Light and Calcium in Salamander Rods

Abstract

Abstract: : Purpose: In darkness, Ca²⁺ enters through cGMP-gated channels in the rod outer segment and exits via Na⁺/Ca²⁺-K⁺ exchange. Light closes the channels and decreases Ca²⁺_i , but the relationship between light, current, and Ca²⁺_i has remained unclear. We have previously shown that intense light evokes a release of Ca²⁺ within the outer segment, most clearly seen when a rod is superfused with a 0Ca²⁺/0Na⁺ solution designed to oppose surface membrane Ca²⁺ fluxes. It seemed possible that this release would contribute to changes in Ca²⁺_i. Methods: Rods isolated from salamanders killed humanely by stunning, decapitation and pithing were loaded with the fluorescent indicator dye fluo 5F and illuminated with a laser to measure free Ca²⁺_i. Electrical responses were measured with suction electrode recording, and solutions were changed as previously described by moving the microscope stage with a stepping motor. Results: After exposure to saturating light, the circulating current, Ca²⁺_i and the light-releasable pool of Ca²⁺ all recover with a nearly identical exponential time course. There is no observable lag in recovery of Ca²⁺_i. Steady background light produces a maintained decrease in current, which is accompanied by a nearly proportional decrease both in Ca²⁺_i and in the light-releasable Ca²⁺ pool. The amount of Ca²⁺ in the light-releasable pool appears to be determined principally by Ca²⁺_i except during bright light exposure. This is most clearly seen if the rod is illuminated in 0Ca²⁺/0Na⁺ solution, since relatively small bleaches in this solution deplete the releasable pool even in the absence of a light-induced decline in Ca²⁺_i, indicating some special effect of pigment bleaching on Ca²⁺ buffering and/or sequestration. This effect is spatially localised, since exposure to a narrow slit of intense light in 0Ca²⁺/0Na⁺ solution eliminates the releasable pool in that location but not in neighbouring unilluminated regions of the outer segment. Conclusions: These experiments show that the major determinant of Ca²⁺_i in the outer segment following just-saturating illumination or in the presence of background light is the rate of influx through the channels, and that the light-releasable pool makes only a minor contribution. Bleaching produces specific depletion of the light-releasable pool, but the functional consequences of this light-induced calcium release for the rods is unknown.

Keywords: photoreceptors • calcium • dark/light adaptation