Effect of cationic amphiphilic drugs on the hydrolysis of acidic and neutral phospholipids by liver lysosomal phospholipase A

Rat liver lysosomal phospholipase A hydrolyzes both acidic and neutral phospholipids. Numerous cationic amphiphilic drugs including imipramine, propranolol, 4,4'-bis(diethylaminoethoxy)-alpha, beta- diethyldiphenylethane and chloropromazine inhibit phospholipase A. Cationic amphiphilic drugs bind readily to acidic phospholipids but much less readily to neutral phospholipids. Formation of drug-lipid complexes is thought to be an important mechanism involved in the inhibition of lysosomal phospholipases. Therefore, we studied the effects of four cationic amphiphilic inhibitors on lysosomal phospholipase A using one acidic and two neutral phospholipid substrates. The concentration of the drugs required to produce 50% inhibition was much higher when phosphatidylinositol was used as substrate. The degradation of phosphatidylethanolamine and phosphatidylcholine was more readily inhibited by these agents than that of phosphatidylinositol. In drug-induced lipidosis, the predominance of acidic phospholipids may be due to redirection of phospholipid metabolism towards the formation of acidic phospholipids with a resultant increased delivery of these lipids to lysosomes. Based on our results, it does not appear to be due to decreased enzymatic hydrolysis of drug-acidic phospholipid complexes, at least when pure phospholipid substrates are used. Lysosomal storage of both acidic and neutral phospholipids appears to be caused by inhibition of lysosomal phospholipase action in view of the probable high intralysosomal levels of these agents.     

Characterization of the phospholipidogenic potential of 4(1H)-pyridone antimalarial derivatives

Drug-induced phospholipidosis (PLD) is characterized by the excessive accumulation of phospholipids in lysosomes. It is accompanied by intracellular retention of drug that could be associated with increased cytotoxicity. Drug-induced PLD is recognized as a significant challenge for drug development, depending on the severity of the effect it could be reversible or caused cell death. Therefore, the identification at early stages of drug discovery of the potential to induce PLD can be advantageous for selecting improved development candidates.PLD has commonly been associated with cationic amphiphilic drugs (CADs) composed by a hydrophobic ring structure and a hydrophilic side chain with a charged amine group. 4(1H)-pyridone derivatives are a family of antimalarial agents that act as potent selective inhibitors of Plasmodium falciparum mitochondrial function and according to their chemical structure might be considered to be CADs.In the present study, the potential of 4(1H)-pyridone derivatives to induce PLD in vitro and their general cytotoxicity properties were investigated. A cell-based fluorescence assay using the fluorescent phospholipid probe NBD-PE [N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt] was established. Five PLD-inducing reference compounds and six negative reference compounds were evaluated in vitro in HepG2 cell line.The pyridones tested were ranked by using a chloroquine-equivalent scale (chloroquine constituting a well-known antimalarial drug that acts as a potent inducer of lysosomal storage of phospholipids in both cell cultures and in vivo studies).The present findings indicate that these novel chemical antimalarial compounds are not PLD inducers despite to be considered structurally as CADs. Furthermore, none of the compounds tested showed significant cytotoxicity at their maximum solubility.     

Ultrastructural, physico-chemical and conformational study of the interactions of gentamicin and bis(beta-diethylaminoethylether) hexestrol with negatively-charged phospholipid layers

Aminoglycoside antibiotics such as gentamicin, which are fully hydrophilic, and cationic amphiphilic drugs such as bis(beta-diethylaminoethylether)hexestrol (DEH), are both known to inhibit lysosomal phospholipases and induce phospholipidosis. This enzymatic inhibition is probably related to the neutralization of the surface negative charges on which the lysosomal phospholipases A1 and A2 are dependent to express fully their activities (Mingeot-Leclerq et al., Biochem Pharmacol 37: 591-599, 1988). Using negatively charged liposomes, we show by 31P NMR spectroscopy that both gentamicin and DEH cause a significant restriction in the phosphate head mobility and, in sonicated vesicles, the appearance of larger bilayer structures. Both DEH and gentamicin increased the apparent size of sonicated negatively charged liposomes (but not of neutral liposomes) as measured by quasi-elastic light scattering spectroscopy. Examination of replicas from freeze-etched samples, however, revealed that gentamicin caused aggregation of liposomes, whereas DEH induced their fusion and the formation of intramembranous roundly shaped structures. Only DEH caused a significant decrease of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, a fluorescent lipid-soluble probe. In addition, DEH, but not gentamicin, interfered with the bilayer to hexagonal phase transition occurring in dioleoyl- and dielaidoylphosphatidylethanolamine liposomes upon warming, and caused the appearance of an isotropic signal suggestive of the formation of inverted micelles. In computer-aided conformational analysis of the molecules at a simulated air-water interface, gentamicin was shown to display a largely-open crescent shape. When surrounded by phosphatidylinositol molecules, it remained as such at the interface which it locally mis-shaped, establishing close contact with the negatively charged phospho groups. In contrast, DEH could be oriented perpendicularly to the interface, with its two cationic groups associated with the phospho groups, and its phenyl- and diethylethandiyl moieties deeply inserted between and interacting with the aliphatic chains. Thus, although both agents cause lysosomal phospholipases inhibition, the differences in their interactions with negatively-charged bilayers is likely to result in a different organization of the phospholipids accumulated in vivo, which could lead to different toxicities.     

Mechanism of aminoglycoside-induced lysosomal phospholipidosis: In vitro and in vivo studies with Gentamicin and Amikacin

Gentamicin, a widely used aminoglycoside antibiotic, is concentrated in lysosomes of proximal tubular cells of the kidney, and induces therein an accumulation of myelin-like material. We show that treatment of rats with Gentamicin (10 mg/kg, 7 days) induces a loss of activity of lysosomal sphingomyelinase and phospholipase A₁, associated with an increase in the amount of total lipid phosphorus in the kidney cortex. In vitro, Gentamicin is shown by gel permeation to bind to phospholipid bilayers (liposomes) under conditions which mimic the lysosomal environment (acid pH and presence of phosphatidylinositol). The reversal of this binding by an increase in the ionic strength (> 0.04) suggests electrostatic interaction between the hydrophilic, polycationic aminoglycoside and the negatively charged phospholipids. Binding of Gentamicin impairs the hydrolysis of phosphatidylcholine present in the bilayer, by lysosomal phospholipases A₁ and A₂ from the liver or kidney. We also show that lysosomal sphingomyelinase is readily and irreversibly inactivated by liposomes in the absence of detergent.The lysosomal phospholipidosis induced by Gentamicin in the kidney, as in cultured cells [Aubert Tulkens et al., Lab. Invest.40, 481 (1979)] appears therefore to be a direct consequence of the lysosomotropic character of this drug and its ability to inhibit therein phospholipid breakdown. Amikacin, a semi-synthetic aminoglycoside, binds more loosely to phospholipid bilayers, induces less inhibition of phospholipases in vitro and is less taken up by tubular cells in vivo. Accordingly, Amikacin does not provoke significant lysosomal phospholipidosis or loss of sphingomyelinase and phospholipase A₁ activities in vivo at the doses and time investigated (0–40 mg/kg, 7 days). Inasmuch as Amikacin is reported to be less toxic to the kidney, we suggest that lysosomal alterations are an early and significant     

Effect of anti-inflammatory drugs on lysosomes and lysosomal enzymes from rat liver

Steroidal and nonsteroidal anti-inflammatory drugs were tested for their capacity to stabilize, in vitro, lysosomes and inhibit lysosomal enzymes. Lysosome membrane stability was measured by determining the effects of drugs on the release of aryl sulfatase and β-glucuronidase from lysosomes which were suspended in a hypo-osmotic sucrose buffer. Lysosomes obtained from a heavy mitochondrial (3500 g) rat liver fraction were found to be highly sensitive to membrane stabilization by naproxen, alclofenac, chloroquine, mefenamic acid, phenylbutazone, hydrocortisone, dexamethasone and methylprednisolone. Ibuprofen and flufenamic acid demonstrated moderate stabilizing activity, while indo-methacin, aspirin and clonixin showed only weak activity. Imuran, as well as other anti-inflammatory drugs, was inactive. In addition to their membrane-stabilizing activity, chloroquine was found to be a potent inhibitor of aryl sulfatase and phenylbutazone an inhibitor of β-glucuronidase activity. Hydrocortisone, dexamethasone and paramethasone inhibited aryl sulfatase activity, while no steroid tested was effective as an inhibitor of β-glucuronidase. The data in this report support the hypothesis that anti-inflammatory drugs inhibit the release of enzymes from lysosomes. In addition, several of these drugs may act as inhibitors of lysosomal enzyme activity.     

Biochemical mechanism of aminoglycoside-induced inhibition of phosphatidylcholine hydrolysis by lysosomal phospholipases

Aminoglycosides such as gentamicin are hydrophilic, polycationic drugs which bind to negatively-charged phospholipid bilayers, inhibit the activities of the lysosomal enzymes involved in the degradation of the major phospholipids and cause, in kidney in vivo or in cultured cells, a lysosomal phospholipidosis. In the present study, we show that the hydrolysis of phosphatidylcholine induced in liposomes by lysosomal extracts at pH 5.4 in vitro is critically dependent on the negative charges carried by the bilayer. This hydrolysis, which is predominantly carried on by phospholipases A1 and A2, markedly increases when the phosphatidylinositol content is raised from 10 to 30% of the total phospholipids, i.e. in a range found in natural membranes. Addition of gentamicin decreases the activity of these enzymes in a non-competitive fashion, but the effect is inversely proportional to the amount of phosphatidylinositol present in the bilayer. Gentamicin and bis(beta-diethylaminoethylether)hexestrol (DEH), a cationic amphiphile which also binds to phospholipid bilayers, are equipotent inhibitors when added to negatively-charged liposomes at equinormal concentrations. Although direct aminoglycoside-enzyme interactions cannot be excluded, these results strongly suggest that gentamicin impairs the activities of the lysosomal phospholipases towards phosphatidylcholine by decreasing the available negative charges required for optimal activity.     

A high content screening assay for identifying lysosomotropic compounds

Lysosomes are acidic organelles that are essential for the degradation of old organelles and engulfed microbes. Furthermore, lysosomes play a key role in cell death. Lipophilic or amphiphilic compounds with a basic moiety can become protonated and trapped within lysosomes, causing lysosomal dysfunction. Therefore, high-throughput screens to detect lysosomotropism, the accumulation of compounds in lysosomes, are desirable.Hence, we developed a 96-well format, high content screening assay that measures lysosomotropism and cytotoxicity by quantitative image analysis. Forty drugs, including antidepressants, antipsychotics, antiarrhythmics and anticancer agents, were tested for their effects on lysosomotropism and cytotoxicity in H9c2 cells. The assay correctly identified drugs known to cause lysosomotropism and revealed novel information showing that the anticancer drugs, gefitinib, lapatinib, and dasatinib, caused lysosomotropism. Although structurally and pharmacologically diverse, drugs that were lysosomotropic shared certain physicochemical properties, possessing a ClogP > 2 and a basic pKa between 6.5 and 11. In contrast, drugs which did not lie in this physicochemical property space were not lysosomotropic. The assay is a robust, rapid screen that can be used to identify lysosomotropic, as well as, cytotoxic compounds, and can be positioned within a screening paradigm to understand the role of lysosomotropism as a contributor to drug-induced toxicity.     

Characterization of LysoTracker Red uptake by in vitro model cells of the outer blood-retinal barrier: Implication of lysosomal trapping with cytoplasmic vacuolation and cytotoxicity

Lysosomal trapping, a physicochemical process in which lipophilic cationic compounds are sequestered in lysosomes, can affect drug disposition and cytotoxicity. To better understand lysosomal trapping at the outer blood-retinal barrier (BRB), we investigated the distribution of LysoTracker Red (LTR), a probe compound for lysosomal trapping, in conditionally immortalized rat retinal pigment epithelial (RPE-J) cells. LTR uptake by RPE-J cells was dependent on temperature and attenuated by ammonium chloride and protonophore, which decreased the pH gradient between the lysosome and cytoplasm, suggesting lysosomal trapping of LTR in RPE-J cells. The involvement of lysosomal trapping in response to cationic drugs, including neuroprotectants such as desipramine and memantine, was also suggested by an inhibition study of LTR uptake. Chloroquine, which is known to show ocular toxicity, induced cytoplasmic vacuolization in RPE-J cells with a half-maximal effective concentration of 1.35 μM. This value was 59 times lower than the median lethal concentration (= 79.1 μM) of chloroquine, suggesting that vacuolization was not a direct trigger of cell death. These results are helpful for understanding the lysosomal trapping of cationic drugs, which is associated with drug disposition and cytotoxicity in the outer BRB.     

Monitoring the accumulation of fluorescently labeled phospholipids in cell cultures provides an accurate screen for drugs that induce phospholipidosis

A large number of cationic amphiphilic drugs (CADs) are known to cause phospholipidosis (PLD) in vivo. In the present study, we have built upon our previous findings to further qualify the use of a fluorescently labeled phospholipid-based cell-culture assay to detect PLD-inducing drugs. In this paper, we demonstrate that 12 PLD-negative compounds and 11 drugs known to cause PLD in vivo are all correctly identified by using this assay. Interestingly, we found that in cells treated with certain CADs, the fluorescent phospholipid was sequestered in a very specific punctate pattern, which overlapped strongly with the staining pattern seen with a lysosomal marker protein. Our data also show that false positives can be generated with the fluorescence assay when compounds are used at concentrations that cause a >30% decrease in cell number in this assay. Confocal microscopy demonstrated that the staining pattern of fluorescent phospholipids in these cases may be differentiated from those of true positives by the fact that diffuse, rather than punctuate, fluorescence is observed. These studies confirm and expand our previous results showing that the fluorescent phospholipid assay is a highly sensitive, specific tool for detecting PLD-inducing drugs, if care is taken to rule out cytotoxicity-related artifact.     

Monitoring the Accumulation of Fluorescently Labeled Phospholipids in Cell Cultures Provides an Accurate Screen for Drugs that Induce Phospholipidosis

A large number of cationic amphiphilic drugs (CADs) are known to cause phospholipidosis (PLD) in vivo. In the present study, we have built upon our previous findings to further qualify the use of a fluorescently labeled phospholipid-based cell-culture assay to detect PLD-inducing drugs. In this paper, we demonstrate that 12 PLD-negative compounds and 11 drugs known to cause PLD in vivo are all correctly identified by using this assay. Interestingly, we found that in cells treated with certain CADs, the fluorescent phospholipid was sequestered in a very specific punctate pattern, which overlapped strongly with the staining pattern seen with a lysosomal marker protein. Our data also show that false positives can be generated with the fluorescence assay when compounds are used at concentrations that cause a >30% decrease in cell number in this assay. Confocal microscopy demonstrated that the staining pattern of fluorescent phospholipids in these cases may be differentiated from those of true positives by the fact that diffuse, rather than punctuate, fluorescence is observed. These studies confirm and expand our previous results showing that the fluorescent phospholipid assay is a highly sensitive, specific tool for detecting PLD-inducing drugs, if care is taken to rule out cytotoxicity-related artifact.     

Examination of the mechanism(s) involved in doxorubicin-mediated iron accumulation in ferritin: studies using metabolic inhibitors, protein synthesis inhibitors, and lysosomotropic agents

Anthracyclines are potent anticancer agents, but their use is limited by cardiotoxicity at high cumulative doses. The mechanisms involved in anthracycline-mediated cardiotoxicity are still poorly understood, but numerous investigations have indicated a role for iron in this process. Our previous studies using neoplastic and myocardial cells showed that anthracyclines inhibit iron mobilization from the iron storage protein, ferritin, resulting in marked accumulation of ferritin-iron. Although the process of ferritin-iron mobilization is little understood, catabolism of ferritin by lysosomes may be a likely mechanism. Because anthracyclines have been shown to accumulate in lysosomes, this latter organelle may be a potential target for these drugs. The present study demonstrated, using native polyacrylamide gel electrophoresis-59Fe autoradiography, that ferritin-59Fe mobilization is an energy-dependent process that also requires protein synthesis. Depression of lysosomal activity via the enzyme inhibitors E64d [(2S,3S)-trans-epoxysuccinyl-l-leucylamido-2-methylbutane ethyl ester] and leupeptin or the lysosomotropic agents ammonium chloride, chloroquine, and methylamine resulted in a 3- to 5-fold increase in 59Feferritin accumulation compared with control cells. In addition, the proteasome inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leuleucinal (MG132) and lactacystin also significantly increased 59Fe-ferritin levels compared with control cells. These effects of lysosomotropic agents or inhibitors of lysosomal activity were comparable with that observed with the anthracycline doxorubicin. Collectively, our study indicates a role for lysosomes and proteasomes in ferritin-iron mobilization, and this pathway is dependent on metabolic energy and protein synthesis. Furthermore, the lysosome/proteasome pathway may be a novel anthracycline target, inhibiting iron mobilization from ferritin that is essential for vital iron-requiring processes such as DNA synthesis.     

Effect of acidic phospholipids on the activity of lysosomal phospholipases and on their inhibition by aminoglycoside antibiotics--I. Biochemical analysis

Aminoglycoside antibiotics accumulate in lysosomes of kidney and cultured cells and cause an impairment of phospholipid catabolism which is considered to be an early and significant step in the development of their toxicity. Using liposomes, wer previously demonstrated that the activity of lysosomal phospholipases A1 and A2 towards phosphatidylcholine was markedly enhanced by the inclusion of phosphatidylinositol in the bilayer, and that gentamicin impaired this activity by binding to phosphatidylinositol. Since gentamicin-induced inhibition was inversely related to the amount of phosphatidylinositol included in the liposomes, we proposed that gentamicin impairs activity of phospholipases by decreasing the quantity of available negative charges carried by the bilayer surface (Mingeot-Leclercq et al., Biochem Pharmacol 37: 591-599, 1988). We now extend these observations to phosphatidylserine and phosphatidic acid, and compare the inhibition caused by gentamicin, amikacin and streptomycin towards lysosomal phospholipases on the hydrolysis of phosphatidylcholine in the presence of each of these acidic phospholipids. Inclusion of phosphatidic acid in liposomes, and, to a lesser extent, phosphatidylserine, caused a larger increase in phospholipases activity than phosphatidylinositol. In parallel, the three aminoglycosides tested were found less inhibitory towards phospholipases activity measured on phosphatidic acid-or phosphatidylserine-containing liposomes than was previously observed with phosphatidylinositol, even though equilibrium dialysis experiments failed to demonstrate significant difference in binding parameters of the drug towards each of these liposomes populations. Yet, as for phosphatidylinositol-containing liposomes, the inhibition was inversely related to the amount of phosphatidic acid or phosphatidylserine included in the bilayer and the inhibitory potency of the three drugs was consistently gentamicin greater than amikacin greater than streptomycin with the three types of negatively-charged liposomes used. We conclude that impairment of lysosomal phospholipases activity towards phosphatidylcholine included in negatively-charged membranes by aminoglycoside antibiotics is dependent upon drug binding to the bilayer, but that it is modulated by the nature of the acidic phospholipid that binds the drug as well as by that of the drug itself. A companion paper (Mingeot-Leclercq et al., Biochem Pharmacol 40: 499-506, 1990) will examine by computer-aided conformational analysis the parameters (drug-phospholipid energy of interaction, position of the drug in a monolayer and its accessibility to the aqueous phase) which may be important for these effects.     

The binding of drugs to different polar lipids in vitro

For evaluation of the physico-chemical basis underlying the drug-induced generalized lipid storage disease, the equilibrium distribution of radioactively labelled amphiphilic drugs between a water phase and liposomes was determined. Liposomes were prepared from sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS); the extent of binding of the drugs occurred in increasing order SM ～ PC < PE < PS. A strong correlation was found between the octanol-water partition coefficient and the PC-water coefficient for monovalent cationic drugs; the absolute values of both partition coefficients resembled each other very closely, suggesting that the hydrophobic forces are mainly responsible for the binding of amphiphilic drugs to PC. The higher extent of binding to PS does not result from higher afiinities of the drugs to PS but rather from the higher capacity of PS-liposomes as compared with that of PC- or SM-liposomes. The divalent cationic drug chloroquine displayed particularly high binding to the anionic lipids PS and to gangliosides as compared with the monovalent drugs. This observation might help to explain the ultrastructural and biochemical findings that chloroquine induces a remarkable storage of anionic lipids upon chronic treatment of animals.     

Interaction of streptomycin and streptomycylamine derivatives with negatively charged lipid layers. Correlation between binding, conformation of complexes and inhibition of lysosomal phospholipase activities

Aminoglycoside antibiotics induce a lysosomal phospholipidosis in kidney proximal tubules after conventional therapy in animals and man. We have previously demonstrated that these drugs bind to negatively charged phospholipid bilayers at acid pH and inhibit the activity of lysosomal acid phospholipases in vitro and in vivo. A combined biochemical and conformational study [Brasseur et al., Biochem. Pharmac. 33, 629 (1984)] showed major and consistent differences between 6 aminoglycosides in current clinical use with respect to the stability of the complexes they form with phosphatidylinositol, their inhibitory potency towards the activity of lysosomal phospholipases and their current toxicity ranking (e.g. gentamicin greater than amikacin greater than streptomycin). In the present study we have extended this approach to experimental derivatives of streptomycin. The derivatives examined were: dihydrostreptomycin, dideguanyldihydrostreptomycin, streptomycylamine, dideguanylstreptomycylamine, N-butyl- and N-benzyl-dideguanylstreptomycylamine. These compounds were examined for (i) their binding to negatively charged liposomes, measured by gel permeation on Sepharose 4B; (ii) their interactions with phosphatidylinositol assessed by semi-empirical conformational analysis and (iii) their inhibitory effect on the activities of lysosomal phospholipases towards phosphatidylcholine present in negatively charged liposomes. Streptomycin and gentamicin were also used as reference compounds with low and high affinity (and inhibitory potency), respectively. Our observations can be summarized as follows: (i) the replacement of the aldehyde in the streptose ring by a methylamino group strikingly changes the conformation of the molecule, allowing a better interaction with phosphatidylinositol. Thus, streptomycylamine binds much more tightly to phospholipid bilayers and shows a higher inhibitory potency towards phospholipase activity, as compared to streptomycin. The conformational analysis shows, however, that this effect is only partially due to the additional cationic charge carried by streptomycylamine. Other modifications of the streptomycin molecule, such as the replacement of the guanidinium groups by aminogroups or the addition of hydrophobic moieties (butyl or benzyl groups) to the streptose do not markedly further strengthen the interactions of the molecule with phosphatidylinositol. (ii) Even though some derivatives (e.g. dideguanylstreptomycylamine) bind as tightly to phospholipids as gentamicin, they remain much less inhibitory towards lysosomal phospholipases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of purified lysosomal phospholipase A1 by beta-adrenoceptor blockers

Inhibition of rat liver lysosomal phospholipases is one of the main events that leads to accumulation of tissue phospholipids during drug-induced phospholipidosis. Drug inhibition of lysosomal phospholipase A may occur by direct effects of drugs on the enzyme (or substrate) or by drug-induced increases in intralysosomal pH. Although beta-adrenoceptor blockers have not been reported to cause lipid storage, they do inhibit lysosomal phospholipase A. To investigate the structural requirements for drug inhibition, we studied the effects of six beta-adrenoceptor blockers on purified rat liver lysosomal phospholipase A1. The agents studied include: propranolol, timolol, metoprolol, practolol, atenolol and the combined alpha and beta adrenoceptor blocking agent, labetalol. The drugs varied by two logs in their abilities to inhibit phospholipase A1 activity. The relative inhibitory potencies were propranolol greater than labetalol much greater than timolol greater than metoprolol much greater than practolol greater than atenolol. Our studies identify drug hydrophobicity as a key determinant for phospholipase A1 inhibition. A strong negative correlation was noted between the octanol/water partition coefficients and IC50 for phospholipase inhibition (r = -0.91). The ability of propranolol to inhibit phospholipase A1 was identical for the d, l and the d and l stereoisomers.     

Effect of calcium-channel-blocking drugs on lysosomal function in human skin fibroblasts

Recent reports suggest that certain Ca²⁺-channel-blocking drugs reduce the severity of atherosclerosis in cholesterol-fed animals. To determine whether the suppression of atherogenesis is related to altered lipoprotein metabolism, we have assessed the effects of these drugs on the catabolism of plasma low density lipoproteins (LDL) by human skin fibroblasts. The Ca²⁺-channel-blocking drugs verapamil and diltiazem inhibit the lysosomal degradation of LDL by these cells; degradation of epidermal growth factor was also inhibited by the same drugs, suggesting a general effect of these drugs on lysosomal function. In contrast, nifedipine did not affect the degradation of LDL or epidermal growth factor. None of the drugs affected phospholipid or protein synthesis. Entry of LDL into the lysosomes also was not affected. [³H]Diltiazem, which inhibited LDL degradation, accumulated in the lysosome-rich fraction, whereas [³H]nimodipine, a drug structurally and functionally similar to nifedipine, did not accumulate. We suggest that the inhibitory effect of some of the Ca²⁺-channel-blocking drugs on lysosomal function is due to their basic nature, causing them to accumulate in lysosomes, thereby increasing intralysosomal pH.     

Induction of Apoptosis by Cationic Amphiphilic Drugs Amiodarone and Imipramine

Phospholipidosis is the excessive accumulation of intracellular phospholipids in cell lysosomes. Drugs that induce this disease often share common physiochemical properties and are collectively classified as cationic amphiphilic drugs (CADs). Although the cause of phospholipidosis and morphologic appearance of affected lysosomes have been studied extensively, less is known about the physiologic effects of the condition. In the current study, U-937 cells were incubated with the CADs amiodarone (2.5–10 µg/mL) and imipramine (2.5–20 µg/mL). Treatment of U-937 cells with these compounds for 96 h resulted in concentration-related increases in phospholipids, as assessed by flow cytometry using the fluorophore nile red. These results were verified by measuring the concentrations of choline-derived phospholipids, which were significantly increased in drug-treated cells. Cell number in amiodarone (10 µg/mL) and imipramine (20 µg/mL) cultures following the 96-h incubation period were markedly reduced compared to control cultures. These observations suggested that accumulation of cellular phospholipids could inhibit cell proliferation. Flow cytometric analysis revealed a decrease in the percentage of cells in the S-phase of the cell cycle following drug treatment, yet DNA replication still occurred in a significant portion of cells. Interestingly, amiodarone and imipramine induced apoptosis in U-937 cells as shown by annexin V-FITC staining and DNA fragmentation. Enzymatic assays demonstrated that amiodarone and imipramine induced the activity of caspases 2 and 3. These results suggest that disruption of cell lysosomes in U-937 cells followingaccumulation of phospholipids does not cause a cell cycle arrest but instead induces apoptosis by activation of caspase pathways.     

The antimalarials quinacrine and chloroquine induce weak lysosomal storage of sulphated glycosaminoglycans in cell culture and in vivo

The antimalarial agents quinacrine and chloroquine are well known as potent inducers of lysosomal storage of polar lipids (lipidosis) in cell culture and in vivo. In previous experiments on cultured fibroblasts, chloroquine was shown to additionally cause weak lysosomal storage of sulphated glycosaminoglycans (GAGs) thus inducing mucopolysaccharidosis (MPS). In the present study, quinacrine was investigated for this ability, because we wished to know whether or not the acridine ring system in quinacrine would enhance the M PS-inducing potency as compared to chloroquine carrying an isoquinoline ring system. Tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one) known as a potent inducer of MPS served as reference compound. The compounds were compared at a concentration (3 μM) which did not enhance the secretion of the lysosomal enzyme β-hexosaminidase (E.C. 3.2.1.52), since this would be an indication of unspecific drug effects upon the endosomal/lysosomal compartments of the cell. Additionally the liver of quinacrine- and chloroquine-treated rats was examined with the question whether the lysosomal GAG storage induced by either drug in cell culture had an equivalent in intact organisms. Both, in cell culture and in vivo, quinacrine was found to be a more potent inducer of lysosomal GAG storage than was chloroquine. The results suggest that the acridine ring system favours this drug side effect as compared with the bicyclic isoquinoline ring system. On the other hand, quinacrine was significantly less potent than tilorone and the symmetrically substituted acridine derivative 3,6-bis[2-(diethylamino)ethoxy]acridine investigated previously. This suggests that the asymmetric structure of the quinacrine molecule reduces the potency as compared to the symmetrically substituted bisbasic compounds with planary tricyclic ring systems such as tilorone and congeners.     

Effects of the schistosomicide 1,7-bis(p-aminophenoxy)heptane (153C51) on lysosomes and membrane stability

The schistosomicide 1,7-bis(p-aminophenoxy)heptane (153C51) stabilised mouse erythrocytes and rat liver lysosomes against osmotic lysis. Chloroquine was less potent in either system in terms of the maximum stabilisation achieved or the concentration needed to produce the maximum effect. Two lysosomal enzymes, acid phosphatase and β-acetylglucosaminidase, were partially inhibited by 153C51. Binding of [³H]-153C51 by rat liver lysosomes was directly proportional to the drug concentration. The results are discussed in relation to the amphiphilic, cationic properties of the drug molecule and the effect of 153C51 on lysosomes in the tegument of the blood fluke Schistosoma mansoni.