This application has two parts. First, we propose to investigate the mechanisms of bleaching and dark adaptation in photoreceptors. Second, we will investigate how light regulates oxidative metabolism in rods and cones. Electrical responses of isolated cells will be measured with suction micropipettes. Substances will be introduced into isolated photoreceptors via patch pipettes. Oxygen consumption in single cells will be measured with oxygen-sensitive microelectrodes. Specifically: 1a. We shall investigate the role of G-protein activation in bleaching adaptation. Activators (GTP-gamma-s, GMP-PNP) of G-proteins will be introduced via patch pipettes into isolated cells to estimate GTP turnover in darkness, during background light, and following bleaching to investigate the notion, suggested by our preliminary data, that bleached pigment (opsin) activates transducin. 1b. We shall investigate the mechanism of the activation of transducin by bleached pigment. Previous experiments have established that bleached pigment (probably opsin) activates PDE and guanylyl cyclase in rods and cones and is probably responsible for bleaching adaptation. We will compare membrane noise which is activated by dim background light with that associated with bleaching to test whether this activation occurs via recycling of pigment intermediates to R* or activates transducin directly. 1c. We shall investigate the role of changes in [Ca2+]i during bleaching adaptation and dark adaptation. We shall make measurements of [Ca2+]i in dark adapted photoreceptors, bleached photoreceptors, and bleached photoreceptors which have been exposed to 11-cis retinal and certain of its analogues. 2. We shall begin to investigate the role that ganglion cells (and other proximal retinal neurons) play in adaptation. Previously, it has been shown that light too dim to adapt rods, adapts ganglion cells and it is thought that this occurs via synaptic interactions in the retina. We will test to see if this is true by comparing background adaptation (associated with photon noise) and bleaching adaptation (which has a much lower noise component) at the level of ganglion cells. 3. We shall investigate the mechanisms whereby light activates changes in oxygen consumption in isolated photoreceptor cells. We shall do this by simultaneously measuring single cell sensitivity and oxygen consumption during and following the release of Ca2+, ATP, and ADP from caged compounds introduced into the cell from patch pipettes.
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