Atypical Granules in the Eyes of the White Mutant of Drosophila melanogaster Are Lysosome-Related Organelles

In the pigment cells of the white mutant of Drosophila melanogaster, as described earlier, two types of abnormal granules are found by conventional electron microscopy. However, both types of abnormal granules, in addition to those in pigment cell invaginations, are also present in the cytoplasm of the photoreceptor cells. Three enzymes (acid phosphatase, peroxidase, and tyrosinase) are localized within the eyes of wild type and white mutant Drosophila melanogaster by electron microscopy. Peroxidase activity is present in lamellar bodies close to the rhabdomeral microvilli of both fly types. However the organelles containing peroxidase activity are 6-fold more frequent in the wild type than in the mutant. Acid phosphatase is present in lamellar bodies between and at the bases of the rhabdomeral microvilli of the wild type, as well as in ommochrome granules of the photoreceptor cells. In the white mutant, however, acid phosphatase was located in electron lucent vacuoles in the cytoplasm of the receptor cells. These acid phosphatase-positive vacuoles also contained both types of abnormal granules. The latter result indicates that abnormal granules in the receptor cells originate from lysosomal degradation and that targeting of lysosomal enzymes is altered in the white mutant. Due to the tyrosinase activity in the hemolymph of flies, the extracellular spaces are electron dense after DOPA incubation. Since some abnormal granules within the photoreceptor cells are not surrounded by an extracellular space, they can be assumed to originate within the photoreceptor cells.     

Direct Regulation of Na+-Dependent myo-Inositol Transport by Sugars in Retinal Pigment Epithelium: Role of Phorbol Ester and Staurosporin

An Na⁺-dependent active process for myo-inositol (MI) uptake, sharing a common carrier system with glucose and sensitive to phlorizin, was previously established in primary cultures of bovine retinal pigment epithelial (RPE) cells (26, 32). The present report further examines the nature of glucose-induced inhibition of MI transport in primary cultures of RPE cells. RPE cells were grown in supplemented Dulbecco's modification of Eagle's medium (DMEM) containing 5 mM D-glucose (basic growth media) or 40 mM D-glucose or its nonmetabolizable analogue, α-methyl-D-glucoside (αMG); 1–5 mM nonradioactive MI, pyruvate, or lactate; or 0.2–20 µM phorbol 12-myristate 13-acetate (TPA) or straurosporin (modified growth media), for up to 4 weeks. The capacity of RPE cells to accumulate ³H-MI (ratios of intracellular transported radioactive MI, [MI]_i, to external free MI concentration, [MI]_i/[MI]₀) decreased by up to 41% or 34% when cells were grown for 10 days or longer with 40 mM D-glucose or 40 mM αMG, respectively, compared to cells grown in basic growth media. The rate of uptake of ³H-MI also was reduced to 63 ± 15% or 48 ± 8% of the control values when cells were fed 1 or 5 mM nonradioactive MI, respectively. In addition, cellular capacity to bind to [³H]phlorizin was reduced to 52 ± 7%, 61 ± 5%, or 38 ± 6% of the controls when RPE cells were fed 40 mM D-glucose, 40 mM αMG, or 5 mM nonradioactive MI, respectively. Growth media containing either pyruvate or lactate, the glucose metabolites, did not suppress the ability of RPE cells to accumulate MI. An 18 ± 8% reduction in [³H]thymidine incorporation into DNA occurred when cells were grown in 40 mM glucose for 12–14 days, compared to cells grown with 5 mM glucose. Chronic treatment (12–14 days) of the cells with phorbol ester, an activator of protein kinase C, caused up to twofold increase in MI uptake, [³H]phlorizin binding, cell number, and DNA synthesis. However, when the rates of MI uptake into cells grown in basic growth media or TPA-treated media were normalized to cell number, no significant difference in MI uptake was found between the treated and untreated cells. Addition of staurosporin, a protein kinase C inhibitor, together with TPA, in the growth media reversed the phorbol-induced increase of MI uptake. In contrast to its chronic effect, a 60-min incubation (acute effect) of cells in the presence of TPA, with or without inclusion of stauropsorin, did not alter the uptake of ³H-MI into RPE cells, regardless of glucose levels in the growth media. These studies indicated that glucose itself, and not glucose metabolites, regulated uptake of MI into primary cultures of RPE cells. In addition, glucose-induced down-regulation of MI uptake was not mediated through the protein kinase C pathway, but the staurosporin-inhibited, TPA-stimulated protein kinase C was partly responsible for growth and proliferation of RPE cells.