Studies on the mechanism of phospholipid storage induced by amantadine and chloroquine in Madin Darby canine kidney cells

Previous studies suggested that hepatic lipidosis caused by cationic amphiphilic drugs in rats is related to the capacity of these drugs to concentrate in liver lysosomes. These drugs inhibit lysosomal phospholipases, causing phospholipid accumulation. Amantadine, an inhibitor of influenza A virus replication, is a cationic amphiphilic drug which concentrates in the lysosomes of the Madin Darby canine kidney (MDCK) cell. In the present study, amantadine and chloroquine were shown to inhibit soluble lysosomal phospholipases isolated from MDCK cell in. vitro. Both amantadine and chloroquine concentrated in MDCK cell lysosomes. These drugs caused phospholipid storage in cultured MDCK cells, and the amounts of the respective agents required to cause phospholipid storage correlated with the capacities of the agents to inhibit lysosomal phospholipases. The mechanisms involved in this phenomenon are discussed, and a three-step hypothesis is presented predicting which agents will cause phospholipidosis.     

Localization of d-tubocurarine in rat liver lysosomes

Summary In the rat d-tubocurarine is taken up by the liver and excreted in bile. A fraction of the drug is taken up very rapidly by lysosomes. This lysosomal localization of the drug was demonstrated by purification of Triton WR 1339 loaded lysosomes (tritosomes) on a sucrose density gradient by flotation; ³H-labeled d-tubocurarine was accumulated in the same fractions as acid phosphatase activity. Lysosome-bound d-tubocurarine is not available for biliary excretion and remains in the lysosomes even when the cytosolic concentration decreases to very low levels. The biliary excretion rate was linearly related to the amount of d-tubocurarine present in the cytosol. Lysosomal uptake of d-tubocurarine was decreased or prevented by the basic drug quinacrine in vivo. The lysosomal storage of d-tubocurarine is discussed in relation to its relevance for the clinical use of this and related drugs.     

Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics I. Differential centrifugation fractionation B16F10 cells and use to study the intracellular fate of HPMA copolymer - doxorubicin

Polymer therapeutics are being designed for lysosomotropic, endosomotropic and transcellular drug delivery. Their appropriate intracellular routing is thus crucial for successful use. For example, polymer-anticancer drug conjugates susceptible to lysosomal enzyme degradation will never deliver their drug payload unless they encounter the appropriate activating enzymes. Many studies use confocal microscopy to monitor intracellular fate, but there is a pressing need for more quantitative methods able to define intracellular compartmentation over time. Only then will it be possible to optimise the next generation of polymer therapeutics for specific applications. The aim of this study was to establish a subcellular fractionation method for B16F10 murine melanoma cells and subsequently to use it to define the intracellular trafficking of N-(2-hydroxyproplylmethacrylamide) (HPMA) copolymer-bound doxorubicin (PK1). Free doxorubicin was used as a reference. The cell cracker method was used to achieve cell breakage and optimised to reproducibly achieve approximately 90% breakage efficiency. This ensured that subsequent subcellular fractionation experiments were representative for the whole cell population. To characterise the subcellular fractions obtained by differential centrifugation, DNA (nuclei), succinate dehydrogenase (mitochondria), N-acetyl-beta-glucosaminidase (lysosomes), alkaline phosphatase (plasma membrane) and lactate dehydrogenase (cytosol) were selected as markers and their assay was carefully validated. The relative specific activity (RSA) of the fractions obtained from B16F10 cells were: nuclei (2.2), mitochondria (4.1), lysosomes (3.7) and cytosol (2.5). When used to study the intracellular distribution at non-toxic concentrations of PK1 and doxorubicin, time-dependent accumulation of PK1 in lysosomes was evident and the expected nuclear localisation of free doxorubicin was seen. Live cell fluorescence microscopy and confocal co-localisation studies gave qualitative corroboration of these results, but by using this method, we were unable to accurately define organelle localisation. In conclusion, the B16F10 subcellular fractionation method developed here provides a useful tool to allow comparison of the intracellular trafficking of other polymer conjugates.     

The potential role of lysosomes in tissue distribution of weak bases

The potential importance of lysosomes as a site of accumulation of weak bases in tissues is discussed. A simple mathematical treatment predicts the quantitative significance of lysosomal trapping for monoacidic and diacidic weak bases. The features which are characteristics of lysosomal trapping are discussed, particularly in comparison with active transport and intracellular binding mechanisms. These features include: linear accumulation at low concentrations; nonlinearity at higher concentrations; dependence on structural integrity of tissue; energy dependence and competition with other weak bases. Subcellular distribution studies have previously shown that weak bases accumulate extensively in membranes; however, the dependence of accumulation on the structural integrity of tissue suggests that this is not the only significant mechanism of accumulation. The results of a range of studies of tissue distribution of weak bases are discussed to illustrate that these findings are consistent with accumulation in lung and liver being attributable to a combination of lysosomal trapping and accumulation in membranes whereas, in muscle, accumulation in membranes is the predominant mechanism of accumulation. The possible pharmacokinetic significance of lysosomal trapping of weak bases is also discussed.     

Lysosomal targeting strategies for design and delivery of bioactive for therapeutic interventions

Lysosomes are of particular interest for the design and delivery of pH-dependent pro-drugs, enhancing selectivity and developing strategies to inhibit drug degradation inside the cells. There is great potential to bring intracellular drug delivery and distribution using nanotherapeutic approaches to target lysosomes for therapeutic interventions. Lysosomal targeting strategies involve two contrasting facets. One aspect is to directly target therapeutics to the lysosome through receptor-mediated endocytosis and the other facet involves strategies, which ensure escape from the lysosome in order to prevent their degradation, so that therapeutics may remain intact and available in the cytosol for their further action. It provides a unique opportunity to explore novel treatment strategies and design future drugs for the effective treatment of lysosome-related diseases especially lysosomal storage disorders (LSD), cancer, inflammatory, neurodegenerative conditions (Parkinson's, Alzheimer's and Huntington's diseases) and autoimmune diseases. In this review, we illustrate the fundamentals of membrane trafficking, subcellular organisation, strategies to target lysosomes and its implications for the advance design of efficient drug delivery vectors for safe and effective therapies.     

Drug-drug interactions involving lysosomes: mechanisms and potential clinical implications

INTRODUCTION: Many commercially available, weakly basic drugs have been shown to be lysosomotropic, meaning they are subject to extensive sequestration in lysosomes through an ion trapping-type mechanism. The extent of lysosomal trapping of a drug is an important therapeutic consideration because it can influence both activity and pharmacokinetic disposition. The administration of certain drugs can alter lysosomes such that their accumulation capacity for co-administered and/or secondarily administered drugs is altered. AREAS COVERED: In this review the authors explore what is known regarding the mechanistic basis for drug-drug interactions involving lysosomes. Specifically, the authors address the influence of drugs on lysosomal pH, volume and lipid processing. EXPERT OPINION: Many drugs are known to extensively accumulate in lysosomes and significantly alter their structure and function; however, the therapeutic and toxicological implications of this remain controversial. The authors propose that drug-drug interactions involving lysosomes represent an important potential source of variability in drug activity and pharmacokinetics. Most evaluations of drug-drug interactions involving lysosomes have been performed in cultured cells and isolated tissues. More comprehensive in vivo evaluations are needed to fully explore the impact of this drug-drug interaction pathway on therapeutic outcomes.     

Ethambutol-induced toxicity is mediated by zinc and lysosomal membrane permeabilization in cultured retinal cells

Ethambutol, an efficacious antituberculosis agent, can cause irreversible visual loss in a small but significant fraction of patients. However, the mechanism of ocular toxicity remains to be established. We previously reported that ethambutol caused severe vacuole formation in cultured retinal cells, and that the addition of zinc along with ethambutol aggravated vacuole formation whereas addition of the cell-permeable zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), reduced vacuole formation. To investigate the origin of vacuoles and to obtain an understanding of drug toxicity, we used cultured primary retinal cells from newborn Sprague-Dawley rats and imaged ethambutol-treated cells stained with FluoZin-3, zinc-specific fluorescent dye, under a confocal microscope. Almost all ethambutol-induced vacuoles contained high levels of labile zinc. Double staining with LysoTracker or MitoTracker revealed that almost all zinc-containing vacuoles were lysosomes and not mitochondria. Intracellular zinc chelation with TPEN markedly blocked both vacuole formation and zinc accumulation in the vacuole. Immunocytochemistry with antibodies to lysosomal-associated membrane protein-2 (LAMP-2) and cathepsin D, an acid lysosomal hydrolase, disclosed lysosomal activation after exposure to ethambutol. Immunoblotting after 12 h exposure to ethambutol showed that cathepsin D was released into the cytosol. In addition, cathepsin inhibitors attenuated retinal cell toxicity induced by ethambutol. This is consistent with characteristics of lysosomal membrane permeabilization (LMP). TPEN also inhibited both lysosomal activation and LMP. Thus, accumulation of zinc in lysosomes, and eventual LMP, may be a key mechanism of ethambutol-induced retinal cell death.     

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.     

Transport of anthracyclines and mitoxantrone across membranes by a flip-flop mechanism

The objectives of the present work are to characterize the transport of mitoxantrone and three anthracyclines in terms of binding to the membrane surface, flip-flop across the lipid core of the membrane, and release into the medium. Mitoxantrone and anthracyclines are positively charged amphipathic molecules, and as such are located at the surface of membranes among the headgroups of the phospholipids. Therefore, their transport across membranes occurs by a flip-flop mechanism, rather than by diffusion down a continuous concentration gradient located in the lipid core of the membrane. Flip-flop rates have been estimated with liposomes labeled at their surface with 7-nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD) moiety attached to the headgroup of phosphatidylethanolamine. Flip-flop of mitoxantrone, doxorubicin, daunorubicin, and idarubicin occurred with half-lives of 6, 0.7, 0.15, and 0.1min, respectively. Partition of the drugs into the membrane occurred with lipid phase/aqueous medium coefficients of 230,000, 8600, 23,000, and 40,000 for mitoxantrone, doxorubicin, daunorubicin, and idarubicin, respectively, which are much higher than their corresponding octanol/aqueous medium values. There was no direct correlation between the lipophilicity of the drugs and their lipid phase/aqueous medium partition coefficient or their flip-flop rate. Mitoxantrone exhibited the highest affinity toward liposome membranes, but the slowest flip-flop across the lipid core of the membranes. Simulation of drug uptake into liposomes revealed that transmembrane movement of the mitoxantrone and anthracyclines is determined by their flip-flop rate and affinity toward membranes.     

GTP gamma S-stimulated lysosomal lysis dependent on the assembly of adaptor protein on lysosome

Cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) destroys the dextran-loaded lysosomes (J. Biochem., 123, 637 (1998)). The transfer of the ADP-ribosylation factor (ARF) from the cytosol to the lysosomal membrane is necessary for this lysis to occur. The role of ARF in the biogenesis of the Golgi complex is to generate high-affinity membrane-binding sites for the heterotetrameric adaptor protein complex in Golgi membranes. We have found that ARF also recruits the adaptor protein to lysosomes. The recruitment of protein coats for vesicles is necessary for the GTPgammaS-stimulated lysis of lysosomes. The GTPgammaS-induced lysis proceeded via a process similar to that for the assembly of coated proteins to coated vesicles, which serve to transport proteins between intracellular organelles.     

Cationic amphiphilic drugs cause a marked expansion of apparent lysosomal volume: implications for an intracellular distribution-based drug interaction

How a drug distributes within highly compartmentalized mammalian cells can affect both the activity and pharmacokinetic behavior. Many commercially available drugs are considered to be lysosomotropic, meaning they are extensively sequestered in lysosomes by an ion trapping-type mechanism. Lysosomotropic drugs typically have a very large apparent volume of distribution and a prolonged half-life in vivo, despite minimal association with adipose tissue. In this report we tested the prediction that the accumulation of one drug (perpetrator) in lysosomes could influence the accumulation of a secondarily administered one (victim), resulting in an intracellular distribution-based drug interaction. To test this hypothesis cells were exposed to nine different hydrophobic amine-containing drugs, which included imipramine, chlorpromazine and amiodarone, at a 10 μM concentration for 24 to 48 h. After exposure to the perpetrators the cellular accumulation of LysoTracker Red (LTR), a model lysosomotropic probe, was evaluated both quantitatively and microscopically. We found that all of the tested perpetrators caused a significant increase in the cellular accumulation of LTR. Exposure of cells to imipramine caused an increase in the cellular accumulation of other lysosomotropic probes and drugs including LyosTracker Green, daunorubicin, propranolol and methylamine; however, imipramine did not alter the cellular accumulation of non-lysosomotropic amine-containing molecules including MitoTracker Red and sulforhodamine 101. In studies using ionophores to abolish intracellular pH gradients we were able to resolve ion trapping-based cellular accumulation from residual pH-gradient independent accumulation. Results from these evaluations in conjunction with lysosomal pH measurements enabled us to estimate the relative aqueous volume of lysosomes of cells before and after imipramine treatment. Our results suggest that imipramine exposure caused a 4-fold expansion in the lysosomal volume, which provides the basis for the observed drug interaction. The imipramine-induced lysosomal volume expansion was shown to be both time- and temperature-dependent and reversed by exposing cells to hydroxypropyl-β-cyclodextrin, which reduced lysosomal cholesterol burden. This suggests that the expansion of lysosomal volume occurs secondary to perpetrator-induced elevations in lysosomal cholesterol content. In support of this claim, the cellular accumulation of LTR was shown to be higher in cells isolated from patients with Niemann-Pick type C disease, which are known to hyperaccumulate cholesterol in lysosomes.     

Inhibition by vitamin E of drug accumulation and of phospholipidosis induced by desipramine and other cationic amphiphilic drugs in human cultured cells.

1. Cationic amphiphilic drugs (CADs) are widely used in chronic pharmacotherapies in spite of frequently observed side effects connected with lysosomal phospholipid (PL) storage. 2. It has recently been shown that alpha-tocopherol (alpha-Toc) inhibits drug- and PL accumulation in cell cultures chronically exposed to the CAD, amiodarone. 3. The mechanisms of alpha-Toc action on drug kinetics and PL storage were studied in human cultured fibroblasts exposed to single and repetitive doses of desipramine and other CADs. 4. alpha-Toc did not influence the initial, pH-dependent rapid phase of drug uptake. It inhibited, in a dose-dependent manner, the slow and the cumulative phases of drug uptake and coincidently the accumulation of cellular PLs. 5. The inhibitory effects of alpha-Toc on CAD and PL accumulations depends on the ratio between CAD and alpha-Toc concentrations in the medium. This points to competition between alpha-Toc and CADs for PL complex formation. 6. Effectiveness of alpha-Toc on drug uptake varies among different CADs. It depends on its structural integrity but is independent of stereoisomerism. The inhibitory action is restricted to the piggyback slow drug uptake and therefore related to the proportion of membrane-mediated transport to permeation into lysosomes (rapid uptake). This proportion differs among CADs. 7. alpha-Toc prevents lysosomal membrane-PL storage, accelerates disintegration of PL-stores and normalizes drug-related increased membrane fluidity. This strongly suggests that alpha-Toc restores membrane recycling, impaired by CAD exposure. 8. It remains to be tested in vivo whether alpha-Toc reduces CAD side effects without interfering with drug effectiveness.     

Lysosomal trapping as an important mechanism involved in the cellular distribution of perazine and in pharmacokinetic interaction with antidepressants

Perazine, a piperazine-type phenothiazine neuroleptic, is the most frequently chosen drug for combination with antidepressants in the therapy of complex or ‘treatment-resistant’ psychiatric illnesses. The aim of the present study was to investigate the contribution of lysosomal trapping to the total tissue uptake of perazine, and the pharmacokinetic interaction between the neuroleptic and antidepressants. Experiments were carried out on slices of different rat organs regarded as a system with functional lysosomes. To distinguish between lysosomal trapping and tissue binding, the experiments were performed in the absence or presence of ‘lysosomal inhibitors’, i.e. the lysosomotropic compound ammonium chloride or [H⁺] ionophore monensin, which abolish the pH-gradient of lysosomes. Under steady-state conditions, the highest tissue uptake of perazine was observed for the adipose tissue, which descended in the following order: the adipose tissue>lungs>liver>heart=brain>kidneys>muscles. The contribution of lysosomal trapping to the total tissue uptake amounted to about 40% in the liver, brain and muscles, to 30% in the kidneys, and to 25% in the heart and lungs. In the adipose tissue, no lysosomotropism of perazine was observed. Of the psychotropics studied, perazine was the only drug showing such a high degree of lysosomal trapping in muscles and distinct lysosomotropic properties in the heart. Perazine and the antidepressants used, both tricyclic (imipramine, amitriptyline) and selective serotonin reuptake inhibitors (fluoxetine, sertraline), mutually decreased their tissue uptake. The potency of imipramine to decrease perazine uptake was similar to that of the ‘lysosomal inhibitors’. Other antidepressants seemed to exert a somewhat weaker effect. The above interactions between perazine and antidepressants were not observed in the presence of ammonium chloride, which indicates that they proceeded at the level of lysosomal trapping. The adipose tissue in which the drug uptake was not affected by the ‘lysosomal inhibitors’ was not the site of such an interaction. Ammonium chloride did not affect the drug metabolism in liver slices; other tissues displayed only a negligible biotransformation of the psychotropics studied. A parallel metabolic interaction between perazine and tricyclic antidepressants took part in liver slices (i.e. perazine and antidepressants mutually inhibited their metabolic pathways), but the influence of such an interaction on the lysosomal uptake of the parent compounds in liver slices did not seem to be great. A substantial decrease in concentrations of the drugs in lysosomes (depot form) observed in vitro may lead to an increase in the concentration in vivo of the neuroleptic and antidepressants at the site of action, which, in turn, may increase the risk of cardiotoxic and anticholinergic side-effects of tricyclic antidepressants and sedative and extrapyramidal effects of the neuroleptic.     

Modified lipophilic vitamin--IV. Stabilizing effect of -tocopherol and tocopheronolactone on mouse liver lysosomes in vivo and in vitro

The effect of α-tocopherol and tocopheronolactone on the stability of mouse liver lysosomal membrane in vitro and in vivo was examined by measurement of the enzyme activity released from lysosomes. Tocopheronolactone is a stronger inhibitor of the release of acid phosphatase from lysosomes in vitro than is α-tocopherol, but when given in vivo, α-tocopherol was as effective as tocopheronolactone in stabilizing the lysosomes. There was no clear-cut structure-activity correlation with α-tocopherol and its derivatives, tocopheronolactone, α-tocopherylquinone, and 6-hydroxy-2-carboxyethyl- 2,5,7,8-tetramethylchroman, with respect to stabilization of lysosomal membrane. Examination of the variation of potency with the amount given indicated that tocopheronolactone showed a stronger stabilization effect with increasing concentration, whereas α-tocopherol showed a biphasic effect, effecting stabilization at a low concentration of 10⁻⁶ to 10⁻⁴ M but labilization at a higher concentration of 5 × 10⁻⁴ M. Acid phosphatase and β-glucuronidase, which were derived similarly from the lysosomes, differed in the degree of inhibition of their release by tocopheronolactone. This fact seems to suggest that these two enzymes are present in different lysosomal particles, in different parts of the same particle, or are activated by different mechanism.     

Contribution of Lysosomal Trapping to the Total Tissue Uptake of Psychotropic Drugs

Abstract: The present study was aimed at assessing individual contributions of the phospholipid binding and lysosomal trapping to the total tissue uptake of psychotropic drugs with different chemical structures, such as promazine, imipramine, amitriptyline, fluoxetine, sertraline (basic lipophilic drugs) and carbamazepine (lipophilic, but not basic). We also tried to find out whether lysosomal trapping may be involved in the pharmacokinetic interactions in clinical combinations of psychotropics. Uptake experiments were carried out on slices of various rat tissues as a system with intact lysosomes. Initial concentration of each drug was 5 μM. The results were compared with those obtained in the presence of the "lysosomal inhibitors", ammonium chloride or monensin. The basic lipophilic psychotropics showed high uptake in tissues known for the abundance of lysosomes, mainly the lungs. The highest drug accumulation was found for promazine and amitriptyline. "Lysosomal inhibitors" significantly decreased the uptake of the basic lipophilic drugs, particularly in the lungs and liver. The most potent effect was observed for amitriptyline, imipramine and promazine. The brain showed moderate accumulation of basic lipophilic psychotropics and the effect of the "lysosomal inhibitors" was significant only in the case of amitriptyline, imipramine and sertraline. The only exception to the above regularity were imipramine and sertraline which were taken up more extensively by the adipose tissue than by lysosome-rich tissues such as the lungs or liver. Carbamazepine did not show lysosomotropism. Amitriptyline and promazine mutually decreased their uptake by lung slices when the drugs were incubated jointly. In the presence of ammonium chloride the interaction did not occur. In conclusion, the obtained results show that (1) the lysosomal trapping is an important factor determining the distribution of the basic lipophilic psychotropics; however (2) their tissue uptake depends more on the phospholipid binding than on the lysosomal trapping; (3) the lysosomal trapping may be involved in the pharmacokinetic interactions between psychotropics.     

The role of lysosomes in the cellular distribution of thioridazine and potential drug interactions.

The purpose of the present study was to investigate the contribution of lysosomal trapping to the total tissue uptake of thioridazine and to potential drug distribution interactions between thioridazine and tricyclic antidepressants (imipramine, amitriptyline) or selective serotonin reuptake inhibitors (SSRIs; fluoxetine, sertraline). The experiment was carried out on slices of various rat tissues as a system with intact lysosomes. Thioridazine and antidepressants (5 microM) were incubated separately or jointly with the tissue slices in the absence or presence of "lysosomal inhibitors," i.e., ammonium chloride or monensin. The results show that the contribution of lysosomal trapping to the total tissue uptake of thioridazine is as important as phospholipid binding. A high degree of dependence of thioridazine tissue uptake on the lysosomal trapping is the cause of substantial distributive interactions between thioridazine and the investigated antidepressants at the level of cellular distribution. Thioridazine and the antidepressants, both tricyclic and SSRIs, mutually decreased their tissue uptake. The potency of antidepressants to decrease thioridazine uptake was similar to that of lysosomal inhibitors. In general, the observed interactions between thioridazine and antidepressants occurred only in those tissues in which thioridazine showed lysosomotropism (the lungs, liver, kidneys, brain, and muscles) but were not observed in the presence of ammonium chloride. The above finding provides evidence that the interactions proceeded at the level of lysosomal trapping. In the adipose tissue and heart no lysosomal trapping of thioridazine was detected and those tissues were not the site of such an interaction. Since the organs and tissues involved in the distributive interactions constitute a major part of the organism and take up most of the total drug in the body, the interactions occurring in them may cause a substantial shift of the drugs to organs and tissues poor in lysosomes, e.g. the heart and muscles. An in vivo study into the thioridazine-imipramine interaction showed that joint administration of the drugs under study (10 mg/kg ip) increased drug concentration ratios of lysosome-poor tissue/plasma and lysosome-poor/lysosome-rich tissue. Considering serious side effects of thioridazine and tricyclic antidepressants (cardiotoxicity, anticholinergic activity), the thioridazine-antidepressant combinations studied should be approached with respect to the appropriate dose adjustment.     

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.     

The uptake and subcellular distribution of gold in rat liver cells after in vivo administration of sodium aurothiomalate.

Using the techniques of differential centrifugation, sucrose sedimentation-gradient and isopycnic-gradient centrifugation and gel chromatography through Sephadex G 75 and G 200, the uptake and subcellular distribution of gold in rat liver has been studied over a period of 1 hr to 36 days after intraperitoneal administration of 0.7 mg kg^?1 to 80 mg kg^?1 sodium aurothiomalate (“Myocrisin”). After 1 hr, gold concentrated in the lysosomes of liver cells and, under certain conditions, it was estimated that probably more than 90 per cent of the cytoplasmic gold was associated with these organelles. After longer time intervals partial redistribution of the gold took place, possibly due to sequestration into telolysosomes. The majority of the lysosomal gold appeared to be membrane-bound and increased with increasing aurothiomalate dose and time after injection.The distribution of gold among the proteins of the cytosol and the lysed granule-fraction supernatant has been investigated and compared with that in the plasma. Whereas in the latter more than 87 per cent of the gold was bound to albumin, the gold in the lysosomal supernatant was bound to molecules over a wide range of molecular weights, and that in the cytosol appeared to be bound to at least three macromolecules, one ~ 300,000, one ~ 40,000 (possibly ligandin) and one ~ 10,000. In addition 10–15 per cent of the cytosol gold appeared to be a much lower molecular weight species (less than 3,000), which was not found in the lysosomal supernatant or the plasma.     

Effects of vasoactive drugs on lysosomal stability in vitro

The effects of methylprednisolone, chlorpromazine, phenoxybenzamine and phentolamine on thermolabilized lysosomes were studied by determining the unsedimentable, fragile lysosomal and total lysosomal activities of β-glucuronidase and acid phosphatase in centrifuged fractions. At concentrations of 10^?5-10^?3 M, methylprednisolone stabilized the lysosomes. Chlorpromazine and phenoxybenzamine at 10^?3 M had a labilizing effect. The changes in the enzyme activities due to phentolamine could not be explained as changes in membrane permeability. The glucocorticoids appear to protect the cell membranes directly and thus counteract the adverse effects of circulatory shock. The same mechanism may explain the salutary effects which chlorpromazine at low concentrations reportedly possesses. The improvement of microcirculation in shock after the administration of phenoxybenzamine or phentolamine is due to their α-adrenergic blocking activity; this in turn leads to vasodilatation and indirectly to a membrane protection.