Incubation Of Lysosomes Research Articles

IN VITRO EFFECT OF L-CARNITINE ON THE ACTIVITY OF LYSOSOMAL CYSTEINE PROTEASES AND THE STATE OF LYSOSOMAL MEMBRANE

The influence of L-carnitine in vitro on the lysosomal cysteine proteinase activity and stability of the lysosomal membrane of the liver homogenates of intact sexually Mature female rats of Wistar line weighing 280-330 g were studied. In the experimental groups isolated lysosomes were incubated in vitro in a solution of L-carnitine during 1, 2 and 4 hours, in the control groups in vitro incubation was carried out in a medium of isolating solution. The activity of ca-thepsins B, L and H was investigated by spectrofluorimetric method of Barrett & Kirschke in two fractions - lysosomal and outside of lysosomes. The activity of acid phosphatase was used as the main marker of a membrane labilization. In vitro incubation of lysosomes showed that carnitine at a concentration of 5 mM increases the total activity of cathepsin B in a one-hour incubation at 73,2% (p=0,008), cathepsin L in a two- and four-hour incubation - at 77,7% (p=0,005) and 42,3% (p=0,013) respectively, and reduces the overall activity of the cathepsin H in a one-hour incubation at 200,0% (p=0,008), in a two-hour - by 67,9% (p=0,05), in a four-hour -27,1% (p=0,02). In addition, incubation in 5 mM L-carnitine solution leads to an increase of unsedimentable activity and fall sedimentaries activity for cathepsin L in a two-hour, and for acid phosphatase - in a two - and four-hour exposure. 5 mM L-carnitine in one - and two-hour incubation stabilizes lysosomal membrane (whereas increase in incubation time up to 4 hours leads to its damage) and increases the selective permeability of the lysosomal membrane for the studied cathepsins, to the greatest extent - for cathepsin H.

Homeostatic cell cycle and the origin of autophagosomal vesicles

The autophagosomes were identified in the viable cycloheximide (CHX)-treated cells which had an incapacitated translational process and thus disabled synthesis of endoplasmic reticulum (ER)-derived vesicular transporters. They were found devoid of the proteins transported from ER to cell organelles, were unable to fuse with ER, Golgi or mitochondria, and displayed affinity with lysosomes. The analysis of autophagosomes, derived from the CHX cell organelles, revealed that their lipid composition resemble that of the maternal organelle. Thus, the ER-derived autophagosomes were marked with the presence of phosphatidylinositol (PI), Golgi-derived vesicles contained sphingomyelin (SM) and glycosphingolipids (GLL), and the mitochondria-derived autophagosomes contained phosphatidylglycerol (PG) and cardiolipin (CL). The incubation of the vesicles with intact lysosomes afforded their and the lysosome membrane lipids degradation. The analysis of the products derived from incubation of lysosomes and autophagosomes with radiolabeled SM, in the presence and the absence of TritonX100, allowed us to conclude that during autophagosome degradation the lysosomal enzymes are not released to cytosol, and that only lysosomes contain the enzymes degrading membrane lipids. In summary, our findings allowed us to authenticate the vesicles generated in the CHX-treated cells as organelle-specific autophagosomes and to determine that complete cycle of cell restitution and debridement includes intralysosomal degradation of the lysosomal membrane engulfing the autophagosomes vesicles.

Ultrastructural analysis of lysosome reactions to inside-out cell membrane vesicles in a cell-free system

Lysosome reactions were ultrastructurally analyzed using a cell-free system with inside-out cell membrane vesicles (IOVs) prepared from rat erythrocyte ghosts in an alkaline buffer and with wheat germ agglutinin-coated colloidal gold particles (WGA-CGs). The submembranous surface coat in the ghosts was depleted from the IOVs' outer surfaces. When lysosomes from rat liver were incubated with these IOVs, some of the trilaminar membranes of the lysosomes and IOVs came into close contact and formed a five-laminar structure without an intermembranous gap. In other reactions, the membranes of both structures formed one continuous trilaminar membrane along the margin of contact and ruffling five-laminar structures in other regions. Several lysosomes exhibited invaginating hollows or projections that entrapped or encircled the IOVs. Similar five-laminar structures were seen at a few points of contact between the IOVs and the hollowing or projecting membranes. In contrast, such reactions were much rarer when IOVs with reconstituted spectrins and actins on their outer surface were used. The formation of tubuliform pits with membrane-bound WGA-CGs was also observed after the incubation of lysosomes with WGA-CGs. These observations suggest that lysosomes fuse with cytoskeleton-depleted IOVs, wrap arm-like projections around them, enclose them by invagination or incorporate their membrane-bound macromolecules through the process of tubuliform invagination. Furthermore, the fusion and wrapping processes are not necessarily independent.

Effects of methylcyclodextrin on lysosomes.

The cholesterol complexing agent methyl-cyclodextrin (MCD) provides an efficient mean for the removal of cholesterol from biological membranes. In order to study the effects of this agent on the lysosomal membrane in situ, we treated HepG2 cells with MCD and studied the effects of this treatment on lysosomes in isolated fractions. We found that lysosomes prepared from treated cells are more sensitive to various membrane perturbing treatments such as: incubation of lysosomes in isotonic glucose, in hypotonic sucrose or in the presence of the lytic agent glycyl-L-phenylalanine 2-naphthylamide. The lysosomal membrane is also less resistant to increased hydrostatic pressure. Centrifugation methods were used to analyse the effect of MCD on lysosomes. Isopycnic centrifugation in sucrose density gradients demonstrates that the drug induces a reversible density increase of the lysosomes. Our study indicates that extracellularly added MCD can modify the properties of the lysosomal membrane in living cells. It suggests that MCD could be an effective tool to modulate the physical properties of lysosomes within intact cells and to monitor the cellular responses to such modifications.

Cationic lipids destabilize lysosomal membrane in vitro

Addition of cationic lipids to plasmid DNA considerably increases the efficiency of transfection. The mechanism has not yet been elucidated. A possibility is that these compounds destabilize biological membranes (plasma, endosomal, lysosomal), facilitating the transfer of nucleic molecules through these membranes. We have investigated the problem by determining if a cationic lipid N-(1-(2,3-dioleoxy)propyl)- N, N, N,-trimethylammonium methyl-sulfate (DOTAP, Boehringer, Mannheim, Germany) affects the integrity of rat liver lysosomal membrane. We have measured the latency of β-galactosidase, a lysosomal enzyme, and found that incubation of lysosomes with low concentrations of DOTAP causes a striking increase in free activity of the hydrolase and even a release of the enzyme into the medium. This indicates that lysosomal membrane is deeply destabilized by the lipid. The phenomenon depends on pH, it is less pronounced at pH 5 than at pH 7.4. Anionic compounds, particularly anionic amphipathic lipids, can to some extent prevent this phenomenon. It can be observed with various cationic lipids. A possible explanation is that cationic liposomes interact with anionic lipids of lysosomal membrane, allowing a fusion between the lipid bilayers which results in a destabilization of the organelle membrane.

Endogenous lipolytic activities during autolysis of highly enriched hepatic lysosomes

High enriched (50- to 70-fold) fractions of "native" lysosomes were isolated using continuous flow electrophoresis from livers of rats which had not been pretreated with Triton WR-1339. Incubation of lysosomes for 30 min at pH 5.0 in the presence of 5 mM EDTA resulted in a dramatic loss in the content of fatty acids bound to triacylglycerols (137 down to 10 mumol/mg protein) and to phospholipids and an elevation in the level of unesterified fatty acid. Phosphatidylcholine, phosphatidylethanolamine and sphingomyelin concentrations decreased whereas those of lysophosphatidylethanolamine (0.8 up to 8.5% of total lipid-P) and lysophosphatidylcholine (1.9 up to 16.7%) rose in a manner parallel to their respective, fully acylated lipids. Other phospholipids, including phosphatidylinositol, did not change in concentration during incubation. These results indicate that lysosomal phospholipase A, sphingomyelin and triacylglycerol lipase are activated by incubation at acid pH, enabling them to hydrolyze endogenous lysosomal lipids. However, lysosomal phosphatidylinositol-directed phospholipase C is apparently unable to interact with phosphatidylinositol of the lysosomal membrane.

The role of lysosomes in the hepatic accumulation and release of beryllium

The toxic metal beryllium (Be) can produce hepatic necrosis and is known to be concentrated by the liver after i.v. injection of particulate and soluble Be compounds. In the present study the accumulation and release of Be by a hepatic lysosome fraction of the rat has been examined after i.v. administration of sublethal doses of particulate Be phosphate (12.5 and 150 μmoles/kg) or the more hepatotoxic soluble BeSO 4 (12.5 and 25 μmoles/kg). Maximal lysosomal Be content is produced within 2 hr or 5 hr after the injection of Be phosphate or BeSO 4 respectively and in both cases this is followed by a gradual decrease in the lysosomal Be concentration of the next 7 days to 30–40 per cent of the maximal values, which is consistent with the loss of Be observed for whole liver. The release of Be from lysosomes has been examined by measurement in vitro of the liberation of Be into a suitable incubation medium from lysosomes prepared from Be treated animals. The results indicate that the liberation of Be was maximal for lysosomes isolated 5 hr after injection of either form of Be and was accompanied by a significant release of lysosomal β-glucuronidase activity, particularly at the higher doses of Be used, and therefore indicates that some loss of lysosomal integrity occurred. Rupture of the lysosomal membrane could not be demonstrated in vitro by incubation of lysosomes from untreated animals with externally added Be compounds. It is concluded, however, that any release of hydrolytic enzymes into the cytosol occurring in vivo may not be the major cause of cell necrosis produced by Be and it is suggested, therefore, that the role of lysosomes in Be hepatotoxicity is primarily in the intracellular accumulation and subsequent release of Be and that the main cytotoxic target for Be is probably extralysosomal.

Effects of selected chelating agents and metals on the stability of liver lysosomes

Diethyldithiocarbamate (DDC), penicillamine and sodium ethyldiaminetetraacetate (Na-EDTA) in 1 mM concentrations significantly stabilized the membrane of isolated liver lysosomes, as shown by a decreased release of β-glucuronidase upon incubation at 37° and pH 5·0. Thiol compounds which are readily auto-oxidized (cysteine, glutathione and dithiothreitol) did not affect lysosomal fragility when subjected to 1 mM concentration; at a higher concentration (5 mM), these substances increased the release of β-glucuronidase. Stabilization by DDC and penicillamine, both of which contain sulphydryl-like groups, may be related to their nonauto-oxidizability, although their chelation effect cannot be excluded. Various chelating agents relatively specific for Fe 2+ or Fe 3+, such as l,10-phenanthroline, 5,6-dimethyl-l,10-phenanthroline, 2-2′-dipyridyl, Desferal, and pyrocatechol-3,5-disulfonic acid (Tiron), had no effect on lysosomal stability. Zinc ions (0·05–2·5 mM) significantly stabilized lysosomal membranes; this stabilization was concentration dependent, was not affected by thiol compounds, and was potentiated by equimolar complex with 8-hydroxyquinoline (8-HQ) ; 8-HQ alone (1 mM) labilized lysosomes. Calcium ions had no effect, whereas Cu 2+ and Hg 2+ labilized the lysosomes. Labilization by Cu 2+ was reversed by chelating agents and decreased by thiol compounds. Incubation of lysosomes in nitrogen and dark prevented labilization by copper, and did not affect stabilization by zinc. The effects of certain chelating agents (8-HQ, DDC, penicillamine, EDTA) or metals (Zn 2+, Cu 2+, Hg 2+) on the stability of the lysosomal membranes are discussed in terms of the reactivity of these agents with components of the membrane, or interference with metal-catalyzed lipid peroxidation.

Effects of anti-inflammatory drugs on the stability of rat liver lysosomes in vitro

Certain steroidal and nonsteroidal anti-inflammatory drugs were found to stabilize in vitro lysosomes obtained from a heavy mitochondrial rat liver fraction. Membrane stabilization was measured by determining the effects of drugs on the release of acid phosphatase and β-glucuronidase from lysosomes. Specific experimental conditions were necessary for the measurement of drug-induced stabilization. The 3500 g liver fraction, prepared in a Ten Broeck tissue grinder and incubated at 37° in sucrosetris acetate buffer (pH 7.4) free of sodium ion, was found to be the most suitable source of lysosomes in these studies. Chloroquine, hydrocortisone, acetylsalieylic acid, phenylbutazone, flufenamic acid and niflumic acid exhibited good lysosome membrane-stabilizing activity, while indomethacin showed only moderate activity. Mefenamic acid, ibufenac, gold, Imuran and Cytoxan as well as dozens of drugs from other therapeutic classes were inactive. When lysosomes were incubated in a buffer containing sodium acetate rather than tris acetate or at 45° rather than 37°, anti-inflammatory drugs no longer stabilized but actually enhanced the labilization of lysosomes. The results of these studies indicate that considerable caution must be exercised in the preparation, handling and incubation of lysosomes in order to reveal the stabilizing capacity in vitro of anti-inflammatory drugs.