Phenylethanolamine-N-methyltransferase (PNMT) activity is located in a subpopulation of amacrine cells in the inner nuclear layer of the rat retina. Kainic, quisqualic, and ibotenic acids, all of which are analogues of glutamic acid, were injected intravitreally to the right and saline to the contralateral left eyes of adult male rats in order to determine the effect of these agents upon retinal PNMT activity. Animals were sacrificed 1 week later for tissue removal. The effect of these agents was measured by radiometric assay for PNMT. The fall in PNMT activity was used to measure the sensitivity of the PNMT-containing cells to these agents. Kainic acid was the most potent, producing the greatest reduction in PNMT activity in the smallest doses. Quisqualic acid was intermediate in potency to that of kainic and ibotenic acids. Ibotenic acid reduced PNMT activity only in extremely high doses. The PNMT-containing cells are sensitive to the toxic actions of kainic and quisqualic acids, but relatively insensitive to the actions of ibotenic acid.

Vision requires the photoreceptors in the eye to rapidly respond to changes in light intensity. These processes are accomplished within rod photoreceptors by the visual pigment rhodopsin that initiates a downstream signaling cascade called phototransduction. Rhodopsin is composed of an apoprotein opsin that is covalently bonded with light sensitive 11-cis retinal. Rhodopsin is activated when 11-cis retinal is photoisomerized into all-trans retinal. This isomerization initiates the phototransduction cascade that culminates in a change in current at the plasma membrane. Rhodopsin, once activated ("bleached"), can no longer absorb photons to activate phototransduction, and must be regenerated through the visual cycle. To enable the photoreceptors to respond to rapid changes in light intensities, phototransduction must terminate in a timely manner. Deactivation involves phosphorylation of activated rhodopsin by rhodopsin kinase, and then binding of visual arrestin. Exposing rods to daylight bleaches a large proportion of rhodopsin molecules. This exposure leads to desensitization of the photoreceptors and phosphorylation of bleached rhodopsin. Full recovery of receptor sensitivity is achieved when rhodopsin is recycled and regenerated through a series of steps to its ground state. The last step in this process is the dephosphorylation of rhodopsin. This dissertation focuses on how rhodopsin dephosphorylation affects rod sensitivity. I exploited a novel observation; mouse retinae when isolated from the retinal pigment epithelium (and eye cup), display blunted rhodopsin dephosphorylation. Isoelectric focusing followed by Western blot analysis of retinal homogenate from bleached isolated retinae showed little dephosphorylation of rhodopsin for up to four hours in darkness, even under conditions when rhodopsin was completely regenerated. Microspectrophotometric measurements of rhodopsin spectra show that regenerated phospho-rhodopsin has the same molecular photosensitivity as unphosphorylated rhodopsin and that flash responses measured by trans-retinal electroretinogram or single cell suction electrode recording displayed dark-adapted kinetics. Single quantal responses displayed normal dark-adapted kinetics, but rods were only half as sensitive as those containing exclusively unphosphorylated rhodopsin. I propose a revised model in which light-exposed retinae contain a mixed population of phosphorylated and unphosphorylated rhodopsin. Moreover, complete dark-adaptation can only occur when all rhodopsin has been dephosphorylated, a process that requires more than three hours in complete darkness.

PURPOSE: Previous studies have reported an increased prevalence of ocular diseases in patients with obstructive sleep apnea. The purpose of this study was to examine the link between such ocular diseases as diabetic retinopathy, diabetic macular edema, retinal vein occlusion, central serous chorioretinopathy, age-related macular degeneration, non-arteritic anterior ischemic optic neuropathy, and glaucoma. METHODS: This meta-analysis was conducted through a search using PubMed, Web of Science, Scopus and EMBASE. We identified both retrospective and prospective studies. RESULTS: The final meta-analysis looked at 30 studies and 7 ocular diseases. The data showed a high prevalence of obstructive sleep apnea for diabetic retinopathy and diabetic macular edema patients. Data for glaucoma and non-arteritic anterior ischemic optic neuropathy patients did not show a statistical increase. There was not enough data for retinal vein occlusion, central serous chorioretinopathy and age-related macular degeneration to calculate statistical significance. CONCLUSION: These data suggests that patient populations with diabetic retinopathy and macular edema show increased rates of obstructive sleep apnea. Data suggest that hypoxia is an important part of the pathophysiology of diabetic retinopathy and diabetic macular edema. Because obstructive sleep apnea has been shown to affect the progression of the ocular diseases included in this study, ophthalmologists should screen for the presence of obstructive sleep apnea to better help their patients. / 2021-06-14T00:00:00Z

The interest of the writer was directed toward the phenomena of color vision in 1927 as a result of attending concurrently two courses in the University of California touching upon the subject, but treating it quite differently with results far from mutually consistent. Prof. R. S. Minor, in his course on physical optics, discussed color vision from the physicist’s standpoint, making use of the Young-Helmholtz theory; while Prof. G. M. Stratton gave the psychological treatment, based upon the Ladd-Franklin theory, as a part of his general psychology. Several of the more obviously points of conflict impressed the writer so strongly that he was led to bring the matter to the attention of the professors concerned. At the resulting conference between the two, a number of demonstrative experiments were performed and from the discussion of these in terms of the various theories the writer profited much. However, many facts remained unreconciled, so the writer resolved to investigate the subject further should opportunity present itself. The following research is a beginning of that study.

No description available.

El diacilglicerol (DAG) es un importante intermediario en la síntesis de muchas clases de lípidos, y es, además, un segundo mensajero producido en respuesta a diversos estímulos extracelulares, capaz de modular la actividad de numerosas enzimas. Una de las vías de metabolización del DAG es la fosforilación llevada a cabo por las DAGK, para dar ácido fosfatídico (PA). El ácido fosfatídico es también un importante segundo mensajero lipídico involucrado en una gran variedad de respuestas celulares. Dada la importancia fisiológica del DAG y del PA como segundos mensajeros lipídicos, y, teniendo en cuenta los numerosos resultados que indican la presencia de las vías de señalización responsables de la síntesis y degradación de estos segundos mensajeros a nivel nuclear, iniciamos nuestros estudios analizando la actividad de las DAGK en los núcleos de la retina bovina, proyectando la investigación hacia la interpretación de su participación en las funciones esenciales de la retina, la recepción y transmisión de la luz. Además, analizamos a nivel nuclear, en las células fotorreceptoras de la retina, los efectos de la insulina, un reconocido protector del sistema nervioso central (SNC) y modulador de la actividad DAGK en otros tipos celulares del SNC. Mediante un protocolo diseñado en nuestro laboratorio, obtuvimos a partir de la retina entera una fracción nuclear enriquecida en los núcleos de las células fotorreceptoras (FNF). En esta fracción nuclear, se pudo detectar actividad DAGK, transformadora del DAG endógeno y exógeno. Se determinó linealidad en función del tiempo de ensayo, el contenido proteico de la fracción nuclear y se analizaron parámetros cinéticos aparentes (Km y Vmax para cada sustrato). Los resultados con detergentes y sustratos diferentes permitieron sugerir la coexistencia de varios tipos de DAGK. Esto fue confirmado por Western Blot (WB) y se detectaron, además, efectos significativos en el contenido nuclear de estas isoformas por efecto de la luz (aumento de la DAGKδ y disminución de las DAGKe, ß y B). Nuestros hallazgos se reforzaron con ensayos enzimáticos en los que se utilizaron condiciones selectivas (preferenciales) para medir la actividad de las isoformas δ y e.En ellos se observó, por efecto de la exposición de la retinas a la luz, una fuerte correlación entre los cambios en el contenido y la actividad de ambas isoformas en la FNF. Se demostró asimismo que el aumento de la actividad DAGK nuclear en respuesta a la luz es dependiente de la actividad PIP2-PLC, y se observó que dicho incremento en la condición lumínica no es debido exclusivamente a un aumento del sustrato de la reacción enzimática, DAG, sino que también participa en la activación el PIP2. También demostramos la presencia de la PKCα en la FNF, PKC de tipo convencional que es activada en presencia de Ca2+ y DAG. La exposición de las retinas a la luz produjo un aumento del estado de fosforilación de la PKCα. Dado que la fosforilación de PKC puede ser mediada por la activación de PDK1, que es dependiente de la activación previa de PI3K, enzima participante de la vía de señalización de insulina, el siguiente objetivo fue determinar la presencia de los principales componentes de las vías de señalización de insulina en la FNF. Nuestros resultados demostraron la presencia de Akt total y en su estado fosforilado (proteína quinasa de la via de la PI3K), y de ERK1/2, pERK1/2 y p38 fosforilada (quinasas correspondientes a la vía de las MAPK). Nuestros estudios demostraron, además, que la exposición de las retinas bovinas a la luz produce un aumento en el estado de fosforilación de Akt, y que induce la translocación de ERK1/2 activada a la FNF. Por otro lado, al analizar los efectos directos e indirectos de la insulina sobre la actividad DAGK nuclear en la FNF de retinas expuestas a la luz o a la oscuridad, fue posible demostrar que la insulina es capaz de modular la actividad DAGK nuclear tanto de forma directa (incubación de los núcleos aislados con insulina) como de manera indirecta (incubación de las retinas bovinas con insulina y posterior análisis de la actividad DAGK en la FNF). Además, se observó que la insulina cumple un rol en los cambios de la actividad DAGK nuclear en respuesta a la luz, y que los efectos de la insulina sobre la actividad DAGK son dependientes de su concentración (los efectos se incrementan con un aumento concomitante de la concentración de insulina empleada en el ensayo enzimático). Teniendo en cuenta los efectos directos de la insulina sobre la actividad DAGK nuclear, analizamos la presencia del receptor de insulina en la FNF por WB e inmunofluorescencia (IF) y demostramos la presencia del mismo en los núcleos de las células fotorreceptoras. Un hallazgo de particular interés fue el de haber demostrado que el contenido del RI aumenta en la FNF cuando las retinas bovinas son expuestas a la luz, lo cual sugiere que la luz puede ser un estímulo capaz de promover la translocación del RI al núcleo de las células fotorreceptoras. Por último, analizamos si la insulina es capaz de participar en la translocación del receptor de insulina al núcleo de las células fotorreceptoras y de mediar la activación de las vías de señalización activadas por la misma a nivel nuclear. Nuestros resultados indicaron que la insulina produce un aumento en el contenido del receptor de insulina nuclear con respecto a la condición luz en ausencia de hormona. Además, la insulina produjo un aumento de ERK1/2 activado en la FNF. En conclusión, nuestros resultados demostraron por primera vez que la exposición de las retinas bovinas a su estímulo natural, la luz, paralelamente a la activación de la típica vía de la fototransducción que se inicia en los segmentos externos, induce a nivel nuclear, la activación de distintas vías de señalización responsables de funciones críticas para la célula, y que a nivel nuclear, pueden intervenir en la transcripción de genes. Nuestros resultados demostraron también que la luz y la insulina son capaces de intervenir en la translocación del receptor de insulina desde la membrana plasmática hacia el núcleo de las células fotorreceptoras, y que la insulina promueve la activación de vías de señalización tanto de forma indirecta, actuando sobre la retina entera, como directa a nivel nuclear, sugiriendo para este último caso que mediaría sus efectos a través de la población nuclear de dicho receptor. / Diacylglycerol (DAG) is an important intermediate in the synthesis of several types of lipids, and it is also a second messenger produced in response to various extracellular stimuli, with the ability to modulate the activity of numerous enzymes. One route of metabolism of DAG is phosphorylation by DAGK to yield phosphatidic acid (PA). PA is also an important lipid second messenger that has been involved in a variety of cellular responses. Given the physiological importance of DAG and PA as lipid second messengers and taking into account the many results that indicate the presence of the signaling pathways responsible for the synthesis and degradation of these second messengers at the nuclear level, we initiated our studies assessing the activity of DAGK in the nuclei of bovine retina, projecting them to the interpretation of their participation in the essential functions of the retina, the receipt and transmission of light. We also analyzed at the nuclear level in retina photoreceptor cells, the effects of insulin, a known protector of the central nervous system (CNS) and DAGK activity modulator in other cell types of the CNS. Using a protocol designed in our laboratory, we obtained from the entire retina a nuclear fraction enriched in photoreceptor cell nuclei (PNF). In the nuclear fraction, DAGK activity could be detected, which is responsible for the transformation of endogenous and exogenous DAG. A linear response was obtained as a function of protein content of the nuclear fraction and as a function of time. The apparent kinetic parameters (Vmax and Km for each substrate) were also determined. The results derived from the different substrates and detergents used suggest the coexistence of various types of DAGK. This was confirmed by Western Blot (WB), and significant effects were detected in the nuclear content of these isoforms by the effect of light (increased of DAGKδ and decreased of DAGK E, ß and B). Our findings were strengthened by further enzyme assays using selective conditions to measure the activity of E and δ isoforms in which, due to the exposure of retina to light, strong correlation was observed between changes in the content and the activity of both isoforms in the PNF. It was also demonstrated that the increase in nuclear DAGK activity in response to light is dependent on PIP2-PLC activity and that this increase in light condition is not due only to an increase of the substrate of the enzymatic reaction, DAG, but also to the fact that PIP2 participates in DAGK activation. We also showed the presence of PKCα in FNF, conventional PKC which is activated in the presence of Ca2+ and DAG. Retina light exposure produced an increase in the phosphorylation status of PKCα. In addition, because phosphorylation of PKC could be mediated by the activation of PDK1, which is dependent on the prior activation of PI3K, an enzyme participant of insulin signaling pathway, our second main objective in the present study was to determine the presence of the main components of the insulin signaling pathway in PNF. Our results showed the presence of total Akt and its phosphorylated state (protein kinase of PI3K pathway) and ERK1/2, pERK1/2 and phospho-p38 (components of the MAP kinases pathway). Our results also showed that light exposure to bovine retinas causes an increase in the phosphorylation status of Akt and induces the translocation of ERK1/2 activated to PNF. In assessing the direct and indirect effects of insulin on nuclear DAGK activity in the FNF from retinas exposed either to light or darkness, it was demonstrated that insulin can modulate DAGK activity nuclear both directly (the incubation of isolated nuclei with insulin), and indirectly (the incubation of bovine retinas with insulin and the subsequent analysis of the DAGK activity in the FNF). We also observed that insulin has a role in the changes of nuclear DAGK activity in response to light, and that these effects on DAGK activity are dependent on its concentration (the effects are increased with a concomitant increase in the insulin concentration used in the enzyme assay). Taking into account the direct effects of insulin on nuclear DAGK activity, we analyzed the presence of the insulin receptor in the FNF by WB and IF and we could interest was to have shown that the contents of RI increase in FNF when bovine retinas are exposed to light, thus suggesting that light can be a stimulus capable of promoting the translocation of RI to the nucleus of the photoreceptor cells. Finally, we examined whether or not insulin is able to participate in insulin receptor translocation to the nucleus of the photoreceptor cells and to mediate the activation of signaling pathways related with insulin at the nuclear level. Our results indicated that insulin causes an increase in the content of the nuclear insulin receptor with respect to light condition in the absence of the hormone. Furthermore, incubation of PNF with insulin resulted in an increase of nuclear, activated ERK1/2. Summing up, our results demonstrate for the first time that light exposure of bovine retinas, its natural stimulus, in parallel to the typical activation of phototransduction pathways which starts in the outer segments, induces, at the nuclear level, the activation of different signaling pathways known to be responsible for critical functions to the cell, as gene transcription. Our results also reveal that light and insulin are involved in the translocation of the insulin receptor from the plasma membrane to the nucleus of the photoreceptor cells. Insulin also promotes the activation of cell signaling pathways, thus indirectly acting on the retina, or directly in the nucleus, suggesting that it mediates their effects through a nuclear population of the insulin receptor.

Aims: The electroretinogram (ERG) can be used to independently assess the function of rod and cone photoreceptors within the human retina. The work in this thesis sought to investigate an alternative method of recording the ERG, using the silent substitution paradigm (Estevez and Spekreijse 1982). The aims are separated into two parts, firstly, the isolation and characterisation of the non-dark adapted rod photoreceptor response, and secondly, characterising the ERG response from L-, M- and S-cones. Methods: Rod, L-, M- and S-cone isolating as well as non-isolating sinusoidal flicker and transient square-wave stimuli were generated on a 4 primary LED ganzfeld stimulator to elicit ERGs from non-dark adapted participants with normal and compromised rod or cone function. Results: The results from the rod experiments showed that ERGs elicited by rod isolating silent substitution stimuli exhibit low-pass temporal frequency response characteristics with an upper response limit of 30Hz and saturate beyond 1000ph Td. Responses are optimal between 5 – 8 Hz and between 10-100 photopic Td. There is a significant correlation between the response amplitudes obtained with the silent substitution method and current standard clinical protocols. The results from the cone experiments showed that the L-, M- and S-cone stimulation produced ERGs with very different morphologies. L- and M-cone stimulation is of limited use as an objective measure of colour vision deficiency. Conclusion: Silent substitution provides an effective method for the isolation of human rod and cone photoreceptor function in subjects when stimuli are used within appropriate parameter ranges.

No description available.

No description available.

No description available.