Effects of chloroquine on lysosomes and endocytosis by liver cells in vivo.

1. Chloroquine accumulation in rat liver after a single and repeated drug administration and lysosomal changes resembling some symptoms of lysosomal storage diseases were observed. 2. Repeated chloroquine treatment of rats resulted in increased activity of liver lysosomal enzymes acid phosphatase and beta-galactosidase and a significant enhancement of the activities of cathepsin D and cysteine proteinases were found. 3. No changes in the activity of liver macrophages (as assessed by the colloidal carbon clearance test) or in fluid-phase endocytosis of the marker 125I-polyvinyl-pyrrolidone by hepatocytes in vivo were found.     

Effect of the segregation and accumulation of chloroquine and daunorubicin by L cells (LSM subline) on the activity of lysosomal hydrolases

Kinetics of chloroquine and daunorubicin (DNR) uptake by cultured L cells (subline LSM) has been studied. With their constant concentrations in the medium the uptake of both chloroquine and DNR was characterized as a two phase process. Within 1.5-2 hours, these cells accumulated as much as 90 per cent of the total chloroquine and DNR amounts taken up during the whole incubation period. The segregation and accumulation of these substances took place in lysosomes. Chloroquine and DNR concentrations within lysosomes exceed those in the medium by 1100 and 5000 times, respectively. The chloroquine and DNR accumulation in lysosomes inhibited activities of some lysosomal hydrolases tested: cathepsins B and D, N-acetyl-beta, D-glucosaminidase and acid phosphatase. Unlike, the activity of acid lipase was not affected by chloroquine, and was sufficiently stimulated (by 55%) by DNR. The mechanism of inhibition of lysosomal enzymes by chloroquine and DNR is not yet known, although some suggestions are made. Possible consequences of lysosomal activity inhibition for cell metabolism are discussed in addition to a possible role of lysosomotropic agents as regulators of lysosomal functional activity.     

Inhibition of hepatocyte proteolysis and lactate gluconeogenesis by chloroquine

Chloroquine (50 μm) is rapidly taken up by isolated hepatocytes in a temperature-dependent manner. It inhibits glucose synthesis from lactate, but not from pyruvate or dihydroxyacetone. The inhibition is reversed by lysine or ammonia but not by oleate or carnitine. Ammonia inhibits chloroquine uptake by the hepatocytes but lysine does not. Chloroquine also inhibits urea synthesis, the release of ninhydrin-reacting substances, the accumulation of amino acids, and the lactate-dependent accumulation of glutamate. Ethanol oxidation in the presence of lactate is also inhibited, and this too is reversed by lysine. Chloroquine increases the redox state of the cytosolic compartment, as evidenced by lactate-to-pyruvate ratios, of hepatocytes prepared from both 48-h fasted and meal-fed rats. The above findings are consistent with chloroquine entering the lysosomes of the hepatocytes and inhibiting proteolysis by raising the lysosomal pH. Isolated hepatocytes are deficient in amino acids and, chloroquine inhibition of proteolysis prevents replenishment of the amino acid pools. Thus, chloroquine prevents reconstitution of the malate-aspartate shuttle required for the movement of reducing equivalents into the mitochondrion during lactate gluconeogenesis, ethanol oxidation, and glycolysis. The metabolic competency of freshly isolated hepatocytes, therefore, depends on the replenishment of amino acid pools by lysosomal breakdown of endogenous protein. Furthermore, chloroquine uptake may be an index of lysosomal function with isolated hepatocytes.     

Uptake and degradation of 125I-labelled high density lipoproteins in rat liver cells in vivo and in vitro.

1. The uptake of 125I-labelled high density lipoproteins (HDL) in various organs of the rat was determined after an intravenous injection. The uptake of 125I-labelled polyvinylpyrrolidone in the same organs was determined in order to assess uptake by fluid endocytosis. The uptake/organ was highest for the liver. The adrenals showed the highest uptake/unit weight of the organs studied. The liver, the kidneys and the spleen showed comparable values for uptake/g of tissue. The uptake of 125I-labelled HDL exceeded by far that of 125I-labelled polyvinylpyrrolidone in the liver, the kidneys, the spleen and the adrenals, indicating that the uptake of 125I-labelled HDL was mediated by adsorptive endocytosis. 2. The in vivo uptake of 125I-labelled HDL was determined in purified hepatocytes and non-parenchymal cells prepared by collagenase perfusion of livers from animals after intravenous injections of 125I-labelled HDL. When expressed per cell, the hepatocytes and the non-parenchymal liver cells took up about the same amount of 125I-labelled HDL. 3. The in vitro uptake and degradation of 125I-labelled HDL in isolated rat hepatocytes was studied. The uptake at increasing concentrations of 125I-labelled HDL was saturable indicating uptake mediated through binding sites. 125I-labelled HDL were easily degraded by contaminating proteases from the perfusate. 4. Subcellular fractionation by isopycnic centrifugation indicated that the accumulation of 125I-labelled HDL did not take place in the lysosomes, but rather on the plasma membrane and possibly in the endosomes (phagosomes). 5. 125I-labelled HDL were internalized into the cells and degraded in the lysosomes. Leupetin and chloroquine, inhibitors of the lysosomal function effectively inhibited the formation of 125I-labelled acid-soluble radioactivity by the cells. Chloroquine, but not the protease inhibitor leupeptin, reduced the hydrolysis of the cholesteryl ester moiety of HDL.     

Transplasma membrane electron and proton transport is inhibited by chloroquine

Chloroquine is a weak base which has been shown to inhibit lysosomal acidification. Chloroquine inhibits iron uptake in reticulocytes at a concentration of 0.5 mM. It is also effective in the control of malaria and other parasitic diseases. We now report that chloroquine inhibits NADH diferric transferrin reductase as well as the proton release stimulated by diferric transferrin from liver and HeLa cells. Ammonium chloride which also inhibits endosome acidification does not significantly inhibit the NADH diferric transferrin reduction. NADH diferric transferrin reductase of isolated rat liver plasma membrane is inhibited by chloroquine at concentrations similar to those required for inhibition of diferric transferrin reduction by whole cells. Ferricyanide reduction by whole cells is also inhibited by chloroquine. These observations provide an alternative mechanism for chloroquine control of acidification of endosomes and suggests a new approach to control of protozoal parasites through inhibition of a transmembrane oxidoreductase which controls transmembrane proton movement.     

Antimelanoma Activity of Chloroquine,an Antimalarial Agent With High Affinity for Melanin

The antimalarial agent chloroquine is known for high affinity for melanin. This 4-aminoquinoline derivative was examined for anti-melanoma activity and uptake into melanoma cells. Chloroquine inhibited growth of cultured melanoma cells; the effect was much greater to a moderately pigmented cell line HMV-II than to a nonpigmented HMV-I. Treatment with chloroquine at a dose of 62 mg/kg i.p. for 12 days prolonged by 71% the life span of mice bearing B16 melanoma, while 24-day treatment at 31 mg/kg resulted in a 81% increase in life span. HMV-II cells showed a two-fold increase in up-take of chloroquine as compared with HMV-I cells. Chloroquine, 24 hr after administration to mice implanted s.c. with B16 melanoma, was selectively accumulated in the pigmented tissues, melanoma and eyes. Other nonpigmented tissues such as the liver, lung, and kidney showed rapid uptake (within 1 hr) and release. These results suggest that chloroquine is toxic to pigmented melanoma cells, the process being partly mediated by binding to melanin     

The intracellular distribution of high density lipoproteins taken up by isolated rat hepatocytes

The subcellular distribution of 125I-labelled HDL taken up by rat hepatocytes in vivo and in vitro has been studied with subcellular fractionation techniques: differential centrifugation and isopycnic centrifugation in sucrose gradients. 125I-labelled HDL bind to plasma membranes both in vivo and in vitro and part of the membrane-bound 125I-labelled HDL can be dissociated by the addition of unlabelled HDL. The hepatocytes also internalize 125I-labelled HDL. The 125I-labelled HDL accumulate, however, at different intracellular sites in the in vivo and in vitro situation. The subcellular distribution pattern of 125I-labelled HDL taken up by the cells in vivo is similar to that of the lysosomal marker enzyme acid phosphatase. Peak activity was found at a density of 1.20 g/ml. In vitro 125I-labelled HDL accumulate in an organelle with a medium density of about 1.13 g/ml. This distribution was similar to that of the plasma membrane marker 5'-nucleotidase. The subcellular distribution of radioactivity taken up in vivo was changed to lower density by incubating the cells with chloroquine, a drug known to render the lysosomes more boyant. Chloroquine had no effect on the distribution of 125I-labelled HDL taken up by hepatocytes in vitro.     

Degradation of insulin by human fibroblasts: effects of inhibitors of pinocytosis and lysosomal activity

The role of the pinosome-lysosome pathway in the degradation of 125I-labelled bovine insulin by cultured human fibroblasts was examined by comparing the effects of various known inhibitors of pinocytosis and lysosomal degradation on the uptake and degradation of 125I-labelled polyvinylpyrrolidone, formaldehyde-denatured bovine serum albumin and bovine insulin by these cells. Fibroblasts incubated with polyvinylpyrrolidone steadily accumulate this substrate, whereas incubations with insulin or denatured albumin led to the progressive appearance in the culture medium of [125I]iodotyrosine. Inhibitors of pinocytosis (bacitracin, colchicine and monensin), metabolic inhibitors (2,4-dinitrophenol and NaF), lysosomotropic agents (chloroquine and NH4Cl) and an inhibitor of cysteine-proteinases (leupeptin) decreased the rate of uptake of polyvinylpyrrolidone and denatured albumin very similarly, but only bacitracin had an effect on the processing of insulin. Chloroquine, NH4Cl and leupeptin strongly inhibited the digestion of denatured albumin, but not of insulin. The different responses to the modifiers, with polyvinylpyrrolidone and denatured albumin on the one hand and insulin on the other, suggest that insulin degradation can occur by a non-lysosomal pathway. The very strong inhibitory effect of bacitracin on insulin processing by fibroblasts may point to an important role of plasma membrane proteinases in insulin degradation.     

Inhibition of proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339.

The proteolytic degradation of 125I-labeled low density lipoprotein by monolayers of cultured human fibroblasts was prevented by exposure of the cells to chloroquine, an agent that has been reported previously to inhibit lysosomal degradative processes. Chloroquine did not inhibit the binding of low density lipoprotein to its cell surface receptor. However, the two regulatory actions that normally follow low density lipoprotein binding to its receptor, namely suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and stimulation of cholesteryl ester formation, were both prevented when degradation of the lipoprotein was inhibited by chloroquine. Two other agents affecting lysosomal function, Triton WR 1339 and concanavalin A, also inhibited the proteolytic degradation of low density lipoprotein in intact fibroblasts and simultaneously prevented low density lipoprotein-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and stimulation of cholesteryl ester formation. Unlike chloroquine, however, these two agents also affect the binding of low density lipoprotein to the cells. The inhibitory action of chloropuine, concanavalin A, and Triton WR 1339 could each be reversed by removal of the agent from the culture medium. These in vivo culture data, together with the observation that cell-free extracts of fibroblasts maximally degrade 125I-labeled low density lipoprotein at pH 4 and do not form acid-soluble material above pH 6, are consistent with the hypothesis that the proteolytic degradation of low density lipoprotein by monolayers of fibroblasts occurs within lysosomes. The data also suggest that normal lysosomal function is required in order for low density lipoprotein to regulate cholesterol synthesis and cholesteryl ester formation in the fibroblast system.     

Chloroquine-induced interference with degradation of serum lipoproteins in rat liver, studied in vivo and in vitro.

The effect of chloroquine, an inhibitor of certain lysosomal enzymes including cathepsin B (EC 3.4.22.1), on the degradation of serum lipoproteins in rat liver was studied in vivo and in liver homogenates. Chloroquine had no effect on the clearance from the circulation of 125I-labeled rat or human very low density lipoproteins or human low density lipoproteins. Pretreatment with chloroquine for 3 h, resulted in a 2-2.5 fold increase in 125i-labeled very low density lipoprotein recovered in the liver 45 min after injection of the homologous and heterologous lipoproteins. This effect was evident on both the 125I-labeled protein and 125I-labeled lipid moiety. 30 min after the injection of [3H]-cholesterol linoleate-labeled very low density lipoproteins, 70% of the injected label was recovered in the liver, both in control and chloroquine-treated rats. Since the perl and 20% in the experimental group, it was concluded that chloroquine interferes with the hydrolysis of [3H]cholesterol linoleate. Following injection of 125I-labeled human low density lipoproteins only 4% of the injected lipoprotein was recovered in the liver of control rats and not more than 10% after chloroquine treatment, when about 50% had been cleared from the circulation. Hence, while very low density lipoprotein protein and cholesterol ester are catabolized in the liver, the catabolism of low density lipoproteins occurs mainly in extra-hepatic tissues. Using post-nuclear liver suprnatant, optimal degradation of various serum lipoproteins was found at pH 4.4, and chloroquine inhibited their degradation. Degradation of very low density and low density lipoproteins was completely inhibited at 0.05 M chloroquine, while less pronounced inhibition was seen with high density lipoproteins, apolipoproteins and apolipoprotein AI. These results indicate that liver acid hydrolases in vivo participate in the degradation of serum lipoproteins. Cathepsin B is apparently responsible for the degradation of aplipoprotein B, while other cathepsins might also be active in the degradation of this and the other apolipoproteins.     

Uptake and degradation of asialo-fetuin by isolated rat hepatocytes.

125I-labelled asialo-fetuin was taken up by isolated rat hepatocytes by a saturable process. Half maximum uptake was seen at about 3 . 10(-8) M asialo-fetuin. Rate of uptake of asialo-fetuin exceeded rate of degradation at all concentrations of asialo-fetuin tested. Degradation of asialo-fetuin, as indicated by release of acid-soluble radioactivity from the cells, was inhibited by NH4Cl and chloroquine. The intracellular distribution of labelled asialo-fetuin was studied by differential and density gradient centrifuging. The distribution curves for radioactivity indicated that asialo-fetuin was present in lysosomes about 1 h after the uptake had started. Chloroquine and ammonium ions seemed to inhibit the uptake of asialo-fetuin into the lysosomes, possibly by interfering with the fusion between phagosomes and lysosomes.     

Uptake of ferritin and iron bound to ferritin by rat hepatocytes: modulation by apotransferrin, iron chelators and chloroquine

Rat liver ferritin is an effective donor of iron to rat hepatocytes. Uptake of iron from ferritin by the cells is partially inhibited by including apotransferrin in the culture medium, but not by inclusion of diferric transferrin. This inhibition is dependent on the concentration of apotransferrin, with a 30% depression in iron incorporation in the cells detected at apotransferrin concentrations above 40 micrograms/ml. However, apotransferrin does not interfere with uptake of 125I-labeled ferritin, suggesting that apotransferrin decreases retention of iron taken up from ferritin by hepatocytes by sequestering a portion of released iron before it has entered the metabolic pathway of the cells. The iron chelators desferrioxamine (100 microM), citrate (10 mM) and diethylenetriaminepentaacetate (100 microM) reduce iron uptake by the cells by 35, 25 and 8%, respectively. In contrast, 1 mM ascorbate increases iron accumulation by 20%. At a subtoxic concentration of 100 microM, chloroquine depresses ferritin and iron uptake by hepatocytes by more than 50% after 3 h incubation. Chloroquine presumably acts by retarding lysosomal degradation of ferritin and recycling of ferritin receptors.     

A method to measure cardiac autophagic flux in vivo

Autophagy, a highly conserved cellular mechanism wherein various cellular components are broken down and recycled through lysosomes, has been implicated in the development of heart failure. However, tools to measure autophagic flux in vivo have been limited. Here, we tested whether monodansylcadaverine (MDC) and the lysosomotropic drug chloroquine could be used to measure autophagic flux in both in vitro and in vivo model systems. Using HL-1 cardiac-derived myocytes transfected with GFP-tagged LC3 to track changes in autophagosome formation, autophagy was stimulated by mTOR inhibitor rapamycin. Administration of chloroquine to inhibit lysosomal activity enhanced the rapamycin-induced increase in the number of cells with numerous GFP-LC3-positive autophagosomes. The chloroquine-induced increase of autophagosomes occurred in a dose-dependent manner between 1 microM and 8 microM, and reached a maximum 2 hour after treatment. Chloroquine also enhanced the accumulation of autophagosomes in cells stimulated with hydrogen peroxide, while it attenuated that induced by Bafilomycin A1, an inhibitor of V-ATPase that interferes with fusion of autophagosomes with lysosomes. The accumulation of autophagosomes was inhibited by 3-methyladenine, which is known to inhibit the early phase of the autophagic process. Using transgenic mice expressing 3 mCherry-LC3 exposed to rapamycin for 4 hr, we observed an increase in mCherry-LC3-labeled autophagosomes in myocardium, which was further increased by concurrent administration of chloroquine, thus allowing determination of flux as a more precise measure of autophagic activity in vivo. MDC injected 1 hr before sacrifice colocalized with mCherry-LC3 puncta, validating its use as a marker of autophagosomes. This study describes a method to measure autophagic flux in vivo even in non-transgenic animals, using MDC and chloroquine.     

A method to measure cardiac autophagic flux in vivo

Autophagy, a highly conserved cellular mechanism wherein various cellular components are broken down and recycled through lysosomes, has been implicated in the development of heart failure. However, tools to measure autophagic flux in vivo have been limited. Here, we tested whether monodansylcadaverine (MDC) and the lysosomotropic drug chloroquine could be used to measure autophagic flux in both in vitro and in vivo model systems. Using HL-1 cardiac-derived myocytes transfected with GFP-tagged LC3 to track changes in autophagosome formation, autophagy was stimulated by mTOR inhibitor rapamycin. Administration of chloroquine to inhibit lysosomal activity enhanced the rapamycin-induced increase in the number of cells with numerous GFP-LC3-positive autophagosomes. The chloroquine-induced increase of autophagosomes occurred in a dose-dependent manner between 1 µM and 8 µM, and reached a maximum 2 hour after treatment. Chloroquine also enhanced the accumulation of autophagosomes in cells stimulated with hydrogen peroxide, while it attenuated that induced by Bafilomycin A₁, an inhibitor of V-ATPase that interferes with fusion of autophagosomes with lysosomes. The accumulation of autophagosomes was inhibited by 3-methyladenine, which is known to inhibit the early phase of the autophagic process. Using transgenic mice expressing mCherry-LC3 exposed to rapamycin for 4 hr, we observed an increase in mCherry-LC3-labeled autophagosomes in myocardium, which was further increased by concurrent administration of chloroquine, thus allowing determination of flux as a more precise measure of autophagic activity in vivo. MDC injected 1 hr before sacrifice colocalized with mCherry-LC3 puncta, validating its use as a marker of autophagosomes. This study describes a method to measure autophagic flux in vivo even in non-transgenic animals, using MDC and chloroquine.     

Chloroquine alters the processing of secretin in isolated rat pancreatic acinar cells

In this study we considered the effect of chloroquine on the processing and intracellular distribution of internalized secretin radioligand in acinar cells. Chloroquine (100 microM) had no effect on the total amount of 125I-secretin bound but had marked effects on the processing of this radioligand in acinar cells. After an initial 60 min of radioligand binding in the presence and absence of chloroquine, cells were washed free of unbound radioligand, resuspended and then processed for different times at 37 degrees C. During 60, 120 and 180 min of processing, the amount of internalized radioligand in the presence of 100 microM chloroquine was increased by 116, 194 and 273%, respectively, compared to untreated control samples. Chloroquine also increased the amount of intact 125I-secretin radioligand within the cell as measured by rebinding to pancreatic plasma membranes. After 120 and 180 min of processing, intact peptide within the acinar cell was 25 and 66% greater in the presence of this agent than in control samples (P less than or equal to 0.01). To determine if chloroquine affected intracellular localization of the secretin radioligand, we measured the amount of radioactivity in soluble and particulate fractions of cell homogenates. Chloroquine decreased radioactivity entering particulate fractions of the cell by greater than 35% after 120 and 180 min of processing (P less than or equal to 0.01). This study demonstrates that (1) chloroquine inhibits the intracellular degradation of secretin in acinar cells and (2) chloroquine alters intracellular localization of this peptide during processing.     

Chloroquine Is a Zinc Ionophore

Chloroquine is an established antimalarial agent that has been recently tested in clinical trials for its anticancer activity. The favorable effect of chloroquine appears to be due to its ability to sensitize cancerous cells to chemotherapy, radiation therapy, and induce apoptosis. The present study investigated the interaction of zinc ions with chloroquine in a human ovarian cancer cell line (A2780). Chloroquine enhanced zinc uptake by A2780 cells in a concentration-dependent manner, as assayed using a fluorescent zinc probe. This enhancement was attenuated by TPEN, a high affinity metal-binding compound, indicating the specificity of the zinc uptake. Furthermore, addition of copper or iron ions had no effect on chloroquine-induced zinc uptake. Fluorescent microscopic examination of intracellular zinc distribution demonstrated that free zinc ions are more concentrated in the lysosomes after addition of chloroquine, which is consistent with previous reports showing that chloroquine inhibits lysosome function. The combination of chloroquine with zinc enhanced chloroquine''s cytotoxicity and induced apoptosis in A2780 cells. Thus chloroquine is a zinc ionophore, a property that may contribute to chloroquine''s anticancer activity.     

Chloroquine treatment of ARPE-19 cells leads to lysosome dilation and intracellular lipid accumulation: possible implications of lysosomal dysfunction in macular degeneration

Background

Age-related macular degeneration (AMD) is the leading cause of vision loss in elderly people over 60. The pathogenesis is still unclear. It has been suggested that lysosomal stress may lead to drusen formation, a biomarker of AMD. In this study, ARPE-19 cells were treated with chloroquine to inhibit lysosomal function.

Results

Chloroquine-treated ARPE-19 cells demonstrate a marked increase in vacuolation and dense intracellular debris. These are identified as chloroquine-dilated lysosomes and lipid bodies with LAMP-2 and LipidTOX co-localization, respectively. Dilation is an indicator of lysosomal dysfunction. Chloroquine disrupts uptake of exogenously applied rhodamine-labeled dextran by these cells. This suggests a disruption in the phagocytic pathway. The increase in LAMP protein levels, as assessed by Western blots, suggests the possible involvement in autophagy. Oxidative stress with H₂O₂ does not induce vacuolation or lipid accumulation.

Conclusion

These findings suggest a possible role for lysosomes in AMD. Chloroquine treatment of RPE cells may provide insights into the cellular mechanisms underlying AMD.     

Chloroquine,a novel inhibitor of amino acid transport by rat renal brush border membrane vesicles

Summary Chloroquine is an antimalarial and antirheumatic lysosomotropic drug which inhibits taurine uptake into and increases efflux from cultured human lymphoblastoid cells. It inhibits taurine uptake by rat lung slices and affects the uptake and release of cystine from cystinotic fibroblasts. Speculations on its mode of action include a proton gradient effect, a non-specific alteration in membrane integrity, and membrane stabilization. In this study, the effect of chloroquine on the uptake of several amino acids by rat renal brush border membrane vesicles (BBMV) was examined. Chloroquine significantly inhibited the secondary active, NaCl-dependent component of 10µM taurine uptake at all concentrations tested, but did not change equilibrium values. Analysis of these data indicated that the inhibition was non-competitive. Taurine uptake was reduced at all osmolarities tested, but inhibition was greatest at the lowest osmolarity. Taurine efflux was not affected by chloroquine, nor was the NaCl-independent diffusional component of taurine transport. Chloroquine (1 mM) inhibited uptake of the imino acids L-proline and glycine, and the dibasic amino acid L-lysine. It inhibited the uptake of D-glucose, but not the neutral-amino acids L-alanine or L-methionine. Uptake of the dicarboxylic amino acids, L-glutamic acid and L-aspartic acid, was slightly enhanced. With regard to amino acid uptake by BBMV, these findings may support some of the currently proposed mechanisms of the action of chloroquine but further studies are indicated to determine why it affects the initial rate of active amino acid transport.     

Inhibition by chloroquine of the secretion of very low density lipoproteins by cultured rat hepatocytes

Cultured rat hepatocytes were incubated in medium containing 1.0 mM oleic acid. The incorporation of [3H]glycerol into cell-associated and medium triacylglycerols was measured after 2 h incubation. More than 95% of the secreted [3H]triacylglycerols were recovered in the very low density lipoprotein (VLDL) fraction (d less than 1.006). Chloroquine and other lysosomotropic amines promoted a marked decrease in [3H]triacylglycerol secretion from the hepatocytes while the synthesis was unaffected. At 50-200 microM final concentration, chloroquine inhibited secretion of triacylglycerols by 70-90% of the control. Similar results were obtained when the mass of secreted triacylglycerols was measured. Chloroquine caused decreased secretion of [3H]triacylglycerols after 15-30 min incubation and the inhibitory effect was completely reversible within 1-2 h after washout of chloroquine. The reduced triacylglycerol secretion was not due to increased reuptake of secreted lipoproteins or decreased protein synthesis caused by chloroquine. Electron microscopy of chloroquine-treated cells showed that the inhibition of VLDL secretion occurs at or prior to the level of the Golgi apparatus. These results suggest that chloroquine interferes with crucial steps in the secretory process and/or that lysosomal function could be essential for secretion of VLDL.     

Degradation of microinjected proteins: Effects of lysosomotropic agents and inhibitors of autophagy

HeLa cells, injected with radioiodinated proteins by fusion with RBC ghosts, were exposed to inhibitors of lysosomal proteolysis and autophagy. The degradation of injected [¹²⁵I]bovine serum albumin (BSA) was unaffected by chloroquine, NH₄Cl, nocodazole, colcemid, puromycin, cycloheximide, or enucleation. Although degradation of [¹²⁵I]lactate dehydrogenase (LDH) and [¹²⁵I]pyruvate kinase (PK) was inhibited one-third by chloroquine or ammonia, their degradation was unaffected by the other compounds. In contrast, enhanced degradation of ¹²⁵I-PK resulting from depriving injected HeLa cells of amino acids and serum was inhibited 70% by colcemid and abolished by chloroquine or ammonia. Similarly, degradation of [¹⁴C]sucrose-labeled BSA-polylysine conjugates that entered HeLa cells by endocytosis was inhibited as much as 80% by chloroquine and ammonia. Sensitivity of both enhanced proteolysis and degradation of exogenous proteins to ammonia or chloroquine indicates they are effective inhibitors of lysosomal proteolysis in HeLa cells. Failure of ammonia or chloroquine to inhibit degradation of injected ¹²⁵I-BSA and the modest inhibition of degradation of injected ¹²⁵I-LDH or ¹²⁵I-PK indicates that virtually all BSA molecules and most PK or LDH molecules are degraded by a nonlysosomal proteolytic system. Components of this degradative system are present in vast excess or are long lived, since inhibition of protein synthesis for 20 hr had no effect on the degradation of injected proteins.