Effect of hydroxylamine on the subcellular distribution of arrestin (S-antigen) in rod photoreceptors.
Effect of hydroxylamine on the subcellular distribution of arrestin (S-antigen) in rod photoreceptors.
复制标题
羟胺对视杆光感受器中视紫红质抑制蛋白（S 抗原）亚细胞分布的影响。
DOI：
10.1017/s0952523800002467
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发表时间：
1994
影响因子：
1.9
通讯作者：
Pepperberg,DR
中科院分区：
文献类型：
--
作者：
Mangini,NJ;Garner,GL;Okajima,TI;Donoso,LA;Pepperberg,DR
在绵羊视网膜的光感受器中的抑制蛋白（S-抗原）的免疫细胞化学标记进行了检查后孵育的视网膜与羟胺（NH 2 OH），一种已知的抑制光活化视紫红质的磷酸化的代理。将浸泡在含有20 mM NH 2 OH的培养基中或不含NH 2 OH的对照培养基中的完整、分离的视网膜保持在黑暗中或暴露于强光下3 min（分别为暗适应和光适应条件）;在黑暗中进一步孵育10 min;然后固定并准备冷冻切片。将冷冻切片与抗S抗原单克隆抗体MAb A2 G5、与辣根过氧化物酶偶联的二抗以及作为色原的3-氨基-9-乙基咔唑或二氨基联苯胺孵育。然后使用光显微图像处理系统对冷冻切片中的抗抑制蛋白标记进行光密度分析。在暗适应对照视网膜中，光感受器外节（OS）层的标记密度（0.061 ± 0.004;平均值± S.e.m.）内段（IS）层为0.138 ± 0.011。在光适应对照视网膜中，OS标记密度（0.139 ± 0.007）超过IS标记密度（0.095 ± 0.005）。与NH 2 OH孵育消除了OS标记相对于IS标记的光依赖性增加，即消除了相对OS/IS标记的增加。在NH_2 OH处理的暗适应视网膜中标记密度分别为0.110 ± 0.006（OS）和0.183 ± 0.006（IS），而在NH_2 OH处理的光适应视网膜中标记密度分别为0.078 ± 0.004（OS）和0.182 ± 0.008（IS）。还在暴露于强光3分钟或13分钟然后立即固定的视网膜中检查抗抑制蛋白标记。在NH 2 OH的情况下孵育的视网膜中，OS/IS标记密度的增加在光照3分钟后是明显的，并且光照13分钟的视网膜表现出OS/IS标记的更大的增加。经光照3 min的NH 2 OH处理的视网膜也表现出OS/IS标记的增加;通过与暗适应NH 2 OH处理的对照组（OS/IS标记的平均值：0.60）相比，这些光照视网膜中的OS/IS标记为0.97。然而，OS/IS标记在NH 2 OH处理的视网膜，已被照亮了13分钟（平均值：0.35）低于暗适应的控制。结果表明，在完整的视杆内，NH 2 OH抑制OS/IS抗抑制蛋白标记的光依赖性增加，该标记通常在视觉色素主要漂白后长时间（约10分钟）表达。在NH 2 OH的作用的可能的基础是减少的驱动力的运动arrestin从内段到外段和/或促进arrestin在外段的降解。
The immunocytochemical labeling of arrestin (S-antigen) in photoreceptors of the ovine retina was examined following incubation of the retina with hydroxylamine (NH2OH), an agent known to inhibit the phosphorylation of photoactivated rhodopsin. Intact, isolated retinas bathed in medium containing 20 mM NH2OH, or in control medium lacking NH2OH, were maintained in darkness or exposed to bright light for 3 min (dark-adapted and light-adapted conditions, respectively); further incubated in darkness for 10 min; and then fixed and prepared for cryosectioning. Cryosections were incubated with anti-S-antigen monoclonal antibody MAb A2G5; with secondary antibodies that were conjugated with horseradish peroxidase; and with either 3–amino-9–ethyl carbazole or diaminobenzidine as chromogen. Anti-arrestin labeling in cryosections was then analyzed densitometrically using a light-microscopic image processing system. In dark-adapted control retinas, labeling density of the photoreceptor 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 ± 0.007) exceeded IS labeling density (0.095 ± 0.005). Incubation with NH2OH eliminated this light-dependent increase in labeling of the OS relative 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 ± 0.004 (OS) and 0.182 ± 0.008 (IS) in NH2OH-treated light-adapted retinas. Anti-arrestin labeling was also examined in retinas that were exposed 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 labeling density was evident after 3 min of illumination, and retinas illuminated for 13 min exhibited an even larger increase in OS/IS labeling. An increase in OS/IS labeling was also exhibited by NH2OH-treated retinas that had been illuminated for 3 min; by comparison with dark-adapted NH2OH-treated controls (average value of OS/IS labeling: 0.60), OS/IS labeling in these illuminated retinas was 0.97. However, OS/IS labeling in NH2OH-treated retinas that had been illuminated for 13 min (average value: 0.35) was lower than that of the dark-adapted controls. The results indicate that, within intact rods, NH2OH inhibits the light-dependent increase in OS/IS anti-arrestin labeling that is ordinarily expressed at long times (~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 degradation of arrestin in the outer segment.