RESUMEN
We study the phase behavior of saturated lipids as a function of temperature and tail length for two coarse-grained models: the soft-repulsive model typically employed with dissipative particle dynamics (DPD) and the MARTINI model. We characterize the simulated transitions through changes in structural properties, and we introduce a computational method to monitor changes in enthalpy, as is done experimentally with differential scanning calorimetry. The lipid system experimentally presents four different bilayer phases - subgel, gel, ripple, and fluid - and the DPD model describes all of these phases structurally while MARTINI describes a single order-disorder transition between the gel and the fluid phases. Given both models' varying degrees of success in displaying accurate structural and thermodynamic signatures, there is an overall satisfying extent of agreement for the coarse-grained models. We also study the lipid dynamics displayed by these models for the various phases, discussing this dynamics with relation to fidelity to experiment and computational efficiency.
Asunto(s)
Rastreo Diferencial de Calorimetría/métodos , Membrana Dobles de Lípidos/química , Transición de Fase , Simulación por Computador , Modelos Moleculares , TermodinámicaRESUMEN
Experiments and molecular simulations have shown that the hydrophobic mismatch between proteins and membranes contributes significantly to lipid-mediated protein-protein interactions. In this article, we discuss the effect of cholesterol on lipid-mediated protein-protein interactions as function of hydrophobic mismatch, protein diameter and protein cluster size, lipid tail length, and temperature. To do so, we study a mesoscopic model of a hydrated bilayer containing lipids and cholesterol in which proteins are embedded, with a hybrid dissipative particle dynamics-Monte Carlo method. We propose a mechanism by which cholesterol affects protein interactions: protein-induced, cholesterol-enriched, or cholesterol-depleted lipid shells surrounding the proteins affect the lipid-mediated protein-protein interactions. Our calculations of the potential of mean force between proteins and protein clusters show that the addition of cholesterol dramatically reduces repulsive lipid-mediated interactions between proteins (protein clusters) with positive mismatch, but does not affect attractive interactions between proteins with negative mismatch. Cholesterol has only a modest effect on the repulsive interactions between proteins with different mismatch.
Asunto(s)
Colesterol/farmacología , Simulación de Dinámica Molecular , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Membrana Dobles de Lípidos/química , Transición de Fase/efectos de los fármacos , Unión Proteica/efectos de los fármacosRESUMEN
In this paper, we present a coarse-grained model of a hydrated saturated phospholipid bilayer (dimyristoylphosphatidylcholine, DMPC) containing cholesterol that we study using a hybrid dissipative particle dynamics-Monte Carlo method. This approach allows us to reach the time and length scales necessary to study structural and mechanical properties of the bilayer at various temperatures and cholesterol concentrations. The properties studied are the area per lipid, condensation, bilayer thickness, tail order parameters, bending modulus, and area compressibility. Our model quantitatively reproduces most of the experimental effects of cholesterol on these properties and reproduces the main features of the experimental phase and structure diagrams. We also present all-atom simulation results of the system and use these results to further validate the structure of our coarse-grained bilayer. On the basis of the changes in structural properties, we propose a temperature-composition structure diagram, which we compare with the experimental phase and structure diagrams. Attention is paid to the reliability and interpretation of the model and simulation method and of the different experimental techniques. The lateral organization of cholesterol in the bilayer is discussed.
Asunto(s)
Colesterol/química , Dimiristoilfosfatidilcolina/química , Membrana Dobles de Lípidos/química , Modelos Biológicos , Transición de Fase , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Molecular , Método de Montecarlo , Estrés Mecánico , Temperatura , TermodinámicaRESUMEN
The mouse Pde6d gene encodes a ubiquitous prenyl binding protein, termed PrBP/delta, of largely unknown physiological function. PrBP/delta was originally identified as a putative rod cGMP phosphodiesterase (PDE6) subunit in the retina, where it is relatively abundant. To investigate the consequences of Pde6d deletion in retina, we generated a Pde6d(-/-) mouse by targeted recombination. Although manifesting reduced body weight, the Pde6d(-/-) mouse was viable and fertile and its retina developed normally. Immunocytochemistry showed that farnesylated rhodopsin kinase (GRK1) and prenylated rod PDE6 catalytic subunits partially mislocalized in Pde6d(-/-) rods, whereas rhodopsin was unaffected. In Pde6d(-/-) rod single-cell recordings, sensitivity to single photons was increased and saturating flash responses were prolonged. Pde6d(-/-) scotopic paired-flash electroretinograms indicated a delay in recovery of the dark state, likely due to reduced levels of GRK1 in rod outer segments. In Pde6d(-/-) cone outer segments, GRK1 and cone PDE6alpha' were present at very low levels and the photopic b-wave amplitudes were reduced by 70%. Thus the absence of PrBP/delta in retina impairs transport of prenylated proteins, particularly GRK1 and cone PDE, to rod and cone outer segments, resulting in altered photoreceptor physiology and a phenotype of a slowly progressing rod/cone dystrophy.
Asunto(s)
Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Eliminación de Gen , Hidrolasas Diéster Fosfóricas/deficiencia , Hidrolasas Diéster Fosfóricas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Animales , Dominio Catalítico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Electrorretinografía , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neopreno/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Células Fotorreceptoras de Vertebrados/química , Transporte de ProteínasRESUMEN
PURPOSE: To study mechanisms leading to photoreceptor degeneration in mouse models for autosomal dominant retinitis pigmentosa (adRP) based on the rhodopsin P23H mutation. METHODS: Mice of a transgenic line expressing a rhodopsin triple mutant, V20G, P23H, and P27L (GHL), were mated with rhodopsin (rho) knockout mice. Littermates of various ages and genotypes (GHL+rho+/+, GHL+rho+/-, and GHL+rho-/-) were examined for outer nuclear layer thickness and outer segment formation (histology), fate of mutant rhodopsin (immunocytochemistry), and photoreceptor function (electroretinogram; ERG). RESULTS: Mice expressing GHL-rhodopsin in the absence of wild-type rhodopsin had severe retinopathy, which was nearly complete by postnatal day (P)30. GHL-rhodopsin formed homodimers nearly exclusively on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, whereas wild-type rhodopsin predominantly formed monomers. Expression level of mutant rhodopsin in predegenerate (P10) GHL+rho-/- retinas was low, approximately 10% to 25% of normal levels. No elaboration of disc membrane or outer segment formation was observed at any time point examined. The mutant rhodopsin was found mostly in perinuclear locales (endoplasmic reticulum; ER) as evidenced by colocalization using the antibodies Rho1D4 and calnexin-NT. CONCLUSIONS: GHL-rhodopsin dimerizes, localizes to the ER, and fails to transport and support outer segment formation. Additionally, the mutant protein does not support a scotopic ERG a-wave and accelerates photoreceptor degeneration over that occurring with the rhodopsin knockout alone. These findings indicate a cytotoxic effect of the mutant protein, probably elicited by an unfolded protein response.
Asunto(s)
Mutación , Células Fotorreceptoras de Vertebrados/ultraestructura , Degeneración Retiniana/genética , Rodopsina/genética , Transgenes/genética , Animales , Cartilla de ADN/química , Electroforesis en Gel de Poliacrilamida , Electrorretinografía , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Expresión Génica , Genotipo , Immunoblotting , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Microscopía Inmunoelectrónica , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Rodopsina/metabolismoRESUMEN
PURPOSE: Homozygous inactivation of the mouse gene for GRK1 (G protein-coupled receptor kinase 1, or rhodopsin kinase) causes severe defects in the recovery of cone phototransduction. However, electroretinographic (ERG) analyses of human oguchi patients with defective GRK1 alleles showed normal or slightly abnormal photopic responses. It remains unclear why the loss of GRK1 yields such different phenotypes in the recovery of mouse and human cones. We examined the localization and enzyme activity of GRK7, the human ortholog of the seventh member of the GRK family, in an attempt to understand its potential role in photopic vision. METHODS: Bioinformatic approaches were used to identify the human GRK7 gene. Human and bovine GRK7 cDNAs were isolated by RT-PCR. Recombinant GRK7, expressed in insect cells, was used to phosphorylate activated rhodopsin. Antibodies raised against GRK7 peptides were used to examine the retina specific expression of GRK7 by immunoblotting and its subcellular localization by immunocytochemistry. RESULTS: The human GRK7 gene is located on chromosome 3q21, spans at least 10 Kb and consists of 4 exons. In human, GRK7 is expressed exclusively in the retina and is found in all retinal neurons, and specifically, in cone outer segments. Recombinant human GRK7 catalyzes rhodopsin phosphorylation in a light dependent manner. We provide evidence that GRK1 and GRK7 are co-expressed in human cones. In contrast, mouse GRK7 is expressed in many tissues including retina where photoreceptors apparently do not express GRK7. CONCLUSIONS: The presence of GRK7 in human, but not in mouse, cone outer segments suggests that GRK7 may function to provide the normal photopic vision reported by oguchi patients with a defective GRK1 gene. The absence of GRK7 expression in cone outer segments of mice is consistent with the notion that mouse cones rely solely on GRK1 to shutoff cone visual pigments.
Asunto(s)
Proteínas del Ojo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células Fotorreceptoras Retinianas Conos/enzimología , Secuencia de Aminoácidos , Animales , Bovinos , Pollos , Cromosomas Humanos Par 3/genética , Clonación Molecular , Técnica del Anticuerpo Fluorescente Indirecta , Quinasa 1 del Receptor Acoplado a Proteína-G , Quinasas de Receptores Acoplados a Proteína-G , Ligamiento Genético , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Ratones , Datos de Secuencia Molecular , Fosforilación , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rodopsina/metabolismo , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
BACKGROUND: The relationship between exposure to house dust mite (HDM) allergens and prevalence of sensitization to these allergens in patients with asthma has been confirmed in many studies. Mite population growth is regulated by humidity. Reducing humidity and removing allergen by efficient vacuuming should control mite allergen and reduce symptoms. OBJECTIVE: We sought to investigate the effect of mechanical ventilation and high-efficiency vacuuming on HDM numbers and Der p 1 concentrations in the homes of mite-sensitive asthmatic subjects and to evaluate the effect of any reductions on symptoms. METHODS: The homes of 40 HDM-sensitive asthmatic subjects were randomized to receive (1) mechanical ventilation and a high-efficiency vacuum cleaner (HEVC); (2) mechanical ventilation alone; (3) an HEVC alone; and (4) no intervention. Homes and patients were monitored for 12 months. Change in absolute humidity, mite numbers, Der p 1 concentrations, lung function, bronchial hyperresponsiveness, and symptom scores were analyzed. RESULTS: Homes with mechanical ventilation achieved significantly lower humidity levels than those without (P <.001), with an associated reduction of mite numbers (P <.05) and Der p 1 concentrations (P <.001 ¿in nanograms per gram, P =.006 ¿in milligrams per square meter) in bedroom carpets and some other mite sources in the ventilated areas of the homes. The addition of a vacuum cleaner enhanced this effect. There was a trend for an improvement in histamine PC(20) (P =.085) in the patients whose homes were ventilated. CONCLUSION: The use of a mechanical ventilation system in suitable homes resulted in some reduction in numbers of HDM and Der p 1 concentrations. The addition of an HEVC slightly enhanced the effect but not sufficiently to see an improvement in symptoms.
Asunto(s)
Alérgenos/inmunología , Asma/inmunología , Asma/prevención & control , Ambiente Controlado , Ácaros/inmunología , Ventilación/métodos , Adolescente , Animales , Antígenos Dermatofagoides , Asma/fisiopatología , Niño , Preescolar , Polvo , Femenino , Pisos y Cubiertas de Piso , Volumen Espiratorio Forzado , Glicoproteínas/análisis , Humanos , Humedad , Masculino , Temperatura , VacioRESUMEN
Allergen avoidance is regarded as an important approach to management of atopic asthma. The effect of Intervent bed covering systems on house dust mite (HDM) allergen exposure, asthma symptoms and markers of inflammation was investigated in 31 HDM sensitive asthmatic children. Dust concentrations of Dermatophagoides pteronyssinus allergen 1 (Der p 1) were monitored before and after covering the mattress, duvet and pillow with active and placebo covers for 3 months, in a single-blind, cross-over trial. Twice daily peak expiratory flow rate (PEFR), daily symptom scores and treatment schedule were recorded. Bronchial hyperresponsiveness was monitored by histamine challenge (provocative concentration of histamine causing a 20% fall in forced expiratory volume in one second (PC20)), and inflammation by measuring eosinophil cationic protein (ECP), eosinophil protein X (EPX), eosinophil peroxidase (EPO), and soluble interleukin-2 receptor (sIL-2R) in serum. There was a significant reduction in Der p 1 when the mattress, duvet and pillow were covered with the active bedding. There was no significant improvement in symptoms of asthma, PEFR, bronchodilator usage of PC20. Also, ECP, EPX, sIL-2R concentrations did not change for either treatment. EPO concentrations were significantly lower in the active compared to the placebo period. The active bed covers reduced retrievable Dermatophagoides pteronyssinus allergen 1 (Der p 1) from the bedding, with short term clinical benefit.
Asunto(s)
Alérgenos , Asma/fisiopatología , Ropa de Cama y Ropa Blanca , Ácaros , Hipersensibilidad Respiratoria/fisiopatología , Ribonucleasas , Adolescente , Animales , Antígenos Dermatofagoides , Asma/sangre , Asma/terapia , Proteínas Sanguíneas/análisis , Hiperreactividad Bronquial , Pruebas de Provocación Bronquial , Niño , Preescolar , Estudios Cruzados , Polvo/análisis , Proteínas en los Gránulos del Eosinófilo , Peroxidasa del Eosinófilo , Neurotoxina Derivada del Eosinófilo , Eosinófilos , Femenino , Glicoproteínas/análisis , Histamina , Humanos , Mediadores de Inflamación/análisis , Masculino , Ápice del Flujo Espiratorio , Peroxidasas/análisis , Receptores de Interleucina-2/sangre , Hipersensibilidad Respiratoria/sangre , Hipersensibilidad Respiratoria/terapia , Método Simple CiegoRESUMEN
BACKGROUND: Lung function tests, including forced expiratory volume in one second (FEV1), forced expiratory flow at 25-75% of vital capacity (FEF25-75%) and provocation concentrations of histamine which reduce FEV1 by 20% (PC20), are used as indicators of airway form and function in bronchial asthma. Recently, markers of eosinophil activation in bronchial lavage and serum have been suggested as a measure of eosinophil mediated inflammation in the airways. These include eosinophil cationic protein (ECP), eosinophil protein X (EPX) (also known as eosinophil derived neurotoxin) and eosinophil peroxidase (EPO). Similarly, serum tryptase has been used as a marker of mast cell activation in systemic anaphylaxis. OBJECTIVES: We measured both sets of indices in a group of children with moderately severe asthma to assess the contribution of eosinophil and mast cell mediated events to airflow limitation and bronchial hyperresponsiveness. METHODS: Forty-eight children aged 5-10 years had spirometric assessments, histamine challenges and blood sampling on the same occasion. After analysis of sera, the indices were compared. RESULTS: The eosinophil markers ECP and EPX correlated very well with each other. They showed a moderate negative correlation with PC20 for histamine. EPX was also found to negatively correlate with FEV1 and FEF25-75%. Serum tryptase levels showed no such correlates with airway function. CONCLUSION: These results suggest that serum markers of eosinophil activation correlate with airway function in childhood asthma, and may be of value in assessing the severity of the disease. It further supports the notion that childhood asthma has a similar immunopathology to that occurring in adults, with predominance of eosinophil mediated inflammation.
Asunto(s)
Asma/metabolismo , Asma/fisiopatología , Bronquios/fisiopatología , Eosinófilos/metabolismo , Mediadores de Inflamación/metabolismo , Ribonucleasas , Biomarcadores , Proteínas Sanguíneas/metabolismo , Líquido del Lavado Bronquioalveolar/química , Recuento de Células , Niño , Preescolar , Quimasas , Proteínas en los Gránulos del Eosinófilo , Peroxidasa del Eosinófilo , Neurotoxina Derivada del Eosinófilo , Eosinófilos/patología , Volumen Espiratorio Forzado , Humanos , Mastocitos/metabolismo , Flujo Espiratorio Medio Máximo , Peroxidasas/metabolismo , Serina Endopeptidasas/sangre , TriptasasRESUMEN
Uptake of 3H-serotonin followed by autoradiography, and uptake of the serotonin analog 5,7-dihydroxytryptamine (5,7-DHT), with subsequent staining, were each used to define a unique set of neurons in the retina of the African clawed frog, Xenopus laevis. Both techniques demonstrated the same population of neurons, on the basis of perikaryal size, shape, and position within the retina. Two classes of amacrine cells accumulated 5,7-DHT at the proximal (vitread) margin of the inner nuclear layer; the two classes were distinguished by the size of their perikarya. Two similar populations of cells, observed in the ganglion cell layer with lower frequency, may represent "displaced" counterparts of these two amacrine cell types. A class of bipolar cells whose perikarya were located in middle-to-distal regions of the inner nuclear layer also accumulated 5,7-DHT and 3H-serotonin. Processes of these cells contributed to a dense plexus of fine fibers that appeared evenly distributed throughout the inner plexiform layer. 3H-Serotonin-accumulating cells first appeared in the developing retina at stage 35/36, a time immediately after retinal stratification but before elaboration of either plexiform layer. Electron microscopic analysis permitted an identification of 3H-serotonin-accumulating terminals in the inner plexiform layer. Serotonin-labeled terminals containing conventional contacts, suggestive of amacrine cells, were presynaptic to unidentified processes and postsynaptic to bipolar cells. Labeled terminals containing ribbon contacts, indicative of bipolar cells, were postsynaptic to amacrine cells. The amount of serotonin contained in isolated retinas was 15 pmol/mg protein as measured by HPLC with electrochemical detection. We attempted to stimulate the release of accumulated 3H-serotonin from mature retinas by increasing the K+-concentration in the bathing medium. Although preloaded glycine is readily released from 14C-glycine-accumulating neurons, from the same retinas there was no calcium-dependent, K+-stimulated release of 3H-serotonin. This finding suggests that serotonin and glycine are processed differently by retinal neurons, the consequence of which results in differing responses to 40 mM K+.
Asunto(s)
Retina/metabolismo , Serotonina/metabolismo , Xenopus laevis/metabolismo , 5,7-Dihidroxitriptamina/metabolismo , Animales , Autorradiografía , Retina/citologíaRESUMEN
The emergence of GABA-accumulating neurons was studied from stages 29 to 40 during retinal histogenesis in the chick, covering embryonic (E) days E6-E14, using autoradiographic analysis following incubation of isolated retinas with [3H]GABA (2 microM). Analysis was restricted to central retina which is more advanced in its differentiation than the periphery. On E6 numerous mitotic figures were present along the scleral border of the unstratified neuroepithelium. Specific localization of [3H]GABA was associated initially with somata situated in middle regions of the retinal expanse. Occasionally contiguous pairs of labeled cells were seen. The inner plexiform layer makes its appearance during E7; at that time silver grains were present over cell bodies located in the ganglion cell layer and the proximal portion of the inner nuclear layer, those of probable amacrine cells. As retinal stratification continued, more cells were observed to have elaborated membrane systems for GABA uptake with varying degrees of affinity. By E8, although dividing, non-labeled cells were in close proximity, GABA-labeled cells were observed in positions of horizontal cells. By E14, the pattern of label distribution appeared essentially similar to that reported for adult retina, i.e. [3H]GABA labeling was observed over horizontal cells and their processes, subpopulations of amacrine cells which appear to ramify extensively across the inner plexiform layer, selected perikarya of the ganglion cell layer, and the nerve fiber layer. In addition, a subpopulation of labeled photoreceptors, some identified as cones by virtue of oil droplets, was observed. Thus, preferential accumulation of GABA appears during E6, prior to formation of either inner or outer plexiform layers. The localization of [3H]GABA demonstrates that ganglion and amacrine cell bodies are labeled initially, followed by horizontal cells. Specific accumulation of [3H]muscimol, a potent agonist of GABA receptors, appears about E12 over cells located in proximal regions of the inner nuclear layer; these somata later ramify in sublaminae 2 and 4 of the inner plexiform layer.
Asunto(s)
Retina/embriología , Ácido gamma-Aminobutírico/biosíntesis , Animales , Ácido Aspártico/metabolismo , Embrión de Pollo , Mitosis , Muscimol/metabolismo , Neuronas/metabolismo , Retina/metabolismo , Células Ganglionares de la Retina/metabolismoRESUMEN
A method for culturing adult mammalian retinal neurons in serum-free N2 medium supplemented with nerve growth factor (NGF) is described. Identification of neurons in cultures of dispersed human retina was based upon morphology, immunocytochemical localization of bound tetanus toxin, and autoradiographic localization of 3H-neurotransmitter candidates (gamma-aminobutyric acid, glycine, dopamine) accumulated by high-affinity uptake mechanisms. Neurons would not attach to glass or plastic substrates, consequently the present studies were performed using neurons plated upon a feeder layer. Serum was required for the initial phase of attachment. The feeder layer was derived from retinal cells that had been plated on glass or plastic in the presence of serum and had later been passaged. Since these cells exhibited glial fibrillary acidic protein (GFAP) immunoreactivity, they were tentatively identified as being glial in origin. Under these conditions, neuron- and glia-specific properties were retained up to 28 days. The presence of interstitial retinol-binding protein (IRBP) in medium of cultures of neuronal cells on feeder layers was demonstrated by an immunoblot technique using rabbit antibovine IRBP antibodies. No IRBP was detected in medium in which the feeder layers alone had been cultured. IRBP biosynthesis was demonstrated by incubation of the cultures with [35S]methionine. Immunoprecipitable [35S]IRBP was detected only in medium from cultures containing neurons; cells of the feeder layer did not synthesize and secrete this glycoprotein. These findings are consistent with the hypothesis that IRBP, a 135K constituent of the interphotoreceptor matrix, is synthesized in vivo by a neuronal cell, specifically, the photoreceptors.
Asunto(s)
Retina/citología , Adulto , Autorradiografía , Células Cultivadas , Medios de Cultivo , Humanos , Inmunoquímica , Factores de Crecimiento Nervioso , Neuroglía/citología , Neuronas/citología , Neurotransmisores/metabolismo , Proteínas de Unión al Retinol/metabolismoRESUMEN
Metalloendoprotease activity was identified in retinal homogenates using a synthetic fluorogenic metalloendoprotease substrate and specific metalloendoprotease inhibitors. The requirement of metalloendoprotease activity in neurotransmitter release was examined during the depolarization-induced release of [3H]glycine from the retina of Xenopus laevis. Neurons with high affinity uptake and calcium-dependent, K+-stimulated release of glycine have been described previously in this retina. When isolated retinas preloaded with [3H]glycine are depolarized by 22 mM K+, the usual efflux of [3H]glycine is completely abolished by the metalloendoprotease inhibitor, 1,10-phenanthroline (100 micrograms/ml). The inhibition of [3H]glycine release by 1,10-phenanthroline is dose dependent; furthermore, 1,10-phenanthroline blocks release by chelating metal and not Ca2+, since addition of equimolar calcium does not alter the inhibition. The metalloendoprotease inhibitor, carbobenzoxy (CBZ)-L-phenylalanine, also prevents release, whereas the amino acid, L-phenylalanine, has no effect. Synthetic carbobenzoxy dipeptide amides which are metalloendoprotease substrates (e.g., CBZ-Gly-Leu-amide, CBZ-Ser-Leu-amide, and CBZ-Gly-Phe-amide) also prevent [3H]glycine release in a dose-dependent and reversible manner. The synthetic dipeptide CBZ-Gly-Gly-amide, however, is not a metalloendoprotease substrate and has no effect on release. The ability of synthetic dipeptides to inhibit neurotransmitter release is amino acid specific, dose dependent, reversible, and matches their ability to interact with characterized metalloendoproteases. Depolarization-stimulated transmitter release may therefore require the activity of a metalloendoprotease.
Asunto(s)
Neurotransmisores/farmacología , Fenantrolinas/farmacología , Potasio/fisiología , Inhibidores de Proteasas/farmacología , Retina/metabolismo , Xenopus/metabolismo , Animales , Glicina/metabolismo , Fusión de Membrana , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotransmisores/metabolismo , Fenilalanina/farmacología , Potasio/farmacología , Retina/efectos de los fármacos , Retina/fisiología , Sinapsis/metabolismo , Sinapsis/fisiología , Tritio , Xenopus/fisiologíaRESUMEN
Neurotransmitter-specific properties of glycinergic neurons in the human retina were studied using 11 pairs of eyes from donors ranging from 2 1/2 to 54 years in age. A mean endogenous level of 10.3 nmoles glycine per mg protein was measured by amino acid analysis in retinas isolated within 1 hour postmortem. When retinas were incubated with 3H-glycine (2 microM) and processed for autoradiography, label was found associated with neurons whose somata reside within the inner nuclear layer. Some heavily labeled neurons located at the vitread border of the inner nuclear layer were identified as amacrine cells based on ultrastructural verification of the conventional synaptic contacts made by their processes in distal regions of the inner plexiform layer. In proximal regions of the inner plexiform layer, dendrites of glycine-accumulating amacrine cells were postsynaptic to both ribbon and conventional synaptic contacts, suggesting input from bipolar and other, nonglycinergic amacrine cells. Their density (30 +/- 11 S.D. cells/mm linear retinal expanse) tended to be greater toward the central fundus. A second population of lightly labeled, probable bipolar cells was present in the middle of the inner nuclear layer; the density of this second set of glycine-accumulating cells approximated that of the heavily labeled population from the fovea, centrally, to the ora serrata, peripherally. Release of either accumulated or endogenous glycine was elicited by K+-depolarization in a Ca2+-dependent manner. Tissue fragments exposed for 6 minutes to normal medium, 40 mM K+-substituted medium, or K+-substituted medium with Co2+, release endogenous glycine into each bathing solution in average amounts of 0.6, 2.6, and 0.7 nmoles per mg protein, respectively. Together these data strongly implicate glycine as a neurotransmitter in the human retina.
Asunto(s)
Glicina/fisiología , Retina/fisiología , Transmisión Sináptica , Autorradiografía , Glicina/metabolismo , Humanos , Microscopía Electrónica , Retina/metabolismo , Retina/ultraestructuraRESUMEN
Protein synthesis, glycosylation, RNA synthesis, and neurotransmitter uptake were monitored using biochemical and autoradiographic techniques following in vitro labeling of retinal tissue from a 79-year-old female with sectoral retinitis pigmentosa (RP). Comparisons were made between degenerate and non-degenerate regions of the RP retina, and normal retinal tissues from an age- and postmortem-matched donor. Autoradiographs of non-degenerate retina from the RP eye following 3H-uridine incubation revealed virtually identical silver grain density over nuclei in all retinal strata as compared to normal control retinas. In contrast, a photoreceptor-specific reduction in silver grain density in the non-degenerate RP retina was noted following 3H-leucine incubation. In the normal retina, rod photoreceptor labeling with 3H-mannose was always greater than cone photoreceptor labeling. This pattern of incorporation was reversed in the non-degenerate region of the RP retina where rod photoreceptor labeling was less pronounced than that observed for cone photoreceptors. In non-degenerate regions of the RP retina, a marked accumulation of 3H-GABA by the Müller's cells was observed. Few cells exhibited selective uptake of 3H-muscimol, a GABA analog, indicating that few GABAergic neurons remained in the degenerate retina. 3H-dopamine-accumulating cell terminals were observed in the usual positions in the non-degenerate RP retina. In the degenerate region of the RP retina, heavy and diffuse uptake of 3H-GABA and 3H-muscimol, respectively, into broad cellular processes were noted, whereas 3H-dopamine was accumulated by only a few punctate terminals.
Asunto(s)
Aminoácidos/metabolismo , Coroides , Degeneración Retiniana/metabolismo , Retinitis Pigmentosa/metabolismo , Enfermedades de la Úvea/metabolismo , Anciano , Coroides/metabolismo , Técnicas de Cultivo , Femenino , Humanos , Leucina/metabolismo , Manosa/metabolismo , Células Fotorreceptoras/metabolismo , Degeneración Retiniana/diagnóstico , Retinitis Pigmentosa/diagnóstico , Ácido Tricloroacético/metabolismo , Uridina/metabolismo , Enfermedades de la Úvea/diagnósticoRESUMEN
Human retinal tissue from a newborn was examined autoradiographically for the presence of high-affinity uptake and localization of the following putative neurotransmitters: dopamine, glycine, GABA, aspartate, and glutamate. In addition, the dopamine content of this newborn retina was measured by high pressure liquid chromatography. Our study reveals that specific uptake mechanisms for 3H-glycine, 3H-dopamine, and 3H-GABA are present at birth. However, the number and distribution of cells labeled with each of these 3H-transmitters are not identical to those observed in adult human retinas. Furthermore, the amount of endogenous dopamine in the newborn retina is approximately 1/20 the adult level. Photoreceptor-specific uptake of 3H-glutamate and 3H-aspartate are not observed. These findings indicate that, while some neurotransmitter-specific properties are present at birth, significant maturation of neurotransmitter systems occurs postnatally.
Asunto(s)
Recién Nacido , Neurotransmisores/metabolismo , Retina/metabolismo , Ácido Aspártico/metabolismo , Autorradiografía , Cromatografía Líquida de Alta Presión , Dopamina/metabolismo , Glutamatos/metabolismo , Glicina/metabolismo , Humanos , Tritio , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The utilization of dopamine in the adult human retina was examined by using high-affinity uptake, localization, synthesis, and release as neurotransmitter-specific physiological probes. Autoradiographic and histochemical studies have shown that dopamine-accumulating and dopamine-containing cells of the human retina belong to a population of neurons whose somata are located in the proximal regional of the inner nuclear layer. Some of these are amacrine cells which are pre- and postsynaptic to other amacrine cells exclusively in the inner plexiform layer. However, evidence is presented which indicates the existence of interplexiform dopaminergic neurons which send processes to both plexiform layers of the retina. These neurons contain a high concentration of dopamine, take up 3H-dopamine by a hig-affinity mechanism, and release endogenous or accumulated dopamine by a Ca2+-dependent mechanism upon depolarization with high extracellular K+. An endogeneous level of about 20 pmoles dopamine per mg protein was measured in freshly isolated retina using high-pressure liquid chromatography with electrochemical detection. These results demonstrate that mechanisms for dopaminergic neurotransmission are present in the human retina.