Bleached Pigment Produces a Maintained Decrease in Outer Segment Ca²⁺ in Salamander Rods

A spot confocal microscope based on an argon ion laser was used to make measurements of cytoplasmic calcium concentration (Ca²⁺_i) from the outer segment of an isolated rod loaded with the fluorescent calcium indicator fluo-3 during simultaneous suction pipette recording of the photoresponse. The decline in fluo-3 fluorescence from a rod exposed to saturating illumination was best fitted by two exponentials of approximately equal amplitude with time constants of 260 and 2,200 ms. Calibration of fluo-3 fluorescence in situ yielded Ca²⁺_i estimates of 670 ± 250 nM in a dark-adapted rod and 30 ± 10 nM during response saturation after exposure to bright light (mean ± SD). The resting level of Ca²⁺_i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 ± 5% (SEM, n = 9) of its value in the dark-adapted rod. Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca²⁺_i. These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca²⁺_i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.