obtained, which improves detection of subgingival calculus. Nd:YAG laser gingivectomy results in similar improvement of deep periodontal pocket as conventional flap surgery, with the advantages such as minimal bleeding and post-operative pain, no swelling, and neither sutures nor post-surgical dressing are needed. Both non-surgical and surgical laser treatments are often performed without local anaesthesia, making full-mouth treatment in one session possible. Treatments are well accepted by the patients and require about 50% less time than the conventional therapies. Laser-de-epithelialization for periodontal regeneration has been experimentally attempted, but more controlled studies are needed to establish its usefulness in the clinic. Laser treatment of peri-implantitis is not recommended.
Asunto(s)
Terapia por Láser , Enfermedades Periodontales/cirugía , Cálculos Dentales/cirugía , Gingivectomía/instrumentación , Gingivectomía/métodos , Humanos , Dolor Postoperatorio , Hemorragia Posoperatoria , Curetaje Subgingival/instrumentación , Curetaje Subgingival/métodosRESUMEN
Manipulating the expression of a protein can provide a powerful tool for understanding its function, provided that the protein is expressed at physiologically-significant concentrations. We have developed a simple method to measure (1) the concentration of an overexpressed protein in single cells and (2) the covariation of particular physiological properties with a protein's expression. As an example of how this method can be used, teratocarcinoma cells were transfected with the neuron-specific calcium binding protein calretinin (CR) tagged with green fluorescent protein (GFP). By measuring GFP fluorescence in microcapillaries, we created a standard curve for GFP fluorescence that permitted quantification of CR concentrations in individual cells. Fura-2 measurements in the same cells showed a strong positive correlation between CR-GFP fusion protein expression levels and calcium clearance capacity. This method should allow reliable quantitative analysis of GFP fusion protein expression.
Asunto(s)
Indicadores y Reactivos , Proteínas Luminiscentes , Proteína G de Unión al Calcio S100/metabolismo , Animales , Calbindina 2 , Calcio/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , Humanos , Masculino , Proteína G de Unión al Calcio S100/análisis , Teratocarcinoma , Neoplasias Testiculares , Células Tumorales CultivadasRESUMEN
Brainstem auditory neurons in the chick nucleus magnocellularis (NM) express high levels of the neuron-specific calcium-binding protein calretinin (CR). CR has heretofore been considered a diffusible calcium buffer that is dispersed uniformly throughout the cytosol. Using high-resolution confocal microscopy and complementary biochemical analyses, we have found that during the development of NM neurons, CR changes from being expressed diffusely at low concentrations to being highly concentrated beneath the plasma membrane. This shift in CR localization occurs at the same time as the onset of spontaneous activity, synaptic transmission, and synapse refinement in NM. In the chick brainstem auditory pathway, this subcellular localization appears to occur only in NM neurons and only with respect to CR, because calmodulin remains diffusely expressed in NM. Biochemical analyses show the association of calretinin with the membrane is detergent-soluble and calcium-independent. Because these are highly active neurons with a large number of Ca2+-permeable synaptic AMPA receptors, we hypothesize that localization of CR beneath the plasma membrane is an adaptation to spatially restrict the calcium influxes.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Animales , Núcleo Basal de Meynert/embriología , Núcleo Basal de Meynert/metabolismo , Núcleo Basal de Meynert/ultraestructura , Western Blotting , Calbindina 2 , Embrión de Pollo , Inmunohistoquímica , Microscopía Confocal , Neuronas/metabolismo , Neuronas/ultraestructura , Fracciones Subcelulares/metabolismoRESUMEN
Functional interaction between ionotropic and metabotropic glutamate receptors (iGluR and mGluR respectively) was studied in cerebellar granule cell cultures using quisqualate (QA), the most potent agonist of phosphoinositide hydrolysis coupled mGluR, and N-methyl-D-aspartate (NMDA) or kainate (KA) that activate different classes of iGluR. Two h exposure to NMDA or KA resulted in a marked reduction (about 75%) of QA-evoked PI hydrolysis. The efficacy of the two agonists was about the same, but the potencies were different (IC50 for NMDA about 35 microM and for KA about 70 microM). NMDA-induced depression of QA-stimulated PI hydrolysis was relatively long lasting but reversible. Recovery required protein synthesis. In nominally Ca2+-free medium both NMDA and KA failed to attenuate QA-stimulated PI hydrolysis. The effect of NMDA was prevented by the NMDA receptor antagonist MK801, but not by the wide spectrum protein kinase inhibitor staurosporin nor by the nitric oxide synthase inhibitor N omega-nitro-L-arginine. Cycloheximide and concanavalin A were also ineffective. The effect of KA was prevented by the selective non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX). Voltage sensitive Ca2+ channel antagonists together with MK801 did not counteract the inhibition by KA of the QA response. Both NMDA and KA attenuated PI hydrolysis evoked by the muscarinic receptor agonist carbachol (about 30%), indicating that the activation of iGluRs exerts a relatively general inhibitory effect on the function of different PLC-coupled metabotropic receptors. Consistent with this observation is that treatments either with KA and NMDA induced an inhibition (about 30%) of NaF-stimulated PI hydrolysis which occurs through the direct activation of G proteins. Our observations show that ionotropic glutamate receptor stimulation induces a long lasting suppression of QA-evoked PI breakdown through a Ca2+ dependent mechanism which seems to involve receptor coupled transduction systems downstream from mGluR. Such a Ca2+-dependent cross-talk involving ionotropic and metabotropic receptors may play a role in certain events of synaptic plasticity.
Asunto(s)
Calcio/metabolismo , Cerebelo/metabolismo , Fosfatidilinositoles/metabolismo , Receptores de Glutamato/fisiología , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Animales , Células Cultivadas , Maleato de Dizocilpina/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Proteínas de Unión al GTP/fisiología , Hidrólisis , Ácido Kaínico/farmacología , N-Metilaspartato/farmacología , Quinoxalinas/farmacología , Ácido Quiscuálico/farmacología , Ratas , Ratas Wistar , Receptores de Glutamato Metabotrópico/fisiología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Fluoruro de Sodio/farmacologíaRESUMEN
We studied the effects of chronic K(+)-induced membrane depolarization and treatment with N-methyl-D-aspartate (NMDA) on cerebellar granule cells (CGCs) from weaver mutant mice and non-weaver litter-mates. The weaver mutation is a Gly-to-Ser substitution in a conserved region of the Girk2 G protein-coupled inward rectifying potassium channel [Patil N., Cox D. R., Bhat D., Faham M., Myers R. M. and Peterson A. S. (1995) Nature Genet. 11, 126-129] which induces early death of CGCs. The biochemical differentiation of CGCs was estimated as the rate of 2-deoxy-D-glucose accumulation and the expression of neural cell adhesion molecule (NCAM). High (25 mM) K+ ion concentration or treatment with NMDA greatly promoted the biochemical differentiation of both weaver mutant and non-weaver litter-mate mouse CGCs. In contrast to the marked effect on biochemical differentiation in both weaver and non-weaver mice CGSs, chronic high K+ treatment only had limited effect on survival. The survival of weaver mutant mouse CGCs in medium containing 5 mM K+ ions was very low, only 20% of the plated cells surviving at 7 days after plating, as opposed to the 50% for non-weaver CGCs. Chronic high K+ treatment improved the relative survival of weaver mutant mouse CGCs 1.6 2.2-fold and that of non-weaver CGCs 1.2-1.4-fold; the same number of CGCs (about 20% of the plated cells) were rescued by high K+ in both types of culture. The findings indicate that, in culture weaver mutant mouse, CGCs have a normal response to membrane depolarization and that the normal function of the Girk2 potassium channel is not critical for the survival of differentiated CGCs.
Asunto(s)
Cerebelo/fisiología , Ratones Mutantes Neurológicos/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Senescencia Celular , Cerebelo/citología , Cerebelo/metabolismo , Desoxiglucosa/metabolismo , Electrofisiología , Ratones , N-Metilaspartato/farmacología , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Potasio/farmacología , Valores de Referencia , Factores de TiempoRESUMEN
1. The precursor of eicosanoids is arachidonic acid, which emanates from the cleavage of the sn-2 position of phospholipids by phospholipase A2 (PLA2). Eicosanoids have diverse physiological and pathophysiological effects in the kidney. The regulation of phospholipase A2 has important implications for kidney function. 2. In the current communication we focus our attention on mesangial cell cytosolic PLA2 (cPLA2) and its regulation at the post-translational and post-transcriptional level. 3. At the post-translational level, using site directed mutagenesis of cPLA2 and a dominant negative ras, we have demonstrated that cPLA2 can be phosphorylated by mitogen activated protein (MAP-2) kinase leading to increased cPLA2 enzymatic activity. 4. At the post-transcriptional level we show that the half-life of cPLA2 mRNA in mesangial cells is significantly increased when mesangial cells are stimulated by mitogens. We further demonstrate the presence of three ATTTA motifs in the 3' untranslated region (3' UTR) of the cPLA2 cDNA. 5. Using chimeric constructs bearing the 3' UTR from rat cPLA2 fused downstream of the luciferase reporter, we demonstrate that this region exerts a destabilizing effect on cPLA2. 6. We have isolated and mapped genomic DNA and polymorphic markers for cPLA2 in the human and rat.
Asunto(s)
Mesangio Glomerular/fisiología , Fosfolipasas A/fisiología , Animales , Riñón/metabolismo , Fosfolipasas A2 , Fosfotransferasas/metabolismo , RatasRESUMEN
Cultured cerebellar granule cells become vulnerable to excitatory amino acids, especially to NMDA and kainate, by 9 days in vitro. In the same time, the sensitivity of cells to (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), in terms of AMPA-induced toxicity or 45Ca2+ uptake, was very low. The low AMPA responsiveness was due to receptor desensitization, because agents known to block desensitization, cyclothiazide and the lectins concanavalin A and wheat germ agglutinin, rendered granule cells vulnerable to AMPA and produced a pronounced stimulation of 45Ca2+ accumulation. 45Ca2+ influx was induced specifically by AMPA-receptor stimulation, because it was blocked virtually completely by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX) and the benzodiazepine GYKI 52466 (selective non-NMDA receptor antagonists). Nevertheless, indirect routes activated by cellular responses to AMPA-receptor stimulation contributed significantly to the overall 45Ca2+ influx. These included Ca2+ uptake through NMDA-receptor channels, voltage-sensitive Ca2+ channels, and via Na+/Ca2+ exchange. However, nearly one-fifth of the total 45Ca2+ influx remained unaccounted for and this estimate was similar to 45Ca2+ influx observed under Na(+)-free conditions. This observation suggested that a significant proportion of the Ca2+ flux passes through the AMPA-receptor channel proper, a view supported by Co2+ uptake into nearly all granule cells on exposure to AMPA in the presence of cyclothiazide. Results are discussed in light of the reported AMPA receptor-subunit composition of cerebellar granule cells in vitro.
Asunto(s)
Ansiolíticos , Calcio/metabolismo , Cerebelo/metabolismo , Receptores AMPA/metabolismo , Animales , Benzodiazepinas/farmacología , Benzotiadiazinas/farmacología , Radioisótopos de Calcio , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/efectos de los fármacos , Cobalto/metabolismo , Concanavalina A/farmacología , Aminoácidos Excitadores/antagonistas & inhibidores , Ácido Kaínico/farmacología , N-Metilaspartato/farmacología , Quinoxalinas/farmacología , Ratas , Receptores AMPA/antagonistas & inhibidores , Aglutininas del Germen de Trigo/farmacología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Transmembrane signal transduction is the process whereby a ligand binds to the external surface of the cell membrane and elicits a physiological response specific for that ligand and cell type. It is now appreciated that numerous disease states represent disturbances in normal transmembrane signaling mechanisms. In the current paper, we focus our attention on the mesangial cell of the glomerular microcirculation as a prototypical model system for understanding normal and abnormal transmembrane signaling processes. Among the major receptor and effector mechanisms for transmembrane signal transduction in the mesangial cell, this paper emphasizes the phospholipase effector response to growth factors and vasoactive hormones. The post-translational and transcriptional pathways for regulation of phospholipase C and phospholipase A2 are described, including consideration of perturbations in these systems that characterize two disease models, namely: acute cyclosporine nephrotoxicity and early diabetic glomerulopathy.
Asunto(s)
Enfermedades Renales/fisiopatología , Riñón/fisiología , Transducción de Señal/fisiología , Animales , Membrana Celular/fisiología , Humanos , Riñón/fisiopatologíaRESUMEN
1. The functional expression of two potassium currents in cultured cerebellar granule cells was investigated with the whole cell patch-clamp technique in relation to development and growth condition. Cells were grown in medium containing different concentrations of potassium: 25 mM (K25) and 40 mM (K40), together referred to as "high K+"; 10 mM (K10) or "low K+"; and K10 with 100 microM N-methyl-D-aspartate (KNMDA). All conditions are known to influence maturation and survival of granule cells in culture. 2. At 2 days in vitro (DIV) the membrane capacitance, taken as index of membrane surface area, was the same for cells grown in each growth condition. At 7-9 DIV it had increased in each condition, but to a substantially larger extent in cells grown in KNMDA, K25, and K40 than in cells grown in K10. During development the input resistance only decreased in cells grown in KNMDA and high K+. 3. A delayed potassium current (IK) and a fast transient potassium current (IA) could both be recorded at 2 DIV in each growth condition, although a few neurons only expressed the IK. The IK was partially suppressed by tetraethylammonium (5 mM), whereas IA was predominantly sensitive to 4-aminopyridine (5 mM). 4. Normalized for cell capacitance, the specific IA conductance hardly changed during development in cells grown in high K+ and KNMDA. Cells in K10, however, displayed an IA with totally different properties in 23 of 24 cells; the specific IA conductance in these cells was considerably smaller at 7-9 DIV, suggesting a deletion of these channels during development.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Cerebelo/fisiología , Activación del Canal Iónico/fisiología , Neuronas/fisiología , Canales de Potasio/fisiología , Animales , Células Cultivadas , Cerebelo/citología , Electrofisiología , Agonistas de Aminoácidos Excitadores/farmacología , Potenciales de la Membrana/fisiología , N-Metilaspartato/farmacología , Neuronas Aferentes/fisiología , Técnicas de Placa-Clamp , Potasio/farmacología , Ratas , Ratas WistarRESUMEN
The survival and maturation of differentiating cerebellar granule cells in culture are known to be promoted by excitatory amino acids (EAAs) which, however, compromise the survival of mature cells. In contrast to the trophic effect, the toxic effect of alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropiate (AMPA) could only be elicited when the desensitisation of AMPA receptors was blocked, cyclothiazide being used in this study. Nevertheless, even under these conditions, toxicity induced by AMPA in contrast to kainate was, at 9 DIV, only half of the maximal toxicity attained by 13-16 DIV. Since cellular responses to AMPA depend so dramatically on the maturational stage of granule cells, we examined here whether this characteristic is related to developmental changes in AMPA receptor properties, which may result from changes in the subunit composition of the receptor. In contrast to toxicity, AMPA-induced 45Ca2+ influx (determined in the presence of cyclothiazide and the NMDA receptor blocker MK-801) reached a maximum already at 9 DIV. This also applied to a fraction of the 45Ca2+ uptake which persisted either after Cd2+ application or under Na(+)-free conditions and therefore presumably was mediated directly through AMPA receptor channels. Quantitative analysis of Western blots showed that the amounts of GluR4 and to a lesser extent GluR2/3/4c are substantial already at 2 DIV, remaining fairly constant until 9 DIV, followed by an increase by 16 DIV. However GluR1, which is hardly detectable in granule cells in vivo and is also low early in vitro, increased almost linearly with cultivation time.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Cerebelo/citología , Neuronas/metabolismo , Receptores AMPA/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Células Cultivadas/metabolismo , Medios de Cultivo , Neuronas/ultraestructura , Neurotoxinas/toxicidad , Potasio/farmacología , Ratas , Receptores AMPA/ultraestructura , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/toxicidadRESUMEN
Differentiating granule cells develop survival requirements in vitro which can be met by treatment with high K+ or excitatory amino acids. Promotion of cell survival by N-methyl-D-aspartate (NMDA) or kainate has already been established and here we report that treatment of the cells with alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) or quisqualate (QA) also leads to cell rescue. In comparison with the effect of NMDA, the influence of AMPA/QA is small, resulting in a 20-30% increase in cell survival, with a peak at a very narrow concentration range (0.5-2.0 microM QA and 5-10 microM AMPA). The effect is exclusive to AMPA receptor stimulation, since stimulation of metabotropic glutamate receptors with (1S3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) has no effect. Furthermore, AMPA/QA rescue of cells is blocked by ionotropic non-NMDA receptor antagonists, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzoquinoxaline (NBQX). In addition, both nifedipine and dizolcipline (MK-801) interfered with the cell survival promoting effect of AMPA, suggesting that the influence of AMPA is mediated via calcium influx involving both depolarization-activated voltage sensitive calcium channels and NMDA receptors stimulated as a result of AMPA-induced release of glutamate. Possible reasons for the small cell survival promoting effect of AMPA/QA compared with the influence of high K+ or NMDA are discussed.
Asunto(s)
Neuronas/efectos de los fármacos , Ácido Quiscuálico/farmacología , Receptores AMPA/efectos de los fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Neuronas/citología , Ratas , Ratas Wistar , Receptores de Glutamato Metabotrópico/efectos de los fármacosRESUMEN
The survival of immature nerve cells in a cerebellar culture, predominantly excitatory granule cells, is known to be promoted by chronic exposure to high K+ (> 20 mM) or glutamate (Glu) receptor agonists. These treatments are believed to mimic the in vivo effect of the incoming glutamatergic afferents, the mossy fibres. Here we report that with maturation the cells become vulnerable to excitatory amino acids (EAAs) and that the characteristics of EAA sensitivity are dependent on the environmental influences being either "trophic" (25 mM K+ or 140 microM NMDA, K25 or K10 + NMDA) or "non-trophic" (10 mM K+, K10). Toxicity was assayed routinely at 9 days in vitro (DIV) after 24 h exposure to EAAs. Under all the tested conditions, the effect of Glu was mediated exclusively through NMDA receptors. However, the efficacy and potency of Glu were high in K25- and K10 + NMDA-grown cells compared with K10-grown cells. Growth conditions had the same influence on NMDA as on Glu-induced toxicity, but with the following special features: (1) in comparison with K25 cells, the potency of NMDA was significantly lower in K10 + NMDA cells. The K10 + NMDA cultures behaved as if they were completely insensitive to the NMDA which is present in their growth medium. (2) The K10-grown cells were not vulnerable to NMDA, unless the cell membrane was depolarised by shifting the cells into K25 medium. The efficacy of NMDA became then similar to that in K25 cultures, although the potency was about 7-fold less. Thus NMDA receptors can be activated by the depolarisation of K10 cells, implying the operation of Mg2+ blockade of the channel at normal resting membrane potential. Although non-NMDA receptors did not seem to be involved in Glu toxicity, cells were vulnerable to kainate, which killed significantly more cells than Glu (about 80% vs 70%). This was partly due to the resistance of GABAergic interneurons present in the cultures to Glu- or NMDA-induced toxicity. In contrast to the effects of Glu or NMDA, KA vulnerability was lower in cells grown in K25 or K40 than K10 medium (rank order K10 > K25 > K40). Under our experimental conditions, cultured cells were resistant to AMPA, quisqualate and the selective metabotropic Glu receptor agonist 1S,3R-ACPD. Collectively, the observations indicated that EAA sensitivity of cultured cerebellar interneurons is significantly and differentially influenced by environmental factors, believed to mimic in vivo trophic influences on these cells.
Asunto(s)
Cerebelo/efectos de los fármacos , Aminoácidos Excitadores/toxicidad , Animales , Radioisótopos de Calcio , Células Cultivadas , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Ácido Glutámico/metabolismo , Inmunohistoquímica , Ácido Kaínico/toxicidad , Potenciales de la Membrana/efectos de los fármacos , N-Metilaspartato/toxicidad , Potasio/farmacología , Ratas , Receptores de GABA/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/efectos de los fármacos , Sales de Tetrazolio , TiazolesRESUMEN
The effects of K(+)-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from degenerated cells was removed by prior DNAase treatment. These DNA estimates of cell numbers were comparable with values obtained by direct counting of fluorescein diacetate-stained viable cells. In agreement with previous studies, the survival of rat granule cells was promoted by increasing the concentration of K+ in the medium from 5 to 25 mM throughout a 7-day culture period. In contrast, mouse granule cells survived in culture containing 'low' K+ (5 or 10 mM), as well as in the presence of 'high' K+ (25 mM). On the other hand, several biochemical parameters in mouse granule cells were markedly increased by cultivation in 'high' as compared with 'low' K(+)-containing media, demonstrated by increased fluorescein diacetate esterase activity, enhanced rate of NADPH-dependent tetrazolium reduction, augmented 2-deoxy-D-glucose accumulation and increased N-methyl-D-aspartate-evoked 45Ca2+ influx. It was concluded that although cultivation in 'high' K+ promotes biochemical differentiation in mouse cerebellar granule cells, these cells differ from their rat counterparts in that they do not develop a survival requirement for K(+)-induced membrane depolarization.
Asunto(s)
Neuronas/efectos de los fármacos , Potasio/farmacología , Animales , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , ADN/análisis , Potenciales de la Membrana/efectos de los fármacos , Ratones , Degeneración Nerviosa/fisiología , Neuronas/citología , Ratas , Coloración y EtiquetadoRESUMEN
Arachidonic acid release in cells highly over expressing cytosolic phospholipase A2 has been attributed to mitogen-activated protein kinase phosphorylation of cytosolic phospholipase A2 on serine-505. To investigate the role of cytosolic phospholipase A2 in cellular physiology, we attempted to inhibit cytosolic phospholipase A2 in the intact cell employing an antisense RNA strategy. Swiss 3T3 cells were stably transfected with an antisense cytosolic phospholipase A2 expression vector. A clone of cells with reduced immunodetectable cytosolic phospholipase A2, compared to a vector transfected cell line, was identified by Western blotting and a corresponding decrease in phospholipase A2 activity was confirmed by enzymatic assay in cell free extracts. However, arachidonic acid release from intact cells in response to agonists was not different between antisense and control cell lines. Thus, arachidonic acid release in intact cells with decreased cytosolic phospholipase A2 activity is likely to be modulated by rate limiting factors that are extrinsic to cytosolic phospholipase A2.
Asunto(s)
Ácido Araquidónico/metabolismo , Fosfolipasas A/metabolismo , ARN sin Sentido/farmacología , Células 3T3 , Animales , Western Blotting , Calcimicina/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Citosol/enzimología , Expresión Génica , Vectores Genéticos , Cinética , Ratones , Fosfolipasas A/biosíntesis , Fosfolipasas A2 , Factor de Crecimiento Derivado de Plaquetas/farmacología , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
Previous studies have provided suggestive evidence for an interaction between ras activation and signalling pathways involved in agonist-stimulated arachidonic acid release in a variety of cell systems. In order to clarify this interaction, we have measured epidermal growth factor (EGF)-stimulated arachidonic acid release in rat-1 fibroblasts transfected with the N-17 dominant negative mutation of ras. Cells transfected with the N-17 ras mutant, display a markedly attenuated arachidonic acid-release response to EGF, compared to sham-transfected and non-transfected cells. In contrast, the response to phorbol myristate acetate (PMA) was not attenuated in the N-17-mutant expressing cells. No differences were detected between sham-transfected and N-17 mutant expressing cells in levels of immunodetectable EGF receptor, cytosolic phospholipase A2 or mitogen-activated protein (MAP) kinase. Attenuation of EGF-stimulated arachidonic acid release in the N-17 mutant expressing cells, was accompanied by a marked diminution in EGF-stimulated tyrosine phosphorylation of MAP kinase. We conclude that the signalling pathway involved in epidermal growth factor-stimulated arachidonic acid release is similar to the signalling pathway for mitogenic responses to epidermal growth factor and requires ras activation, likely followed by a downstream cascade of kinases eventuating in MAP kinase activation.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Genes ras/fisiología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/análisis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Línea Celular , Receptores ErbB/análisis , Fibroblastos/química , Regulación de la Expresión Génica/genética , Genes ras/genética , Mutación/genética , Fosfolipasas A/análisis , Fosfolipasas A2 , Fosforilación , Pruebas de Precipitina , Ratas , Acetato de Tetradecanoilforbol/farmacología , Transfección , Tritio , Tirosina/metabolismoRESUMEN
Recent findings have suggested that certain ligand-dependent responses to EGF may be propagated in a manner that is not dependent on the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGF-R, Campos-Gonzalez, R., and Glenney, J. R., Jr. (1992) J. Biol. Chem. 267, 14535-14538) or, alternatively, that these responses may occur through the interaction of the human tyrosine kinase-deficient EGF-R with an as yet unidentified kinase (Selva, E., Raden, D. L., and Davis, R. J. (1993) J. Biol. Chem. 268, 2250-2254). These conclusions represent a significant departure from our current understanding of signal transduction by receptor tyrosine kinases. Therefore we examined the effect of expression of tyrosine kinase-negative human EGF receptor in murine NIH-3T3-2.2 cells on the EGF-dependent phosphorylation of mitogen-activated protein (MAP-2) kinase. In parental cells (NIH-3T3-2.2) that express low levels of endogenous murine EGF-R, there was no demonstrable EGF-dependent coupling to MAP-2 kinase. In NIH-3T3-2.2 cells transfected with tyrosine kinase-negative human EGF-R, there was unexpected EGF-dependent phosphorylation of MAP-2 kinase. Analysis of the tyrosine kinase-negative human EGF-R in these cells revealed significant tyrosine phosphorylation of the EGF-R. A low level of endogenous murine EGF-R present in these cells were also phosphorylated on tyrosine residues and displayed autokinase activity. Similar results were obtained using an unrelated cell line (B82L cells), in which EGF-dependent phosphorylation of MAP-2 kinase was previously attributed to signal propagation through a tyrosine kinase-negative human EGF-R (Campos-Gonzalez, R., and Glenney, J. R., Jr. (1992) J. Biol. Chem. 267, 14535-14538). Taken together, these results suggest that the tyrosine kinase-negative human EGF-R are able to amplify the response to activation of low levels of endogenous murine EGF-R, thus leading to EGF-dependent phosphorylation of MAP-2 kinase in cells expressing tyrosine kinase-negative human EGF-R.
Asunto(s)
Receptores ErbB/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Células 3T3 , Animales , Receptores ErbB/genética , Humanos , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Fosforilación , Pruebas de Precipitina , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Glutamate receptors coupled to polyphosphoinositide (PPI) hydrolysis (metabotropic glutamate receptors, mGluR), are highly efficient during the early stages of postnatal life and are thought to be involved in developmental plasticity. The dramatic decrease with age in mGluR activity suggests the existence of mechanisms that down-regulate this receptor after a certain stage of neuronal maturation. In cultured cerebellar granule neurons grown under conditions that promote the survival and maturation of cells (serum-containing medium with 25 mM K+), enzymatic depletion of extracellular glutamate prevented the age-dependent decrease in mGluR agonist-stimulated PPI hydrolysis that normally occurs after 4 days of maturation in vitro, suggesting that mGluR activity declines as a result of developmental changes affecting homologous desensitization. This was borne out by the observation that glutamate at low concentrations (1-10 microM) readily desensitized mGluR at 7 days but not at 4 days in culture. Furthermore, the critical period during which the high sensitivity to agonist-induced desensitization of mGluR developed coincided with the period when phorbol ester-activated protein kinase C acquired the ability to suppress mGluR activity. The developmental pattern of mGluR agonist-induced PPI hydrolysis was similar in granule cells grown under "trophic" and "nontrophic" conditions (in cultures in 25 mM K+ and in a medium containing "low" K+, in this study, 10 mM, respectively). However, the developmental decline in the response to mGluR stimulation after 4 days in vitro was not prevented in cells grown in 10 mM K+ by the removal of extracellular glutamate; rather, it could be counteracted by treatment with N-methyl-D-aspartate (NMDA) (EC50, approximately 4 microM), which blocked the development of mGluR desensitization. The effect was NMDA receptor mediated and required DNA transcription and protein synthesis. However, NMDA exerted a different effect in cells grown in 25 mM K+, inducing a substantial decrease rather than an increase in mGluR activity. The effect of growth conditions was also examined on mGluR mRNA levels, which were not always correlated with mGluR activity. In general, either increases in the medium K+ concentrations or NMDA supplementation of the cultures resulted in a decrease in mGluR mRNA levels. It is noteworthy that NMDA could also restore mGluR activity after the metabotropic response had reached its peak. This implies that NMDA receptor activation may be involved in the increase in mGluR activity in adult life under conditions that elicit plastic changes in the nervous system.
Asunto(s)
Cerebelo/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Envejecimiento/metabolismo , Animales , Carbacol/farmacología , Células Cultivadas , Cerebelo/citología , Glutamatos/metabolismo , Ácido Glutámico , Hidrólisis , N-Metilaspartato/farmacología , Fosfatos de Fosfatidilinositol/metabolismo , Potasio/metabolismo , Ácido Quiscuálico/farmacología , ARN Mensajero/metabolismo , Ratas , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacosRESUMEN
Cerebellar granule cells in culture develop survival requirements which can be met either by chronic membrane depolarization (25 mM K+) or by stimulation of ionotropic excitatory amino acid receptors. We observed previously that this trophic effect is mediated via Ca2+ influx, either through dihydropyridine-sensitive, voltage-dependent calcium channels (activated directly by high K+ or indirectly by kainate) or through N-methyl-D-aspartate receptor-linked ion channels. Steps after Ca2+ entry in the transduction cascade mediating the survival-supporting effect of high K+ and excitatory amino acids have now been examined. Using protein kinase inhibitors (H-7, polymixin B and gangliosides), and modulating protein kinase C activity by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, we obtained evidence against the involvement of protein kinase C and cyclic nucleotide-dependent protein kinases in the transduction cascade. On the other hand, calmidazolium (employed as a calmodulin inhibitor) counteracted the trophic effect of elevated K+ with high potency (IC50 0.3 microM), which exceeded by approximately 10-fold the potency for the blockade by the drug of voltage-sensitive calcium channels. The potency of calmidazolium in interfering with the N-methyl-D-aspartate rescue of cells was also much higher in comparison with the inhibition of 45Ca2+ influx through N-methyl-D-aspartate receptor-linked channels. Our results indicated that after calmodulin the next step in the trophic effects involves Ca2+/calmodulin-dependent protein kinase II activity. KN-62, a fairly specific antagonist of this enzyme, compromised elevated K+ or excitatory amino acid-supported cell survival with high potency (IC50 2.5 microM). In the relevant concentration range, KN-62 had little or no effect on Ca2+ entry through either voltage- or N-methyl-D-aspartate receptor-gated channels. Combining information on the toxic action of glutamate in "mature" granule cells with the trophic effect of either excitatory amino acids or high K+ treatment on "young" cells, we conclude that after the initial steps involving calcium in both cases the respective transduction pathways diverge. The toxic action of glutamate seems to be mediated through protein kinase C [Favaron et al. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 1983-1987 whereas a Ca2+/calmodulin-dependent protein kinase, which can be inhibited by KN-62 (but is resistant to gangliosides and to inhibitors whose potency is higher for protein kinase C than for Ca2+ calmodulin-dependent protein kinases, such as H-7 and polymixin B), is involved critically in the trophic effect.