EFFECT OF HYDROXYLAMINE ON THE SUBCELLULAR-DISTRIBUTION OF ARRESTIN (S-ANTIGEN) IN ROD PHOTORECEPTORS

The immunocytochemical labeling of arrestin (S-antigen) in photorecept ors of the ovine retina was examined following incubation of the retin a with hydroxylamine (NH2OH), an agent known to inhibit the phosphoryl ation of photoactivated rhodopsin. Intact, isolated retinas bathed in medium containing 20 mM NH2OH, or in control medium lacking NH2OH, wer e maintained in darkness or exposed to bright light for 3 min (dark-ad apted and light-adapted conditions, respectively); further incubated i n darkness for 10 min; and then fixed and prepared for cryosectioning. Cryosections were incubated with anti-S-antigen monoclonal antibody M Ab A2G5; with secondary antibodies that were conjugated with horseradi sh peroxidase; and with either 3-amino-9-ethyl carbazole or diaminoben zidine as chromogen. Anti-arrestin labeling in cryosections was then a nalyzed densitometrically using a light-microscopic image processing s ystem. In dark-adapted control retinas, labeling density of the photor eceptor outer segment (OS) layer (0.061 +/- 0.004; average +/- S.E.M.) was less than that of the inner segment (IS) layer (0.138 +/- 0.011). In light-adapted control retinas, OS labeling density (0.139 +/- 0.00 7) exceeded IS labeling density (0.095 +/- 0.005). Incubation with NH2 OH eliminated this light-dependent increase in labeling of the OS rela tive to that of the IS, i.e. eliminated the increase in relative OS/IS labeling. Densities of labeling were 0.110 +/- 0.006 (OS) and 0.183 /- 0.006 (IS) in NH2OH-treated dark-adapted retinas vs. 0.078 +/- 0.00 4 (OS) and 0.182 +/- 0.008 (IS) in NH2OH-treated light-adapted retinas . Anti-arrestin labeling was also examined in retinas that were expose d to 3 min or 13 min of bright light and then immediately fixed. Among retinas incubated in the absence of NH2OH, an increase in OS/IS label ing density was evident after 3 min of illumination, and retinas illum inated for 13 min exhibited an even larger increase in OS/IS labeling. An increase in OS/IS labeling was also exhibited by NH2OH-treated ret inas that had been illuminated for 3 min; by comparison with dark-adap ted NH2OH-treated controls (average value of OS/IS labeling: 0.60), OS /IS labeling in these illuminated retinas was 0.97. However, OS/IS lab eling in NH2OH-treated retinas that had been illuminated for 13 min (a verage value: 0.35) was lower than that of the dark-adapted controls. The results indicate that, within intact rods, NH2OH inhibits the ligh t-dependent increase in OS/IS anti-arrestin labeling that is ordinaril y expressed at long times (similar to 10 min) after major bleaching of the visual pigment. Among the possible bases for the effect of NH2OH are a reduction in the driving force for the movement of arrestin from the inner to the outer segment and/or a facilitation of the degradati on of arrestin in the outer segment.