Lysosomotropic Compounds Research Articles

Potentiometric detection of N, N′-diethylaminoethanol and lysosomotropic amino alcohols in cation exchange high-performance liquid chromatography systems

Potentiometric detection of N, N′-diethylaminoethanol (DEAE) and a series of physiologically relevant lysosomotropic amino alcohols was demonstrated in cation exchange HPLC. Poly (vinyl chloride) (PVC) based liquid membrane electrode coatings were used which contained the lipophilic cation exchanger tetrakis( p-chlorophenyl)borate (TCPB) either alone or in combination with one of the macrocyclic ionophores hexakis-(2,3,6-tri- O-octyl)-α-cyclodextrin (CDX), dibenzo-18-crown-6 (CRW) or calix[6]arene hexaethylacetate ester (CLX). For all PVC membrane electrodes used, improved sensitivity over UV detection at 210 nm was obtained for the analyzed aliphatic amino alcohols. Especially, addition of CLX to the PVC-based membrane leads to highly enhanced detection limits for the low molecular weight lysosomotropic amino compounds, i.e. aminoethanol and methylamine. The performance of the proposed potentiometric detection method was demonstrated in cation exchange HPLC modes employing two different aqueous mobile phase systems with pH ∗ 2.30 comprising phosphoric or acetic acid with acetonitrile as organic modifier. Detection limits of 1.1×10 −8 and 2.4×10 −8 M (injected concentrations) were achieved, respectively, for N-methylaminoethanol and hexylamine by using a CLX based electrode. Sensitive and reliable determination of DEAE, a local anaesthetic agent, after liquid–liquid or SPE extraction from fortified bovine serum samples was obtained with the HPLC method coupled with the proposed potentiometric detection system. A linear free-energy relationship (LFER) based on a general solvation equation was used to predict detection limits of amino alcohols from their molecular structure in the developed potentiometric detectors employing CLX and CRW.

Involvement of vacuolar proton ATPase in Junin virus multiplication.

The role of vacuolar-proton ATPase (V-H+ ATPAse) on Junin virus (JV) replication was evaluated by analyzing the effect of specific inhibitors of the enzyme activity on different steps of virus multiplication cycle. The presence of the macrolide antibiotics bafilomycin A1 and concanamycin A during the first two hours of infection caused a significant reduction of extracellular infectious virus production and viral protein expression in Vero and BHK-21 cells. The inhibitory action of the compounds was mainly exerted at an early stage of the JV multiplication cycle, without affecting virus attachment to the cell but preventing virus penetration. A correlation between the inhibitory action of the compounds on intracellular compartments acidification and the reduction of JV yield was observed. The addition of concanamycin A at different times after infection indicated that the compound also interferes with the release of infectious particles to the extracellular medium. Although, intracellular transport of JV glycoproteins to the cell membrane, seems not to be affected as revealed by immunofluorescence staining. The results confirm that JV enters into the cell through the endocytic pathway as previously suggested by using lysosomotropic compounds.

Influence of some lysosomotropic compounds on calcium ion desorption process from liposome membrane.

The effect of a group of model lysosomotropic compounds on the process of Ca2+ ion desorption from lecithin liposome membranes was studied. The compounds studied were: hydrochlorides of fatty acids 2-dimethylaminoethyl esters (DM-n) for n = 9, 11, 13 and 15 carbon atoms in the fatty acid alkyl chain and methochloride of 2-dimethylaminoethyl laurate (DMS-11). It was found that all the compounds studied caused increased desorption with increasing concentration of the compound. Most effective was the quaternary ammonium salt, DMS-11. Moreover, it was found that the process of Ca2+ desorption from the membrane depended on pH of the medium. Compound DM-11 was more active at pH 8 than at pH 5. The action of DM-n compounds depended on the alkyl chain length, DM-11 and DM-13 being the most active. Apparently free amines penetrate the phospholipid membranes and incorporate into its hydrophobic core causing structural deformations. Hydrochlorides of fatty acids and the quaternary ammonium salt induce desorption of calcium ions mostly as a result of competitive electrostatic interactions. By quantum chemistry, PM3 method, and methods of molecular modelling we established the higher hydrophilicity of the polar head of DM-n series with respect to the polar head of the DMS-n compounds. DM-n compounds possess both acceptor and donor properties for hydrogen bonding while DMS-n are instrumental as acceptors only. It should be noted, that the results obtained in this paper for model membranes are in accordance with those for biological ones.

"Soft" lysosomotropic compounds as new substrates of the yeast PDR network.

"Soft" lysosomotropic compounds as new substrates of the yeast PDR network.

Hemolytic activity of aminoethyl-dodecanoates.

The effect of new lysosomotropic compounds on red blood cell hemolysis and erythrocyte membrane fluidity has been investigated. In earlier studies it was shown that the compounds inhibit the growth of yeast and plasma membrane H(+)-ATPase activity. The study was performed with eight aminoethyl esters of lauric acid variously substituted at nitrogen atom. Esters of dodecanoic acid were chosen for study because at that chain length dimethylaminoethyl esters showed maximum activity. The hemolytic activity of the substances studied exhibits diversified activity in their interaction with the erythrocyte membrane: they differ in hemolytic activity and affect membrane fluidity differently. Erythrocyte membrane fluidity changes under the effect of those compounds which possess highest hemolytic activity. The hemolytic activity of the aminoesters investigated was found to follow a sequence that depended on basicity (i.e. ability of the protonated form formation) of the compound and its polar head group size. The most active are the compounds that possess not more than four carbon atoms substituted at nitrogen and highest pKa value.

Entry of Mouse Hepatitis Virus into Cells by Endosomal and Nonendosomal Pathways

OBLV60 is an acid-dependent syncytium-forming variant isolated from OBL21 cells persistently infected with the pH-independent mouse hepatitis virus (MHV)-4 strain. The fusion activity of OBLV60 can be strictly regulated by controlling pH and thus provides the means to definitively examine the entry of MHV into cells by endosomal and nonendosomal pathways. Shortly after high multiplicity infection, both MHV-4 and OBLV60 were detected by electron microscopy in endosomal vesicles and were recovered from lysates of cells treated with proteinase K to remove extracellular virus. For OBLV60, but not MHV-4, exposure to lysosomotropic compounds early in infection prevented viral penetration and significantly reduced viral yields. These results suggested that both MHV-4 and OBLV60 utilized the endosomal route of entry into cells, but that MHV-4 did not require acidification of endosomal vesicles. Studies on the entry of virus through fusion at the cell surface were performed by briefly exposing surface-bound OBLV60 to a fusion-permissive pH under conditions that prevent endocytic entry. Acid treatment of surface-bound OBLV60 caused a significant increase in the yields of virus produced in cultures of fusion-sensitive Sac− or DBT cells, demonstrating entry of virus by fusion at the cell surface. No measurable increase in virus production was detected with acid treatment of OBLV60 bound to OBL21 cells, suggesting that entry at the cell surface does not occur in these cells, which are resistant to MHV-induced syncytia formation. These results raise interesting questions concerning how mechanisms of MHV entry influence the selection of fusion variants.

Lysosomotropic agents activate the capacity for calcium dependent pinocytosis in starvedAmoeba proteus: evidence for a mechanism involving phospholipase A2

Pinocytosis induced by Na+ was assayed by phase contrast microscopy in 8–12 days starvedAmoeba proteus. These cultures were inactive with respect to calcium-dependent Na+-induced pinocytosis, but treatment with amino acid methyl and ethyl esters increased their capacity for pinocytosis. Besides promoting pinocytosis these compounds also stimulated calcium-sensitive secretion of lysosomal enzymes from normal, 2–3 days starved, cells. Only uncharged 1-forms of the amino acid esters were effective. Also other lysosomotropic compounds including monodansylcadaverine, glycine-phenylalanine-2-naphthylamide, NH4Cl, and the ionophores monensin and A23187 activated starved cells. The effect of these agents (except A23187) was inhibited by the drug dantrolene suggesting that activation is a consequence of release of Ca2+ from intracellular stores. Several of the lysosomotropic agents also lost their activating effect in the presence of phospholipase A2 (PLA2) inhibitors. To investigate whether or not PLA2 activity in the cell culture could imitate the effect of the lysosomotropic agents, we incubated starved cells with snake venom PLA2s. These enzymes caused rapid, dantrolene-sensitive activation of the cells. Measurement of endogenous PLA2in ldquonormalrdquo cells revealed significant cellular activity but no significant secretion of the enzyme into the culture medium was observed. Together the studies with enzyme inhibitors and dantrolene suggest that the process by which lysosomotropic agents affect pinocytosis involves activation of PLA2 and release of Ca2+ from intracellular stores. (Less)

Pathway of rubella virus infectious entry into Vero cells.

The mechanism and the kinetics of rubella virus (RV) penetration into Vero cells were studied. By using pronase or acid treatment to inactivate virus which had adsorbed to cell membrane but had not been internalized, it was found that a period of 7 h was required in order for all of the adsorbed virus to enter the host cells. Lysosomotropic agents (monensin, methylamine, ammonium chloride and chloroquine) were used to study the mechanism by which RV penetrates host cells. Virus replication was inhibited if treatment of cells with these compounds was performed for at least 9 h after infection. However, if extracellular adsorbed virions were eliminated by acid treatment following removal of the lysosomotropic compounds, RV replication was completely inhibited by treatment with these drugs for any time period after adsorption. This indicated that the prolonged period of treatment with these compounds necessary to inhibit virus replication is due to the slow rate of RV internalization. None of the compounds had any effect on infection initiated by transfection of RV RNA, confirming that these drugs were exerting their inhibitory activity at penetration. The inhibition of RV replication by lysosomotropic compounds indicates that RV penetrates host cells by the endosomal pathway.

The entry of Junin virus into Vero cells.

SummaryThe entry mechanism of Junin virus (JV) into Vero cells was studied analyzing the effect of lysosomotropic compounds and acid pH on JV infection. Ammonium chloride, amantadine, chlorpheniramine and procaine inhibited JV production. The action of ammonium chloride was exerted at early times of infection. Virus internalization was inhibited and viral protein expression was not detected. When the extracellular medium was buffered at low pH, the ammonium chloride induced block on JV infection was overcome. Furthermore, JV was able to induce fusion of infected cells at pH 5.5 leading to polykaryoctye formation. Taken together, these results demonstrate that JV entry occurs through an endocytic mechanism requiring a low pH dependent membrane fusion.

Antileukemic activity of phenylalanine methyl ester (PME): a lysosomotropic peptide methyl ester.

The antileukemic activities of the lysosomotropic compounds, such as phenylalanine methyl ester (PME), have received little attention. In this study, a 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to investigate the antileukemic activity of PME. Leukemic specimens from untreated patients that contained greater than or equal to 75% blasts were used. Leukemic cells were treated with PME at 37 degrees C and 22 degrees C in concentrations ranging from 0.5 to 50 mM. Normal blood mononuclear cells served as controls. At both 37 degrees C and 22 degrees C, the recovery of normal peripheral blood cells was approximately 28% following incubation with 50 mM PME. At 37 degrees C, 50 mM PME caused greater than one log reduction of leukemic cells in 13/16 acute myelogenous leukemia (AML), 7/9 acute lymphocytic leukemia (ALL), and 8/8 in blast crisis of chronic myelogenous leukemia (CML-BC) specimens. PME had less activity at 22 degrees C than at 37 degrees C. PME was compared with 100 micrograms/ml 4-hydroperoxycyclophosphamide (4HC). In contrast to PME, 4HC was associated with a greater than one log reduction of leukemic cells in only 1/13 AML, 1/3 ALL and 0/6 CML-BC specimens. 4HC activity exceeded PME activity in only one case each of ALL and prolymphocytic leukemia (PLL). In a case of CD34+ B cell ALL, synergy of PME and 4HC was demonstrated. These studies indicate 1) PME has antileukemic activity and 2) 4HC has less antileukemic activity than PME.

A potent and specific immunotoxin for tumor cells expressing disialoganglioside GD2.

Monoclonal antibody 14G2a (anti-GD2) reacts with cell lines and tumor tissues of neuroectodermal origin that express disialoganglioside GD2. mAb 14G2a was coupled to the ribosome-inactivating plant toxin gelonin with the heterobifunctional cross-linking reagent N-succinimidyl-3(2-pyridyldithio)propionate. The activity of the immunotoxin was assessed by a cell-free translation assay that confirmed the presence of active gelonin coupled to 14G2a. Data from an enzyme-linked immunosorbent assay demonstrated the specificity and immunoreactivity of the 14G2a-gelonin immunotoxin, which was identical to that of native 14G2a. Assays for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) revealed that these functional properties of the native 14G2a antibody were also preserved in the 14G2a-gelonin immunotoxin. The gelonin-14G2a immunotoxin was directly cytotoxic to human melanoma (A375-M and AAB-527) cells and was 1000-fold more active than native gelonin in inhibiting the growth of human melanoma cells in vitro. The augmentation of tumor cell killing of 14G2a-gelonin immunotoxin was examined with several lysosomotropic compounds. Chloroquine and monensin, when combined with 14G2a-gelonin immunotoxin, augmented its cytotoxicity more than 10-fold. Biological response modifiers such as tumor necrosis factor alpha and interferon alpha and chemotherapeutic agents such as cisplatinum and N,N'-bis(2-chloroethyl)-N-nitrosourea (carmustine) augmented the cytotoxicity of 14G2a-gelonin 4- to 5-fold. The results of these studies suggest that 14G2a-gelonin may operate directly by both cytotoxic efforts and indirectly by mediating both ADCC and CDC activity against tumor cells; thus it may prove useful in the future for therapy of human neuroectodermal tumors.

Early events in arenavirus replication are sensitive to lysosomotropic compounds.

Lysosomotropic compounds (ammonium chloride, chloroquine, amantadine, monensin) effectively inhibited the replication of Pichinde, Mopeia, and Lassa viruses in BHK-21 and Vero cells. The inhibitory effect was dependent upon the time of drug addition and was most effective when the drugs were added 1 h before the viral adsorption. The drugs had no direct effect on the infectious viruses nor on adsorption of the arenaviruses. These results suggest that the arenaviruses enter cells by adsorptive endocytosis with the participation of acidic intracellular vesicules.

Effect of lysosomotropic compounds on early events in foot- and- mouth disease virus replication

The effect of three lysosomotropic compounds, chloroquine, monensin and NH 4C1, on the replication of foot-and-mouth disease virus (FMDV) type A 12 was studied. Viral replication was almost totally inhibited by 0.5 mM chloroquine, 50 μM monensin, or 25 mM NH 4Cl. Monensin and NH 4Cl affected replication when added either before or within the first hour of infection. Chloroquine, however, still inhibited viral replication when added up to 2.5 h after infection. Assays of binding of radiolabeled virus to cells showed that these compounds had no effect on viral adsorption. Neither monensin nor NH 4Cl had any significant effect on cellular protein synthesis, but there was no evidence of viral protein synthesis in cells infected in the presence of these compounds. In contrast, chloroquine inhibited both cellular and viral protein synthesis. Eclipse assays, performed in the presence of the compounds, showed that while chloroquine and NH 4Cl had little effect on cell-induced degradation of incoming virions to 12 S protein subunits, monensin inhibited this reaction. The replication of representative members of all seven serotypes of FMDV was inhibited by monensin although some types were less sensitive to the compound than others. These results are consistent with a model which postulates that viral eclipse is the result of acidification of endocytic vesicles which degrade entrapped virions to 12 S protein subunits resulting in the release of genome RNA.

Effects of various basic compounds on the degradation of formaldehyde-treated 125I-labeled bovine serum albumin in mouse liver lysosomes.

The effect of various basic compounds on degradation of formaldehyde-treated 125I-bovine serum albumin (denatured 125I-BSA) in livers of mice was studied in detail. Five basic compounds (BTTB, chloroquine, methylamine, ammonium chloride, and vinblastine) were tested. All the basic compounds inhibited the degradation of exogenous protein in lysosomes. When p-biphenylmethyl-(dl-tropyl-alpha-tropinium)bromide (BTTB) was used, the most effective inhibition was obtained at the concentration of 3.2 mM. Also, vinblastine, a well-known inhibitor of microtubular function, inhibited the degradation of an exogenous protein to the similar extent as that of chloroquine. The inhibition of protein degradation caused by BTTB closely related to the uptake of BTTB into lysosomes. It is supposed that BTTB accumulates in lysosomes and that it inhibits the hydrolytic enzyme by neutralizing intralysosomal pH. Furthermore, it is supposed that BTTB, a quaternary ammonium compound, becomes a useful tool in the study of protein degradation in lysosomes as well as the typically lysosomotropic compounds (chloroquine, ammonium chloride, and methylamine).

Responses of cultured cardiac myocytes to lysosomotropic compounds and methylated amino acids.

Cardiac myocytes whose lysosomes had been pre-labelled with acridine orange were exposed to either L-amino acid methyl esters (L-leucine or methionine methyl ester) or to 'lysosomotropic' weak bases (chloroquine, methylamine, and NH4Cl) for 1 h. Both types of interventions dilated lysosomes equally and inhibited proteolysis to varying degrees. The weak bases produced no apparent alterations in the acridine orange staining, whereas the methylated amino acids induced a marked redistribution of the fluorescent dye from lysosomes into the myoplasm, suggesting that they may have provoked a change in lysosomal membrane permeability. A brief exposure to weak bases failed to enhance acid proteinase secretion into the culture medium but apparently inactivated cellular cathepsin B activity. In contrast, methylated amino acids induced no alterations in acid proteinase activity or the cellular distribution of the two proteolytic enzymes. Lastly, weak bases markedly elevated intralysosomal pH as measured with fluorescein dextran, while only modest rises were observed after amino acid methyl ester treatment. The present observations imply that amino acid methyl esters represent a new class of reagents with actions distinctly different from those of chloroquine and NH4Cl, and they may provide a unique and valuable means of studying secondary lysosomal function in cell culture.

Processing of human growth hormone by rat hepatocytes in monolayer culture.

The fate of cell-bound [125I]iodo-human GH ([125I]iodo-hGH) was studied in monolayer cultures of hepatocytes from pregnant and nonpregnant rats. Both the binding of [125I]iodo-hGH and its degradation were significantly higher in cells from pregnant than from nonpregnant rats. The positive correlation between the number of binding sites for hGH and the amount of degradation of [125I]iodo-hGH suggests that degradation, at least in part, is a receptor-mediated process. Degradation as a time- and temperature-dependent process was impaired by lysosomotropic compounds such as chloroquine and NH4Cl in a dose-dependent manner. Dinitrophenol, N-ethyl-maleimide, and NaN3 were also effective in preventing degradation, suggesting that an energy-requiring process is involved in degradation of [125I]iodo-hGH. Cell-bound [125I]iodo-hGH became less dissociable as a function of time of association. Degradation of [125I]iodo-hGH accelerated the dissociation of cell-bound radioactivity. Gel-filtration experiments revealed that [125I]iodo-hGH is degraded to smaller molecular species, but only a small portion of the degradation products exists within the cells. These observations suggest that receptor-bound [125I]iodo-hGH is degraded by an internalization process; lysosomal enzymes are probably responsible for the degradation of [125I]iodo-hGH, and the degraded products are rapidly released into the extracellular space.

Antigen presentation by human monocytes: evidence for stimulant processing and requirement for interleukin 1.

We studied the role of stimulant processing and presentation and of IL 1 in monocyte-mediated activation of human lymphoproliferative responses. The effects of two lysosomotropic agents, ammonium chloride and chloroquine, on the capacity of human monocytes to activate T lymphocyte responses to the soluble antigen streptolysin O (SLO) and to the polyclonal stimulant S. aureus protein A (SpA) were investigated. These agents inhibited the presentation of SLO and SpA by human monocytes in a dose-dependent manner. The inhibition occurred if monocytes were treated with ammonium chloride and chloroquine for 1.5 hr, starting only 30 min after exposure to the stimulants, whereas only minimal inhibition occurred when monocytes were treated with the two lysosomotropic compounds 2 hr after pulsing with SLO or SpA. In contrast, cell membrane alloantigen presentation by monocytes in the MLR was not affected by ammonium chloride or chloroquine treatment. Thus, these reversible inhibitors of monocyte phagosome-lysosome functions presumably interfere with intracellular processing of the stimulants but do not seem to interfere with alloantigen presentation at the cell surface. Furthermore, we investigated whether gently fixed monocytes were still capable of passively presenting stimulant or whether active metabolic processes as well as IL 1 were required. We observed that only monocytes treated with paraformaldehyde after SLO or SpA pulsing stimulated a proliferative response by T lymphocytes, provided 50 U/ml of partially purified human IL 1 were added back to cultures. In contrast, monocytes fixed before exposure to SLO or SpA were not able to stimulate T lymphocytes even if supplemented by IL 1. Taken together these data suggest that a finite incubation period is required for human monocytes to become able to present SLO or SpA to T lymphocytes. During this time the soluble stimulants presumably undergo some metabolic process in viable macrophages perhaps at the phagosome-lysosome level, to become recognizable by T lymphocytes.

Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells.

This paper describes the effects of two lysosomotropic compounds, ammonia and chloroquine, on the interaction of mononuclear phagocytes with immune T cells. The uptake and ingestion of Listeria monocytogenes by macrophages were not affected by the drugs; however, the macrophage catabolism of 125I-labeled Listeria was reduced in a dose-dependent way. The macrophage presentation of Listeria to T cells, an I-region-dependent phenomenon, was also inhibited. The degree of inhibition of catabolism paralleled that of antigen presentation. The inhibition of antigen presentation took place if the macrophages were treated before and during Listeria uptake; minimal inhibition took place if the macrophages were treated 30 min after Listeria ingestion, at which time a significant amount of bacteria was already catabolized. Our previous studies had shown that the macrophage-associated antigen recognized by T cells became apparent 30-60 min after uptake of Listeria. We conclude that ammonia and chloroquine affected an intracellular handling step required for the expression of the immunogen relevant for T-cell recognition.

Liposomes as carriers in vivo for methotrexate.

In differential activation, advantage is taken of the different sensitivity of the various organelles towards digitonin which is added in uitro to liver homogenates (HuybrechtsGodin & Jacques, 1974). The method can be applied to the localization of exogenous material without parallel treatment by ancillary lysosomotropic compounds. Table 2 illustrates the digitonin-induced release of latent marker enzymes characterizing several cytoplasmic organelles or substructures, and of two drugs utilized in applied lysosomology : horse-radish peroxidase and suramin. The totality of the intracellular drug could be found within cytoplasmic vacuoles representing either lysosomes, or vesicles deriving from the plasma membrane (heterophagosomes?) or both types of structures, in a tissue whose molecular and cellular dynamics cannot possibly have been altered by the ancillary lysosomotropic compounds required for the centrifugation technique. In addition, these results allow us to discard the hypotheses that, in our experimental conditions, a measurable proportion of any of the two drugs might have concentrated within Golgi structures, the lumen of endoplasmic reticulum or the interstitial compartments of mitochondria. Huybrechts-Godin, G. &Jacques, P. J. (1974) Arch. Int. Physiol. Biochim. in the press Jacques, P. J. (1968) Epuration plasmatique de protiines dtrangt?res, leur capture et leur destinie Jacques, P. J. (1972) in Uptake of Informative Molecules by Living Cells (Ledoux, L., ed.), Jacques, P. J. (1973) in International Aspects of Drug Evaluation and Usage (Jouhar, A. J. & Jacques, P. J. (1974) in Methodological Developments in Biochemistry (Reid, E., ed.), vol. 4, dam l'appareil vacuolaire du foie, Librairie Universitaire, Louvain pp. 277-294, North-Holland, Amsterdam Grayson, M. F., eds.), pp. 347-352, Churchill-Livingstone, London pp. 415-420, Longman, London