Caldwell R B, McLaughlin B J
J Comp Neurol. 1985 Jun 22;236(4):523-37. doi: 10.1002/cne.902360408.
We have studied sterol distribution in the retinal pigment epithelial (RPE) microvillous and outer segment disc membranes of rats with inherited retinal degeneration (RCS; RCS-p/+) and of normal genetic controls (RCS-rdy+, RCS-rdy+-p/+) by using the polyene antibiotic filipin, which binds specifically to 3-B-hydroxy-sterols, and freeze-fracture techniques. Retinas were perfusion-fixed, incubated with filipin in the same fixative, and prepared routinely for freeze-fracture electron microscopy. In the normal retina, the distribution of filipin binding sites on both RPE microvillous and outer segment disc membranes changes during development. Prior to outer segment elongation and the onset of phagocytosis (10 days postnatal), filipin sterol complexes are homogeneously distributed in both microvillous and outer segment membranes. With the onset of phagocytosis (2 weeks postnatal and later) filipin binding in both tissues forms a proximal-to-distal gradient, and binding sites decrease as distance from the cell body increases. In the normal RPE microvillous membranes, binding sites are numerous proximally and sparse on the distal tips. In the normal outer segment disc membranes, binding sites are often present on the basal discs, but are sparse on the intact apical discs prior to shedding. As the discs are cast off and engulfed by the RPE, however, filipin binding increases on both disc and phagosome membranes. In the dystrophic retina, the distribution of filipin binding sites differs from the normal. First, in the microvillous membranes, the proximal-to-distal gradient in filipin binding is rarely present at 2 weeks postnatal and becomes prominent only after the buildup of membranous debris has begun (3-5 weeks postnatal). Second, as the photoreceptors degenerate and the membrane debris disappears (4 months postnatal), filipin binding on the microvillous membranes becomes relatively sparse and homogeneous. Third, filipin binding on the intact disc membranes does not change with outer segment elongation, and numerous filipin binding sites are present on both apical and basal outer segment disc membranes. Fourth, large aggregates of filipin binding sites occupy the vast expanses of particle-free areas of debris membranes which accumulate between the photoreceptors and the RPE. These changes in the amount and distribution of filipin binding sites in the dystrophic retina add to the evidence that the disease process involves outer segment as well as RPE membranes and suggest that alterations in cholesterol distribution could contribute to the phagocytic defect.
我们利用多烯抗生素制霉菌素（其能特异性结合3-β-羟基固醇）和冷冻断裂技术，研究了遗传性视网膜变性大鼠（RCS；RCS-p/+）以及正常基因对照大鼠（RCS-rdy+，RCS-rdy+-p/+）视网膜色素上皮（RPE）微绒毛膜和外节盘膜中的固醇分布。视网膜经灌注固定，在相同固定剂中与制霉菌素孵育，然后常规制备用于冷冻断裂电子显微镜观察。在正常视网膜中，RPE微绒毛膜和外节盘膜上制霉菌素结合位点的分布在发育过程中会发生变化。在外节伸长和吞噬作用开始之前（出生后10天），制霉菌素固醇复合物在微绒毛膜和外节膜中均匀分布。随着吞噬作用的开始（出生后2周及以后），两种组织中的制霉菌素结合形成从近端到远端的梯度，且结合位点随着距细胞体距离的增加而减少。在正常的RPE微绒毛膜中，近端结合位点众多，远端尖端则稀疏。在正常的外节盘膜中，结合位点通常存在于基底盘上，但在脱落前完整的顶端盘上则稀疏。然而，随着盘被RPE脱落并吞噬，盘膜和吞噬体膜上的制霉菌素结合增加。在营养不良的视网膜中，制霉菌素结合位点的分布与正常情况不同。首先，在微绒毛膜中，出生后2周时很少出现制霉菌素结合的近端到远端梯度，只有在膜碎片开始积累后（出生后3 - 5周）才变得明显。其次，随着光感受器退化且膜碎片消失（出生后4个月），微绒毛膜上的制霉菌素结合变得相对稀疏且均匀。第三，完整盘膜上的制霉菌素结合不会随着外节伸长而改变，顶端和基底外节盘膜上都存在大量制霉菌素结合位点。第四，大量制霉菌素结合位点聚集体占据了光感受器和RPE之间积累的碎片膜大片无颗粒区域。营养不良视网膜中制霉菌素结合位点数量和分布的这些变化进一步证明该疾病过程涉及外节以及RPE膜，并表明胆固醇分布的改变可能导致吞噬缺陷。
