Lysosomes are membrane-bound organelles whose matrixes contain many hydrolytic enzymes that are optimally active at an acidic pH.1-3) The intralysosomal environment is maintained at pH 4.5 by membrane-integrated H ϩ -ATPase. 4)Lysosomes receive extracellular macromolecules through the endocytic transport system. Intracellular proteins are sequestered into lysosomes via autophagocytosis. A variety of lysosomotropic amines have been employed to analyze the lysosomal biogenesis and function. [5][6][7] There is general agreement that these amines inhibit protein degradation in lysosomes and dissociation of receptor-ligand complexes in endosomes. Biosynthetic transport of newly synthesized lysosomal enzymes is affected by these amines, causing their secretion into the extracellular space.Chloroquine has been used as an anti-malarial drug and is known as a lysosomotropic amine as well. Previous reports demonstrated that chloroquine is accumulated in lysosomes and consequently often causes a shift of lysosomes to a less dense fraction upon isopycnic centrifugation of a mitochondrial fraction (into a fraction containing lysosomes but not the cytosolic fraction) in a sucrose gradient. 8,9) However, it is not known whether the chloroquine-induced shift of lysosomes to the less dense fraction is caused by a change of lysosomal buoyant density or by the disruption of lysosomes.In the present study, the effects of chloroquine on lysosomal integrity in cultured rat hepatocytes were studied by measuring b-G or lamp-1 in Percoll density gradient fractions, in the cytosolic fraction obtained from cells permeabilized by digitonin or in the cytosolic fraction obtained by conventional cell fractionation. MATERIALS AND METHODS MaterialsMale Wistar rats weighing 200 g were obtained from Shimazu Experimental Animals (Kyoto, Japan). Percoll and the ECL Western blotting detection kit were from Amersham Pharmacia Biotech (Tokyo, Japan). Chloroquine was purchased from Sigma-Aldrich Japan Co. (Tokyo, Japan). Specific anti-rat lamp-1 IgG was prepared in a previous study. Antiserum against rat MPD was prepared in a previous study. Horseradish peroxidase (HRP)-conjugated antirabbit IgG goat IgG was bought from O.E.M. Concepts, INC. (Toms River, NJ, U.S.A.). Eagle's essential medium and Hank's solution were obtained from Nissui Co. (Tokyo, Japan). Complete Mini (tablet containing protease inhibitor) was purchased from Roche. All other chemicals were of reagent grade, and were purchased from various commercial sources.Cultured Rat Hepatocytes Rat hepatocytes were prepared from rat livers by collagenase perfusion as described by Seglen. 10) Hepatocytes were diluted to 3ϫ10 6 per 60-mm tissue culture dish with Eagle's essential medium containing 10% fetal calf serum, then incubated in humidified air containing 5% CO 2 at 37°C for 24 h.Cell Fractionation by Percoll Density Centrifugation Cells incubated in 60 mm tissue culture dishes were washed several times in cold Hank's buffer, then in a cold isotonic sucrose solution (0.25 M sucrose, 1 mM EDT...

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Keratinocytes were cultured on fibroblast-free dermal substitutes made of type I collagen film (collagen dermal substitute) and an extracellular matrix gel film (matrix dermal substitute), each of which was laid on a lyophilized type I collagen sponge. The morphology of the basal keratinocytes in these three-dimensional culture models of the skin was studied ultrastructurally and immunohistochemically to assess their differentiation to basal cells. The basal keratinocytes in the artificial epidermis cultured on the collagen dermal substitute showed poorly organized tonofibril networks and desmosomes. Neither the tonofibril-hemidesmosome complex nor the lamina densa were detected along the interface, where many cytoplasmic projections of basal keratinocytes were noted. There were no detectable antigens of type IV or VII collagen, LDA-1, or laminin in the interface. Bullous pemphigoid (BP) and 1-2B7B antigens and integrins were expressed along the cytoplasmic membrane and the projections of the basal keratinocytes. A high molecular weight keratin (keratin 1, 68 kDa, 34 beta B4) was detected only in part of the uppermost layers of this artificial epidermis. In contrast, basal keratinocytes in the artificial epidermis on the matrix dermal substitute developed tonofibril networks radiating to desmosomes and hemidesmosomes, under which a primitive lamina densa was present. Basement membrane zone antigens, such as type IV and VII collagens, LDA-1 and laminin were noted along the interface as were 1-2B7B and BP antigens and integrins. Laminin and type VII collagen were also detected along or in the membrane of the endoplasmic reticulum of basal keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Recently, it has been questioned whether mevalonate pyrophosphate decarboxylase (MPD) is predominantly located in the peroxisomes or cytosol. We previously reported that a small amount of MPD in the liver of rats fed a CP diet (5% cholestyramine and 0.1% pravastatin) existed in the peroxisomes, although MPD is predominantly located in the cytosol in the liver of rats fed normal chow and a CP diet for 12 days. In the present study, we examined the subcellular distribution of MPD in mouse melanoma cells (B16 and B16F10) treated with or without lovastatin, using digitonin permeabilization and immunoblotting. In permeabilized B16 by digitonin after treatment with or without lovastatin, 95% and 5%, or 98% and 2% of MPD existed in the cytosol and membrane/organelle (M/O) fraction, respectively. Using B16F10 under the same conditions, 80% and 20%, or 91% and 9% of MPD existed in the cytosol and M/O fraction, respectively. These results indicated that MPD was predominantly located in the cytosol in both mouse melanoma cells treated with or without lovastatin.

We examined the change in the subcellular distribution of a lysosomal enzyme, beta-glucuronidase (beta-G), caused by decreased cholesterol levels in mouse melanoma cells using an HMG-CoA reductase inhibitor, lovastatin and lipoprotein-deficient serum (LDS). There was a decrease in the cholesterol content of the cells and increased secretion of the mature form of beta-G located in lysosomes, as documented by Percoll density gradient fractionation, digitonin permeabilization and immunoprecipitation. Furthermore, another lysosomal enzyme, cathepsin H, was found to be released in the medium from cells treated with lovastatin. Both the precursor and mature forms of cathepsin H were detected in the medium of treated cells. Next, when cells were treated with LDS without lovastatin, concomitantly with the decrease in the levels of cholesterol and beta-G activity in the cells, beta-G activity in the medium increased. Also, the ratio of beta-G (3.2-fold) released in the medium from cells treated with Dulbecco's modified Eagle medium (D-MEM) containing lovastatin and LDS was higher than that (2.3-fold) on treatment with D-MEM containing LDS without lovastatin. From these results, it was suggested that the exocytosis of mature enzymes from lysosomes into the medium or mis-sorting of the lysosomal precursor forms to the medium was caused by the lovastatin- and/or LDS-induced decrease in the cholesterol content of the cells, although the mechanism of secretion by lysosomal enzymes differed somewhat.