Bleached pigment activates transduction in isolated rods of the salamander retina.

Abstract

1. We have used suction electrode recording together with rapid steps into Li' solution and 0 5 mM IBMX solution to estimate the rates of the guanylyl phosphodiesterase (PDE) and guanylyl cyclase in isolated rods of the salamander, Ambystoma tigrinum. 2. We show that both the PDE and cyclase velocities are accelerated by steady background light. The steady velocities of both enzymes appear to be saturating functions of background intensity. 3. Bleaching also accelerates both the PDE and cyclase. This effect is maintained long after the bleaching stimulus is removed (up to 2 h) and is reversed only if the photopigment is regenerated with exogenous chromophore. 4. The estimated steady-state PDE and cyclase velocities appear to be linear functions of the amount of pigment bleached, as if each bleached pigment molecule activated the transduction cascade with the same probability and gain. 5. The effectiveness of bleached pigment in activating transduction is only 10-6 to times that of activated rhodopsin (Rh*), but this is sufficient after large bleaches to produce an 'equivalent background' excitation of the rod, which is probably responsible, at least in part, for bleaching desensitization.When the eye is exposed to light bright enough to bleach a significant fraction of the visual pigment, the sensitivity of the visual system is greatly depressed and recovers slowly. This phenomenon, known as dark adaptation or bleaching adaptation, is still poorly understood. Since the recovery of sensitivity has a time course which is approximately the same as the regeneration of visual pigment (Rushton, 1965), it is reasonable to suppose that the loss of pigment is somehow responsible for the loss of sensitivity. However, it has long been realized that the decrease in sensitivity is much greater than would be expected from the decrease in the probablility of light absorption by the photopigment (Campbell & Rushton, 1955). The visual system appears to produce a bleaching signal that elevates threshold much more than simple loss of pigment would predict.To investigate the nature of this bleaching signal, we have recorded from isolated rods of the salamander, Ambystoma tigrinum. Earlier experiments have shown that the bleaching of pigment in these photoreceptors produces a loss of sensitivity which, like that in the visual system as a whole, is considerably greater than expected from the decrease in pigment concentration (Cornwall, Fein & MacNichol, 1990). Furthermore, the light responses of bleached rods at steady state have many of the characteristics of those of rods in the presence of background light: sensitivity is decreased, circulating current is reduced, and the decay time of the response to brief flashes is accelerated. These observations suggest that bleaching may desensitize the rods by producing an 'equivalent background' excitation, as first suggested from psychophysical experiments by Stiles & Crawford (1932).Furthermore, the similarity of responses in the presence of background light and after bleaches suggests t...

Light-Dependent Translocation of Arrestin in the Absence of Rhodopsin Phosphorylation and Transducin Signaling

“…This might not be surprising given that opsin triggers excitation with a 10 6 -10 7 times smaller efficiency than rhodopsin (Cornwall and Fain, 1994) and given that the light intensity threshold for detection of transducin movement after 1 hr of exposure was estimated to be 20 scotopic cd/m 2 , resulting in the bleaching of ϳ0.02% of the rhodopsin content (ϳ10 4 rhodopsin molecules) per second (Sokolov et al, 2002). The restriction of Tr ␣ immunolocalization to the rod outer segments of RPE65 Ϫ/Ϫ retinas excluded the nonspecific mislocalization of proteins in these rods attributable to cytoskeletal disruption.…”

Why photoreceptors die (and why they don't)

“…Only after many months of deprivation do the rods start to degenerate, leading to a permanent loss of vision and eventual blindness. (30) The connection between vitamin A deprivation and constant light exposure was for a long time unclear and only became apparent when Cornwall and I (31) showed that opsin can activate the visual cascade. Our experiments demonstrated that opsin even without chromophore is noisy, occasionally triggering the activation of transducin (32) and collectively producing an electrical response; these observations were later confirmed by biochemical measurement of opsin activity.…”

“…Mammalian opsin is considerably noisier, though it still takes of the order of 10 4 -10 5 opsin molecules to have the effect of one Rh* per second. (34) But since activation is proportional to the opsin concentration, (31) and since there are so many opsin molecules in the outer segment during vitamin A deprivation, the combined effect of the opsin of the entire cell can produce an activation equivalent to that produced by a nearly saturating steady light. That is, the free opsin stimulates the visual cascade and acts effectively like a continuous background illumination, which we shall call an equivalent background.…”

Measurement of Slow Spontaneous Release of 11-cis-Retinal from Rhodopsin

“…In contrast to the opsin formed during Meta-II decay following the photoactivation or thermal activation of Rho, the free opsin resulting from spontaneous PSB breaking should not be phosphorylated nor arrestin-bound. Opsin exhibits a low level (~10 À6 relative to fully activated Meta-II Rho, Table 1) of basal activity (31,32). The total number of opsin (~10 4 to 10 5 ) correspond to~0.01 to 0.1 Meta-II Rho per rod cell per second.…”