The distribution of Man-6-P receptors was determined by immunoperoxidase cytochemistry in Clone 9 hepatocytes cultured in the presence or absence of tunicamycin and chloroquine, agents that perturb lysosomal enzyme sorting and lead to their secretion . In control (untreated) cells, receptors were localized in cis Golgi cisternae, coated vesicles, and in endosomes or lysosomes . After tunicamycin treatment, receptors were found in coated vesicles lined up along the cis cisternae but were not detected in endosomes or lysosomes. After chloroquine treatment, receptors were localized in large vacuolated endosomes or lysosomes but were not usually detected in Golgi cisternae or in coated vesicles . These results demonstrate a redistribution of receptors along the normal Man-6-P-dependent sorting pathway after these treatments . In ligand-deficient (tunicamycintreated) cells, immunoreactive receptors accumulate at the presumptive sorting site in the cis Golgi and are depleted from endosomes and lysosomes. When the intralysosomal pH is increased (by chloroquine treatment) preventing ligand-receptor dissociation, receptors accumulate at the presumptive delivery site (lysosomes and endosomes) and are depleted from the cis Golgi region. The findings also suggest that (a) ligand binding triggers movement of the receptor to endosomes or lysosomes, and (b) ligand dissociation triggers their movement back to the cis Golgi region.Recently we localized the mannose-6-phosphate receptor for lysosomal enzymes in several secretory and absorptive cell types by immunocytochemistry in an attempt to define the Man-6-P-dependent transport route and to identify the site at which sorting of lysosomal enzymes and secretory products takes place (1) . We localized the receptor in cis Golgi cisternae, in coated vesicles, and in endosomes and lysosomes ofseveral cell types. We have assumed that this distribution delineates the Man-6-P-dependent pathway by which lysosomal enzymes are delivered to lysosomes, and that the organelles mentioned represent stations along this pathway which correspond to the sorting site, the carrier, and the delivery site, respectively. On the basis of these results we proposed that normally lysosomal enzymes bearing the recognition marker are removed from the secretory pathway in the cis Golgi for delivery to lysosomes, bypassing the trans Golgi cisternae .Our previous findings applied to the steady-state distribution of Man-6-P receptors in several epithelia (hepatocytes, exocrine pancreas, and epididymis) in which the polarity of the Golgi stacks (cis vs. trans) is clear. In this paper, we have extended our inquiry to cultured cells and have determined the distribution of the receptor both at steady state (untreated cells) and under conditions in which the traffic of lysosomal 320 enzymes is perturbed by tunicamycin (2, 3) or chloroquine (3-6) treatment . In cells incubated with these agents, sorting oflysosomal enzymes does not take place owing either to the production of defective ligand (2, 3) or to a d...

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Recent data indicate that the oxidative stress plays an important role in the pathogenesis of diabetes and its complications such as retinopathy, nephropathy and accelerated atherosclerosis. In diabetic retinopathy, it was demonstrated a selective loss of pericytes accompanied by capillary basement membrane thickening, increased permeability and neovascularization. This study was designed to investigate the role of diabetic conditions such as high glucose, AGE‐Lysine, and angiotensin II in the modulation of antioxidant enzymes activities, glutathione level and reactive oxygen species (ROS) production in pericytes. The activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total glutathione (GSH) was measured spectrophotometrically. The production of ROS was detected by spectrofluorimetry and fluorescence microscopy after loading the cells with 2′‐7′ dichlorofluoresceine diacetate; as positive control H2O2 was used. Intracellular calcium was determined using Fura 2 AM assay. The results showed that the cells cultured in high glucose alone, do not exhibit major changes in the antioxidant enzyme activities. The presence of AGE‐Lys or Ang II induced the increase of SOD activity. Their combination decreased significantly GPx activity and GSH level. Athree times increase in ROS production and a significant impairment of intracellular calcium homeostasis was detected in cells cultured in the presence of the three pro‐diabetic agents used. In conclusion, our data indicate that diabetic conditions induce in pericytes: (i) an increase of ROS and SOD activity, (ii) a decrease in GPx activity and GSH level, (iii) a major perturbation of the intracellular calcium homeostasis. The data may explain the structural and functional abnormalities of pericytes characteristic for diabetic retinopathy.

In this study we investigated the effects of ageing on the carbonyl stress (protein carbonyls and 4‐hydroxy‐2‐nonenal groups) and glutathione antioxidant defense in red blood cells (RBCs) of obese Type 2 diabetic patients with/without hypertensive complications. To this purpose the following methods were used: spectrophotometry (protein carbonyls, glutathione and glutathione peroxidase assays), immunofluorescence (4‐hydroxy‐2‐nonenal localization), western blotting (immunodetection of carbonylated proteins). The results showed that compared to RBCs of healthy subjects, in obese Type 2 diabetics, ageing is associated with: (i) an increase in the concentration and expression of carbonylated proteins, a marker of oxidative stress; (ii) a decrease of both non‐enzymatic and enzymatic endogenous glutathione defenses; (iii) a severely disturbed oxidant/antioxidant balance when obesity was associated with hypertension. The simultaneous insults of high blood pressure, obesity, and diabetes conducted to the highest carbonyl strss, exposure of 4‐hydroxy‐2‐nonenal Michel adducts at the outer leaflet of RBCs plasmalemma, and the lowest glutathione antioxidant potential, particularly in elderly patients. These results can explain the gradual age‐dependent diminishment of the detoxification potenital of RBCs that at the old age can not overcome the deleterious effects of the high systemic oxidative stress.

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