NADPH-diaphorase histochemistry reveals cone distributions in adult human retinae.

NADPH-diaphorase histochemistry was used to identify and analyse the topography of cones in adult human retinae (in the age range 44 to 68 years). Retinae were treated for NADPH-diaphorase reactivity, present in vascular walls, amacrine cells and cone and rod outer segments; a subpopulation of less intensely reactive profiles, morphologically resembling cones, possibly represents the blue cone population. Regularly spaced pairs of cones (one intensely labelled and one weakly labelled cone) were also apparent throughout the retina, and were most common along the horizontal meridian, particularly towards the periphery. The diameters and distributions of labelled cone outer segments were assessed using image analysis. Cone density in the adult retina ranged from 2000 per mm2 in the temporal periphery, to 82,000 to 120,000 per mm2 at the fovea centralis. Distribution patterns confirmed the presence of a cone streak, extending from the foveal region into nasal retina, but no evidence of superior-inferior asymmetry was detected.

Retinal pigment epithelial cells of the posterior pole have fewer Na/K adenosine triphosphatase pumps than peripheral cells.

The density of Na/K adenosine triphosphatase (ATPase) pumps in retinal pigment epithelial (RPE) cells in different retinal regions was quantified by measuring the binding of 3H-ouabain to RPE in cow and human eyecups. In bovine eyes, pump density was estimated in RPE samples isolated from three retinal regions outlined with a 7-mm trephine: one from the posterior pole in the area centralis and two from the superior, equatorial retina representing unpigmented (in the tapetum) and pigmented zones. In human eyes, RPE samples were isolated from a posterior region centered around the macula and one superior region. Ouabain binding to RPE of the posterior pole of both species was approximately 40-60% lower than binding to RPE of more peripheral regions in the same eyes. For bovine eyes, ouabain binding did not differ between pigmented and unpigmented cells of the superior retina, suggesting that reduced binding in the relatively amelanotic posterior cells was not related to levels of pigmentation. For human RPE, binding to posterior cells was lower in eyes from donors of all ages (range, 17-90 yr). The data suggest that Na/K ATPase pump site density is lower in posterior RPE cells of both bovine and human eyes, perhaps due to a regional difference in requirements for ionic regulation.

Distribution of carbonic anhydrase among human photoreceptors.

The distribution of carbonic anhydrase (CA) among human photoreceptors has been a topic of dispute. In our experiments, by modifying an established enzyme histochemical technique, reproducible staining was observed. Of the cones in the peripheral retina, 91% were positive for CA. The CA-negative (CA-) cones were absent within approximately 8 arc min of the foveal center and their density peaked at 2 arc deg. No CA activity was found in the rods. Morphologic differences were noted between the CA-positive (CA+) and CA- cones. Compared to the CA+ cones, the CA- cones had longer and more nearly columnar inner segments, more nearly spherical nuclei, and more generous amounts of perikaryal cytoplasm. In the peripheral retina, the distance between CA+ to CA+ nearest neighbors were larger compared to CA- to CA+ nearest neighbors (P less than 0.0001). The frequency distribution and morphology of the CA- cones suggest that they are the blue-sensitive cones. As such, this study demonstrates a biochemical similarity between blue cones and rods that may provide insight into the function and phylogeny of the blue cones.

Regional comparisons of cathepsin D activity in bovine retinal pigment epithelium.

Cathepsin D is the lysosomal protease in the retinal pigment epithelium which is presumed to be the major enzyme involved in the degradation of shed discs during the photoreceptor renewal process. In this study, the cathepsin D activity in RPE cells from the posterior area centralis was compared to the activity in cells from the equatorial region of the same bovine eyes. Enzyme activities were measured both in paired fresh RPE isolates from the two retinal regions and in paired regional RPE cultures. Cultures were further analyzed for changes in enzyme activity with time in vitro from 3 to 11 weeks. Analysis of freshly isolated RPE cells from 30 eyes indicated that cells from the area centralis have significantly higher cathepsin D activity than cells from the more peripheral retina. Paired cultures of RPE from the two regions did not express the intraeye topographical differences in enzyme activity which were observed in fresh isolates. There were significant variations in enzyme activity in cultured RPE cells with time in vitro, but activity levels did not show progressive increases or decreases with in vitro aging. After 11 weeks in vitro, but not at earlier times, the enzyme activities in the paired regional cultures from the same eye were highly correlated. The data suggest that the higher levels of cathepsin D activity observed in the freshly isolated RPE from the area centralis result from modulators of enzyme activity which are not present in culture.