The project aims to assess the importance of changes in intracellular free calcium concentration, [Ca2+]i, in the response to light of intact vertebrate rod photoreceptors. We hope to determine (1) what is [Ca2+]i in rod outer segments (ROS) in darkness and light; 2) if [Ca2+]i does change on illumination, as current theories predict, does it correlate with stimulus intensity and with the conductance change in ROS plasma membranes; 3) to what extent are [Ca2+]i changes driven by plasma mambrane fluxes or by internal stores; 4) can [Ca2+]i be altered by means other than light, and if so, how are rod properties affected? [Ca2+]i will be measured by highly Ca2+-specific indicators loaded into rod cytoplasm by intracellular hydrolysis of labile membrane-permeant esters. Our newest indicator, """"""""Fura-2"""""""", is likely to be good enough to permit physiological measurements of [Ca2+]i, through major efforts will continue towards the synthesis of yet better chelators working at deep red or infra-red wavelengths. Toad or bullfrog rods will be loaded with the [Ca2+]i indicator, whose fluorescence will be monitored along with the mass receptor potential or the outer segment current of one representative rod. We would hope to correlate [Ca2+]i with input variables such as stimulus light intensity and duration, extracellular ion concentrations (especially of Ca2+ and Na+), pharmacological agents including ionophores for Ca2+ and modifiers of cyclic nucleotide metabolism, and indicator content varing from non-buffering levels to high levels which should damp down any [Ca2+]i transients. [Ca2+]i would also be compared with the simutaneously measured electrical responses and with intradiskal calcium contents directly observed in separate experiments by new electron-microscopic techniques. Such experiments could help greatly in defining what [Ca2+]i does in rods and in deciding between models for phototransduction in vertebrate retina. If [Ca2+]i measurements succeed, closely analogous measurements of cytosolic pH and free Na+ should be possible using other indicators developed by the P.I.
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