David S. Williams

Isolation of rod outer segments on percoll gradients: effect of specific protease inhibition

Rat rod outer segments (ROSs) were isolated by vortexing retinas and separating the detached components on performed Percoll gradients. A lighter band of 20 x 10(6) unsealed ROSs per ten retinas, and a heavier band of 60 x 10(6) sealed ROSs per ten retinas were obtained from each 12 ml gradient. The yield of sealed ROSs (but not unsealed ROSs) was increased up to twofold in the presence of the specific cysteine protease inhibitor, Ep-475. Aprotinin, pepstatin, PMSF, TPCK and EGTA plus EDTA had no effect. These results indicate that during isolation, ROSs are vulnerable to damage by cysteine protease activity either from damaged retinal cells or from within.

Effects of low sodium, ouabain, and strophanthidin on the shedding of rod outer segment discs

Summary1.To test if the light-evoked hyperpolarization of the rods or retinal pigment epithelium (RPE) is important for the light-evoked component of rod disc shedding, eyecups of the anuran,Xenopus, were incubated in media that have been reported to have different effects on the membrane potential of these cells.2.Hyperpolarization was induced by transferral to medium with Na+ entirely replaced by choline+, or mostly replaced by Li+. Shedding in darkness was increased 4-fold in both cases (Figs. 1, 2 and 3).3.To prevent light-evoked hyperpolarization, eyecups were transferred to medium containing 0.5 mmol/l ouabain. However, light-evoked shedding was not inhibited; instead, it was activated further (Fig. 4). Moreover, ouabain increased shedding in darkness by about 9-fold (Fig. 5).4.Ouabain likewise activated shedding without light in eyecups of the frog,Rana (Fig. 6). InRana, shedding is entirely light-evoked, so that, unlike the case represented byXenopus eyecups, there was no question that inhibition of the light-evoked component could have been masked by over-whelming stimulation of an endogenous component.5.Strophanthidin, which, in contrast to ouabain, binds reversibly to Na+-K+-ATPase, also activated shedding inXenopus eyecups kept in darkness, even when it was washed out 20 min after light onset (Fig. 7).6.These results suggest that a particular change in the membrane potential of the rod or RPE cells is not directly responsible for the occurrence of light-evoked rod disc shedding.7.In addition, the results identify pharmacological treatments that activate shedding in darkness. These treatments should provide a useful tool for future studies of rod disc shedding. It is noted that common effects of these treatments possibly include: (1) inhibition of Na+, K+-ATPase activity, with a resulting increase in ATP; (2) an increase in intracellular Ca2+; (3) alteration of melatonin levels.

Gene Expression of the Neuropeptide-Processing Enzyme Carboxypeptidase E in Rat Photoreceptor Cells

Abstract: We characterized the spatial expression of mRNA for the enzyme Carboxypeptidase E (CPE) in the Long‐Evans rat retina. CPE is involved in the processing of neuroactive peptides to a mature form. A cDNA encoding the 3’ terminus of CPE mRNA was cloned by polymer‐ase chain reaction amplification of rat retina single‐stranded cDNA. The sequence of this cDNA was identical to a rat genomic clone for CPE and nearly identical (130/ 132 nucleotides) to a cDNA for rat brain CPE. In addition, the cDNA hybridized to a single allele on Southern blots and to a 2.1‐kb mRNA on northern blots of both rat brain and retina. These data support the conclusion of others that CPE is a single‐copy gene in the rat. In cell fractionation experiments, the majority of CPE mRNA fractionated with rod opsin mRNA, suggesting that CPE is expressed predominantly in rod photoreceptors. The high abundance of CPE mRNA in photoreceptors was confirmed by in situ hybridization studies, although CPE was detected at lower levels in other retinal cell types as well. The presence of abundant levels of the mRNA of a neuro‐peptide‐processing enzyme in photoreceptor cells suggests that photoreceptors may utilize neuropeptides for normal function.