The action of 11-cis-retinol on cone opsins and intact cone photoreceptors

Abstract

11-cis-retinol has previously been shown in physiological experiments to promote dark adaptation and recovery of photoresponsiveness of bleached salamander red cones but not of bleached salamander red rods. The purpose of this study was to evaluate the direct interaction of 11-cis-retinol with expressed human and salamander cone opsins, and to determine by microspectrophotometry pigment formation in isolated salamander photoreceptors. We show here in a cell-free system using incorporation of radioactive guanosine 5'-3-O-(thio)triphosphate into transducin as an index of activity, that 11-cis-retinol inactivates expressed salamander cone opsins, acting an inverse agonist. Similar results were obtained with expressed human red and green opsins. 11-cis-retinol had no significant effect on the activity of human blue cone opsin. In contrast, 11-cis-retinol activates the expressed salamander and human red rod opsins, acting as an agonist. Using microspectrophotometry of salamander cone photoreceptors before and after bleaching and following subsequent treatment with 11-cis-retinol, we show that 11-cis-retinol promotes pigment formation. Pigment was not formed in salamander red rods or green rods (containing the same opsin as blue cones) treated under the same conditions. These results demonstrate that 11-cis-retinol is not a useful substrate for rod photoreceptors although it is for cone photoreceptors. These data support the premise that rods and cones have mechanisms for handling retinoids and regenerating visual pigment that are specific to photoreceptor type. These mechanisms are critical to providing regenerated pigments in a time scale required for the function of these two types of photoreceptors.

FIGURE 1.

Effect of 11-cis-retinol and -retinal on opsin activation and pigment regeneration in salamander red rods. A, transducin activation by expressed salamander red rod opsin (open circles), with 11-cis-retinol (filled circles) and with 11-cis-retinal (triangles). Each sample was assayed in triplicate and averaged; error bars reflect mean ± S.E. and often were smaller than the symbol size. At 5.5 min, samples with 11-cis-retinal and 11-cis-retinol were illuminated with light for 12 s as described under “Experimental Procedures” and illustrated in the figure with the vertical line. B, absorption spectra of isolated salamander red rods measured microspectrophotometrically in native state (gray circles, n = 28), measured ∼130 min following a complete (>90%) bleach (black filled circles, n = 3), measured 130–190 min after treatment with 11-cis-retinol following a complete bleach (open circles, n = 4), and measured 140–200 min after treatment with 11-cis-retinal following a complete bleach (triangles, n = 9). Retinoids were suspended in lipid vesicles or ethanolic solution. Continuous curves fitted to the data are sums of templates for A1 and/or A2 visual pigments (see “Experimental Procedures”).

FIGURE 2.

Effect of 11-cis-retinol and -retinal on salamander opsin activation and pigment regeneration in red cones. A, transducin activation by expressed salamander red cone opsin (open circles) with 11-cis-retinol (filled circles) and 11-cis-retinal (triangles). Each sample was assayed in triplicate and averaged; error bars reflect mean ± S.E. and often were smaller than the symbol size. At 5.5 min, samples with 11-cis-retinal and 11-cis-retinol were illuminated with light for 12 s as described under “Experimental Procedures” and illustrated in the figure with the vertical line. B, absorption spectra measured on salamander red cones in the native dark-adapted state (gray circles, n = 41), about 44 min after >90% bleach (black open circles, n = 23), and after treatment of bleached cones for 50–100 min with 11-cis-retinol (filled black circles, n = 44). Inset, normalized absorption spectra of salamander red cones before and after bleaching and regeneration with 11-cis-retinol (circles, n = 44) or 11-cis-retinal (triangles, n = 4).

FIGURE 3.

Effect of 11-cis-retinol and -retinal on opsin activation and pigment regeneration in salamander blue cones and green rods. A, transducin activation by expressed salamander blue cone/green rod opsin (open circles) with 11-cis-retinol (filled circles) and 11-cis-retinal (triangles). Each sample was assayed in triplicate and averaged; error bars reflect mean ± S.E. and often were smaller than the symbol size. At 5.5 min, samples with 11-cis-retinal and 11-cis-retinol were illuminated with light for 12 s as described under “Experimental Procedures” and illustrated in the figure with the vertical line. B, averaged spectra of single salamander blue cones: dark adapted (gray circles, n = 8), 1–9 min after >90% bleach (open circles, n = 13), and 60 min following exposure to 11-cis-retinol applied 85 min following the bleach (black filled circles, n = 7). Inset, normalized absorption spectra of salamander blue cones before and after bleaching and regeneration with 11-cis-retinol (circles, n = 7) or 11-cis-retinal (triangles, n = 5). C, spectra of a single dark-adapted salamander green rod before bleaching (gray circles), after bleaching measured at 2 (black dashes) and 32 min (open circles), and after treatment with 11-cis-retinol in vesicles for 90 min (black filled circles).

FIGURE 4.

Effect of 11-cis-retinoids on expressed human rod and cone opsin activities. Transducin activation by expressed human (A) rod, (B) red cone, (C) green cone, and (D) blue cone opsins (open circles) with 11-cis-retinol (filled circles) and with retinal (triangles). At 5.5 min, the samples with 11-cis-retinoids were illuminated for 12 s, and only opsins with 11-cis-retinal showed a light-dependent increase in transducin activation. Each sample was assayed in triplicate and averaged; error bars reflect mean ± S.E. and often were smaller than the symbol size. At 5.5 min, samples with 11-cis-retinal and 11-cis-retinol were illuminated with light for 12 s as described under “Experimental Procedures” and illustrated in the figure with the vertical line.

FIGURE 5.

Schematic representation of separate visual cycles involving the retinal pigment epithelium and the neural retina (Müller cells). See text for details.