Abstract
Purpose: :
The recovery of sensitivity following significant pigment bleaching requires the quenching of the catalytic intermediates in phototransduction. The reduction of all-trans retinal to all-trans retinol is key in this process. Studies of the buildup of retinol fluorescence after bleaching show that the rate decreases in a gradient going from the base to the tip of the rod, and broadly matches the time course of the recovery of sensitivity. This gradient in fluorescence must result from a gradient in a key component in retinal reduction. The goal of these experiments is to determine the metabolic constraints on the recovery of sensitivity, particularly the role of the co-factor NADPH on retinal reduction.
Methods: :
The outer segment membrane current from isolated salamander rods were recorded with suction electrodes while cells were superfused with Ringer solution at 22oC. Light responses were evoked by 30 ms flashes of full-field illumination (max ~ 520 nm) or by a narrow slit to stimulate selectively the base or tip of the rod. Simultaneous whole-cell voltage clamp recordings were made from the tip of the outer segment for 1-2 minutes prior to bleaching to dialyze the cell with 5 mM NADPH, or a control solution, to investigate how NADPH alters the rate of retinal reduction and the recovery of sensitivity.
Results: :
After a 50% bleach of the visual pigment rod photoreceptors remained in saturation for nearly 10 minutes. We measured the recovery of dark current and sensitivity elicited by light flashes focused at the base and tip of the outer segment as a function of the post-bleach time. We found that the base recovered sensitivity and responsiveness to steady-state, with a time constant of ~ 200 s, but the tip recovered more slowly with a time constant of ~ 700 s. Whole-cell dialysis of 5 mM NADPH into the rod prior to bleaching: (1) accelerated the time at which the post-bleach responses began to recover by about ~ 2 min, and (2) surprisingly slowed the rates of recovery at both the base and tip of the of the OS by ~ 2-fold.
Conclusions: :
The earlier recovery of the dark current following dialysis of NADPH suggests that it depends on the reduction of all-trans retinal and is limited by the cellular NADPH concentration. An explanation for the secondary effect of NADPH slowing the rate of recovery of sensitivity is more problematic, but suggests that NADPH affects rod recovery by multiple mechanisms, one which may involve an NADPH-dependent slowing of an additional factor that controls sensitivity.
Keywords: photoreceptors • metabolism • photoreceptors: visual performance