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J. Cell Sci. 74119-135 (1985) 119 Printed in Great Britain © Company of Biologists Limited 1985 RESPONSES OF CULTURED CARDIAC MYOCYTES TO LYSOSOMOTROPIC COMPOUNDS AND METHYLATED AMINO ACIDS R. S. DECKER, M. L. DECKER Department of Medicine, Cell Biology &Anatomy, Northwestern University Medical School, 303 E. Chicago Ave, Chicago, Illinois 60611, U.SA. V. THOMAS Department of Cell Biology, The University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235, U.SA. AND J. W. FUSELER Department of Biology, Tulane University, New Orleans, Louisiana 70112, U.SA. SUMMARY 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 NH4CI) 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 NH4CI, and they may provide a unique and valuable means of studying secondary lysosomal function in cell culture. INTRODUCTION The exposure of partially purified lysosomal fractions from rat liver to amino acid methyl esters markedly reduces the latency and sedimentability of lysosomal enzyme activity in such preparations (Goldman, 1973; Goldman & Kaplan, 1973; Goldman & Naider, 1974; Goldman, 1976). This phenomenon is presumably mediated by the hydrolysis of the freely permeable methyl ester within the lysosomal compartment (Reeves, 1979), with the accumulation of the polar, less-permeable amino acid product culminating in an osmotic rupture of the lysosomes (Goldman, 1976; Reeves, 1979). Perfusion of intact beating rat hearts with leucine methyl ester provokes a similar rise in non-sedimentable cathepsin D activity, which is also accompanied by a modest inhibition of cardiac protein degradation (Reeves, Decker, Crie & Wildenthal, Key words: amino acid methyl esters, secondary lysosomes, cardiac myocytes. 120 R. S. Decker, M. L. Decker, V. Thomas andjf. W. Fuseler 1981; Long, Chua, Lautensack & Morgan, 1982). These biochemical alterations coincide with a marked dilation of secondary lysosomes in situ (Reeves et al. 1981), possibly followed by disruption of some of the organelles after long periods of exposure to high concentrations (^50mM) of the methylated amino acids (Miller, Griffiths, Lenard & Firestone, 1983; unpublished observations). Since other subcellular membranous systems that transiently house lysosomal enzymes appear unperturbed by exposure to amino acid methyl esters (Reeves et al. 1981), these compounds may specifically alter secondary lysosomes. In an attempt to characterize further the principal events accompanying the lysosomal changes provoked by amino acid methyl esters and lysosomotropic weak bases, we have studied the effects of these agents on primary cultures of foetal rat myocytes preincubated with acridine orange to label the lysosomes (Allison & Young, 1964; De Duve et al. 1974). In a parallel set of experiments, lysosomal function was monitored by measuring changes in lysosomal proteinase activity, intralysosomal pH and 'accelerated' protein degradation. Structural alterations in foetal myocyte lysosomes were also examined by acid phosphatase cytochemistry. Our observations indicate that amino acid methyl esters rapidly and selectively dilate secondary lysosomes housing acridine orange in a fashion distinct from lysosomotropic agents; such probes should be useful in elucidating the role of secondary lysosomes in regulating proteolysis under conditions of serum deprivation. MATERIALS AND METHODS Myocyte isolation and culture Primary cultures of isolated myocytes were prepared from hearts of foetuses of timed pregnant Sprague-Dawley rats (Holtzman, Madison, WI). Whole hearts obtained from 16-day-old embryos were minced in adhesion salt buffer (0-2g/lMgSO4-7H 2 O, 6-8g/lNaCl; O4g/1KC1; l-5g/lNaH 2 PO 2 -H 2 O; 1 g/1 glucose; 4-76g/l HEPES, pH7-S) before transferring them into a sterile 25 ml spinner flask, which contained 20 ml of digestion medium (0-6mg/ml Pancreatin, Gibco, Grand Island, NY, in adhesion salts buffer). The solution containing the hearts was stirred at 75rev./min for 60min at 37°C (Nath, Shay & Bollan, 1978). The supernatant from this initial digestion was collected and the pancreatin was neutralized with Dulbecco's Modified Eagle Medium (DMEM, Gibco, Grand Island, NY) supplemented with 15 % horse serum, 5 % foetal calf serum (K.C. Biological, Lenexa, KS) and 1 % antibiotics (Gibco, Grand Island, NY). The isolated cells were concentrated by centrifugation (1000 rev./min for 3 min), resuspended in 1 ml of fresh tissue culture medium, and placed in an ice bath. This procedure was repeated until 90-95 % of the heart tissue was digested. The myocytes were partially purified from non-myocyte (endothelial and fibroblast) cells by differential adhesion (Kastan, 1973). The cold cell suspensions from the sequential digestions were transferred into a T-75 Corning culture flask (Corning, NY), which contained 10 ml of prewarmed (37°C) DMEM culture medium and incubated in a CO2 incubator for a minimum of 4h. The myocytes remained in suspension, whereas the non-myocytes rapidly adhered to the flask surface. This method resulted in a population of cells that consisted predominantly of myocytes that could be maintained beating in culture for several weeks. The myocyte preparation was then plated onto sterile coverslips previously placed in 35 mm Petri dishes (Corning, NY). The cells in the drops were allowed to adhere to the coverslips for 24 h before the culture dish was flooded with 2 ml of culture medium. Remaining non-myocytic contaminants were destroyed by exposing the cultures to medium containing lO/M-cytosine-l/3-D-arabinofuranoside (Sigma Chemical Co., St Louis, MO) for 3-5 days (Moses &Claycomb, 1982). Other 35 mm dishes were plated at densities of 1 X 10* cells per dish; these cultures were used for biochemical assays of lysosomal enzymes, creatine kinase and Lysosomal effects of amino acid methyl esters 121 measurements of protein degradation. Myocytes were maintained in culture for 5-7 days before being treated with either lysomotropic compounds or methylated amino acids. Vital staining of cardiac lysosomes with Acridine Orange The lysosomes in the cultured myocytes were labelled with 1 fcu-Acridine Orange (AO) in DMEM for IS min. Such an exposure was sufficient to label maximally the lysosomal complement in the contracting myocytes. After labelling with AO, the cells were washed free of the fluorescent dye with Hank's balanced salt solution (Gibco) and returned to normal DMEM plus serum. The AO fluorescence of the lysosomes was stable for 24 h after removal of the dye. Other coverslips were cultured in the presence of neutral red (100(JM) or Lucifer Yellow (lmg/ml) for 15min or 24h, respectively, before examination. The labelled myocytes were viewed on a Leitz Orthoplan microscope (E. Leitz, Rockleigh, NJ) equipped for phase and epi-fluorescence microscopy with a Ploem illuminator. The excitation source was an XBO ISO watt Xenon lamp (Osram) filtered with a Leitz BG38 red suppression filter and a KP490 FITC excitation filter. The barrier filter was a Leitz K530. Photographic records were made with the Leitz Orthomat automatic camera system using Kodak Ektachrome Professional film (ASA 200). Magnification was calibrated by photographing a standard stage micrometer through the optical system. Treatment reagents of myocyte cultures with methylated amino acids and lysosomotropic Cardiac lysosomal alterations were induced by exposure of the labelled myocytes to L-leucine methyl ester (L-Leu-OMe) or L-methionine methyl ester (L-Met-OMe) (Sigma Chemical Co., St Louis, MO). The normal DMEM was removed, and the cells on the coverslips were exposed to serum-free DMEM containing 10mM-amino acid methyl esters (Reeves el al. 1981). Control cultures were exposed to serum-free medium containing 10mM-D-methionine methyl ester (DMet-OMe), an analogue that is not hydrolysed by lysosomal enzymes and has no effect on lysosomal integrity (Goldman & Kaplan, 1973; Reeves, 1979). At intervals (20, 40, 60 min) coverslips were prepared for fluorescent or phase observations, and culture dishes were used for biochemical experiments or ultrastructural study. In other experiments myocytes were exposed for identical periods to chloroquine (100/fltf), ammonium chloride (10mM) or methylamine (10min), instead of the amino acids. It is well-documented that each of these lysosomotropic reagents can alter lysosomal structure and function (De Duve et al. 1974; Ohkuma & Poole, 1978, 1981). Electron microscopy and cytochemistry Cultured myocytes were fixed in situ in 2 % glutaraldehyde and 1 % paraformaldehyde buffered with O-lM-cacodylate, (pH7-4) for 3-4h. The cells were then washed, post-fixed in osmium tetroxide, dehydrated and flat-embedded in Epon. Myocytic fields selected by phasecontrast microscopy were then cored from the Epon and flat-embedded in 'Beem' capsules. Thin sections were prepared, stained with uranyl acetate and lead citrate, and viewed in a Philips 200 electron microscope. Some cultures were fixed for 30 min, washed and incubated in Gomori's acid phosphatase medium, as modified by Barka & Anderson (1962), for 30 min to reveal the distribution of this lysosomal enzyme. Controls were incubated in an identical fashion except that the substrate (/S-glycerophosphate) was removed or lOmM-sodium fluoride was added to the medium. Lysosomal proteinase activity and proteolysis The media and cells were assayed for lysosomal cathepsins B and D (Barrett & Heath, 1977) and creatine kinase (Szasz, Busch & Taroks, 1970). The medium was removed and directly assayed for all three enzymes. The cultured myocytes were rinsed in Hank's balanced salt solution, scraped from the dishes and extracted with 2 ml of 0-1 % Triton X-100 before assay. Protein was measured by the method of Lowry, Roseborough, Farr&Randall (1951), and enzyme activity was expressed in units of activity per mg protein per h ± standard error of the mean. Analysis of statistical significance for 122 R. S. Decker, M. L. Decker, V. Thomas andj. W. Fuseler observed differences in enzyme activities was made by Student's two-tailed Meat for unpaired samples. Another measure of lysosomal function was to assess quantitatively the rate of proteolyais. Culture myocytes were labelled for 24h with 0-5/iCi of L-[HC]tyrosine (530mCi/mmol, Amersham) in complete, serum-supplemented medium. The cultures were then rinsed for 1 h in serum-free DMEM supplemented with cold 2 mM-L-tyrosine to remove unincorporated amino acid and to wash out the label derived from proteins that turn over rapidly. Cultures were again rinsed twice in Hank's balanced salt solution, placed in serum-free DMEM plus cold 2 mM-L-tyrosine and samples of medium were precipitated with 10% trichloracetic acid (TCA) at 30-min intervals. The samples were delipidated and solubilized in NCS (Amersham) and counted in a Beckmann liquid scintillation spectrometer calibrated with external standards. After 4 h the experiments were terminated and the cells were scraped into 10 % TCA and a sample was counted while another sample was assayed for protein content according to the procedure of Lowry et al. (1951). The relative rate of protein degradation was expressed as the release of acid-soluble counts per mg of cell protein over the duration of the experiment. Lysosomal pH Lysosomal function was also evaluated by monitoring how each methylated amino acid or weak base altered intralysosomal pH. Lysosomal pH was measured in myocytes that had been loaded for 24h with 1 mg/ml fluorescein dextran (40 X 103A/r; Sigma Chemical Co., St Louis, MO). The cells were rinsed in two changes of Hank's balanced salt solution and cultured for 4 h in serum containing DMEM to ensure that the endocytosed fluorescent dextran reached the lysosomal compartment (Decker, Decker, Canon & Thomas, 1982). The coverslips were then rinsed three times in Hank's balanced salt solution, mounted into Rose chambers, maintained at 37° C, and perfused with serumfree DMEM supplemented with either methylated amino acids or weak bases at the concentrations outlined above. The pH was determined at various intervals by calculating the ratio of fluorescent emission at 525 nm when individual myocytes were alternatively excited at 490 nm and 450 nm for 1 s on a Leitz Orthoplan fluorescent microscope equipped with a Leitz MPV compact microscope photometer (Maxfield, 1982; Tycko & Maxfield, 1982). The excitation ratios were compared with those obtained from fluorescein dextran loaded myocytes that had been fixed and equilibrated at various pH values (Decker & Fuseler, 1984). Since the fluorescent emission of fluorescein dextran is exquisitely sensitive to pH (Ohkuma & Poole, 1978; Poole & Ohkuma, 1981), this probe provides a relatively simple and accurate method of continuously monitoring lysosomal acidity (Decker & Fuseler, 1984) and, therefore, indirectly lysosomal activity, throughout the duration of our experiments. Figs 1-4 The effect of 10mM-L-leucine methyl ester (L-Leu-OMe) on acridine-orangepositive granules in cultured foetal myocytes. Fig. 1 shows the distribution of red-orange granules in myocytes not exposed to L-Leu-OMe. A brief exposure to L-Leu-OMe (20 min) dilates lysosomes, shifting their fluorescent excitation toward yellow (Fig. 2). A few peripherally located particles (perhaps endosomes) still exhibit an orange hue. After a longer treatment (40 min) most myocytes disclose green granules. The myoplasm also exhibits a green tint and a small number of orange granules are still apparent in the cell periphery (Fig. 3). A 1-h exposure to L-Leu-OMe enhances the cytoplasmic staining and eliminates almost all granular staining, creating large 'black holes' (arrows) in the myocytes (Fig. 4). Fig. 1, X 850; Figs 2-4, x 425. Figs 5, 6. The effects of NH4CI (Fig. 5) and chloroquine (Fig. 6) on myocytes prelabelled with acridine orange. Exposing cells to either of the weak bases for 1 h swells most but not all AO-positive granules. The weak bases do not induce a subcellular redistribution of acridine orange like the methylated amino acids, although chloroquine-loaded lysosomes emit in the green region of the spectrum because they have loaded vast quantities of the weak base. Note that little cytoplasmic fluorescence is apparent after exposure to either weak base. Fig. 5, X 850; Fig. 6, X 425. Lysosomal effects ofamino acid methyl esters 123 RESULTS Incubation of primary cultured myocytes with 1 jtai-Acridine Orange (AO) for 15min maximally labelled a large population of granules whose properties and distribution resembled lysosomal populations described previously in other types of cells (Allison & Young, 1964, 1969; Canonico & Bird, 1969; Poole, 1977). Even though these vacuoles load only small quantities of the fluorescent dye and were not structurally altered, the AO fluorescence emitted from these lysosomal granules displayed a brick-red or reddish-orange hue in contractile myocytes (Fig. 1). The distribution of AO within the cardiac lysosomes remained stable for at least 1 day after this brief labelling period. Generally, fluorescent granules were observed in the perinuclear cytoplasm and at the cell margins, but in many myocytes orange lysosomes also appeared to be arrayed along developing myofibrils (Fig. 1). Addition of either L-leucine (L-Leu-OMe) or L-methionine (L-Met-OMe) methyl ester to cultures prelabelled with AO dramatically altered the structure of the granules and their fluorescent spectrum. Within 20min myocytic granules dilated and the fluorescent emission of the granules shifted toward the yellow region of the spectrum (Fig. 2). After a 40-min exposure to either of the L-methylated amino acids, many phase-lucent vacuoles were observed, displaying pale green fluorescence (Fig. 3). At 1 h, myocytes treated with L-amino acid methyl ester and viewed under fluorescent microscopy revealed immense vacuoles or 'black holes' in the perinuclear cytoplasm (Fig. 4). These large vacuoles emitted no generalized fluorescence but frequently contained minute fluorescent particles. At this juncture, the cytoplasm of these myocytes disclosed a vivid green tint, suggesting that a redistribution of the AO had occurred from the lysosomal compartment into the myocyte cytoplasm. Only a few AO-positive granules could be visualized in myocytes exposed to the methylated L-amino acids for one or more hours and this staining may reflect the localization of the dye within endosomes. The treatment of cultures with AO and the amino acid methyl esters did not seriously affect the viability of the myocytes, for they continued to contract throughout the 1-hour observation period, albeit at a reduced rate when compared with untreated control myocytes or cells exposed to D-methionine methyl ester. The D isomer, which is reportedly not hydrolysed by lysosomal enzymes (Goldman & Naider, 1974; Reeves, 1979), failed to elicit any changes either in the apparent structure and distribution of lysosomes or in their AO emission profile over the course of the 1-h exposure. Ultrastructural cytochemistry confirmed that many of the black holes observed in the fluorescent microscope were dilated secondary lysosomes (Fig. 8). Exposure of myocytes to either L-Leu-OMe or L-Met-OMe led to progressive swelling of lysosomes and a reduction of their acid phosphatase content. After 40 min of treatment, only small deposits of reaction product could be visualized (Fig. 8); by 60 min still less was apparent (Fig. 8, inset). The reaction product, when visible, was associated with the lysosomal membrane or membranous debris confined to the swollen vacuole (Fig. 8). In contrast, 1 h of exposure to the D isomer failed to alter the location or intensity of the lead phosphate reaction product (Fig. 7). Other sub- 124 R. S. Decker. M. L. Decker. V. Thomas and 7. W. Fuseler Figs 7-8 Lysosomal effects ofamino acid methyl esters 125 cellular organelles remained structurally unaltered in L-Leu-OMe-exposed myocytes compared to myocytes cultured in the absence of the amino acid methyl ester or in the presence of the D isomer (compare Fig. 7 with Fig. 8). The endoplasmic reticulum, the Golgi apparatus, Golgi-endoplasmic reticulum-lysosome complex (GERL) and small coated vesicles, all of which are believed to be involved in lysosomal processing (Friend & Farquhar, 1967; Novikoff et al. 1971; Holtzman, 1976), were not altered by either L-methylated amino acid. In contrast to the response provoked by the methylated amino acids, other lysosomotropic reagents that also dilate myocytic lysosomes did not appear to influence their AO content. Myocytes briefly labelled with AO displayed little apparent vacuolar swelling (Ohkuma & Poole, 1981) even though the organelles fluoresced brilliantly (Fig. 1). Application of non-fluorescing weak bases like ammonium chloride or methylamine to such cells yielded a pronounced dilation of the stained vacuoles, which appeared larger and more numerous than the black holes of amino acid methyl ester-treated cells; however, these organelles continued to emit the redorange fluorescence characteristic of the acridine dyes (Fig. 5). Moreover, neither weak base induced a redistribution of the dye into the cytoplasm of responding myocytes. Chloroquine provoked a similar swelling of labelled vacuoles but, because of the fluorescent properties of this base (Ohkuma & Poole, 1978) and the tremendous amounts of the drug that are concentrated within lysosomes (i.e. between 50 and 100 mM) during the 1-h exposure (De Duve et al. 1974; Ohkuma & Poole, 1978, 1981), the emission spectrum of the labelled granules shifted into the green region (Fig. 6). Nevertheless, after treatment, the vacuoles remained highly fluorescent, with little indication of cytoplasmic staining. All of the organic bases induced severe contractile depression, most myocytes ceasing to beat within 5 min of treatment. Alterations in a variety of other lysosomal properties further demonstrated that the methylated amino acids and the weak bases elicited different lysosomal responses. Over the brief period during which the myocytes were exposed to the weak bases, no evidence of enhanced lysosomal proteinase secretion could be demonstrated, but it was readily apparent that the weak bases inactivated 80-90% of the cathepsin B activity without markedly influencing the distribution or activity of lysosomal cathepsin D (Table 1). Moreover, the ratio of cathepsin B activity in the medium to total activity indicated that cell-associated lysosomal cathepsin B appeared to be the principal form of the enzyme that was inactivated. Conversely, L-Leu-OMe or L-MetFigs 7, 8. The localization of acid phosphatase reaction product in control (Fig. 7) and L-Met-OMe-treated myocytes (Fig. 8). Myoctyes exposed to D-Met-OMe (Fig. 7) or left untreated exhibit acid phosphatase-positive lysosomes (arrows) throughout their cytoplasm. Some, but not all larger vacuoles, reveal lead phosphate precipitates (arrowheads); the latter may represent endosomal compartments. Exposure of myocytes to lOmM-L-Met-OMe for 40 min (Fig. 8) swells many acid phosphatase-positive lysosomes (v). However, not all vacuoles (v) display reaction product. By 60min (Fig. 8, inset) many of the vacuoles (y) apparently fuse with one another (arrows) but few reveal reaction product. Note that no other structural alteratipns are induced by exposing the cultured myocytes to methylated amino acids. G, Golgi apparatus; n, nucleus; my, myofibrils. Figs 7, 8, X 25 200; Fig. 8, inset, X 18000. Medium Cells M+ M+C Medium Cells Cathepsin D & tyrosine/ mg protein per h Creatine kinase p o l ATP/ mg protein per h 59f 4 95 f 5 0.38 61f 6 98 f 7 0.38 N.D. 0-47 f 0.05 0.10 0.10 N.D.t 0.49 f 0.05 84 7 712 f 60 + D-Met-OMc 78 f 8 698 f 65 DMEM + N.D. 0.42 0.04 0.40 62 f 5 93f6 0-12 9 5 f 10 711 f 58 L-Met-OMe + 58 + N.D. 0.48 0.05 0.39 57 5 91f 4 + 0.11 724 W f 11 ~-Leu-oMe N.D. 0.49 f 0.07 0.37 57 f 5 9 8 f 10 0.37 83f 9 1 4 2 f 15 Chloroquine N.D. 0.44 f 0.04 0.39 6Of 7 %f 7 0.30 75 f 7 167 f 18 NH4CI + Cultures were exposed to each compound for 1 h in serum-free DMEM and each value represents the average of 10 cultures f 1 S.E.M. 'Percentage of the distribution of lysosomal activity between the medium (M) and cells (C) was determined by dividing the activity in M by the total activity (M C). t N.D., not detectable. Enzyme activity was sipficantly different at a P < 0.01 compared to either DMEM or L-Leu-OMe-treated cultures. Medium Cells Me M+C Distribution Cathepsin B nmol 2-napthal.1 mg protein per h Enzyme Table 1 . Effects of amino acid methyl esters and weak bases on the speafic activities of lysosomal cathepsins B and D and m a t i n e kinase in rat myocyte cultures Lysosomal effects of amino acid methyl esters 127 OMe failed to alter either the activity or the distribution of either acid proteinase (Table 1). The inactivation of cathepsin B, furthermore, appeared to be unrelated to viability of the cultures, for no creatine kinase could be measured in the culture medium following exposure of the myocytes to either class of agents (Table 1). Accelerated proteolysis (Dean, 1980) was dramatically inhibited by both classes of reagents; but, perhaps somewhat surprisingly, L-Leu-OMe and L-Met-OMe suppressed degradation to a greater degree than the weak bases (Fig. 9). After a 1-h exposure to the methylated amino acids, the release of acid-soluble, radiolabelled tyrosine into the culture medium was reduced some 50%, whereas chloroquine and 30 90 120 150 Time (min) 180 210 240 Fig. 9 reveals the effects of L-Leu-OMe (10mM), NH4CI (10mM) and chloroquine (10/JM) on the degradation of long-lived proteins, which were labelled for 24 h before a 1-h exposure to each of these compounds. Proteolysis was measured by determining the amount of acid-soluble radiolabelled L-[14C]tyrosine released into the culture medium over 30-min intervals. The arrow indicates the point at which the drugs were removed and cultures were rinsed before recovery. After a 1-h exposure to L-Leu-OMe, the release of [I4C] tyrosine was inhibited about 50%; if exposure was continued for an additional 3h release was reduced to 40 % of control values. Chloroquine inhibited proteolysis 25 % and NH4CI inhibited it 30% after a 1-h treatment. Continued exposure to chloroquine or NH4CI for a total of 4 h increased the inhibition of proteolysis 55 % and 45 %, respectively. Each value represents the mean of five separate cultures; the mean range, ± 5 - 7 % . (O O) DMEM; (O <J) chloroquine; (© C) NH 4 C1; ( • • ) L-Leu-OMe. 128 R. S. Decker, M. L. Decker, V. Thomas andjf. W. Fuseler 009 DMEM Out *" 0-17 7 0-38 6 0-75 5 009 8 D-Met-OMe 017 7 ij 0-38 6 .2 0-75 5 5. I e 009 q L-Met-OMe ° 47 7 I jjj 0 38 6 C 0-75 5 c 0-09 8 L-Leu-OMc 0-17 7 0-38 0-75 40 20 20 40 60 120 180 240 Time (min) Fig. 10. The temporal change in the fluorescence intensity ratio and intralysosomal pH prior to and after exposure of fluorescein dextran-labelled myocytes to L-Leu-OMe (lOmM), L-Met-OMe (IOITIM), or D-Met-OMe (10mM). Cultures were perfused for 40 min in DMEM and then at 'In' the perfusate was changed to DMEM plus a methylated amino acid for 1 h. At 'Out' the perfusion was returned to normal DMEM, which was continued for an additional 3 h. Each experiment traces the A pH of an individual myocyte with the values plotted representing examples of 5-10 separate cells. ammonium chloride inhibited proteolysis 25 % and 30%, respectively, even though myocytic lysosomes appeared equally dilated by the drug treatments. Prolonging the exposure of the myocytes for 4 h altered the percentage of proteolysis inhibited by L-Leu-OMe to 40 % of control levels, while the NH4CI inhibited degradation by about 45 %. The suppression of proteolysis mediated by chloroquine continued to increase, reaching a maximum of 55% at 4h. This apparent anomalous behaviour of the myocytes is presently being pursued. An appearance of creatine kinase in the medium Lysosomal effects of amino acid methyl esters 129 after 4 h of treatment with chloroquine suggests that foetal myocytes are extremely sensitive to the toxic effects of the weak base over prolonged periods. Therefore, protein degradation was inhibited only 1 h before the cultures were rinsed free of the methylated amino acids and placed in serum-free DMEM plus cold tyrosine. The rate of release of labelled tyrosine returned to control values within 1 h after treatment with L-Leu-OMe. In contrast, several hours were required for proteolysis to resume reasonable rates after the myocytes were exposed to NH+C1; even after 4h of 009 DMEM In Out 0-17 0-38 0-75 009 NH4C1 8 °- 1 7 1 0-38 I S3 •a 009 Methylamine .S 8 0-17 g 0-38 o 7 1 3 E 0-75 009 Chloroquine 0-17 0-38 0-75 40 20 20 40 60 120 180 240 Time (min) Fig. 11. The fluorescent intensity ratio and the A pH of fluorescein-dextran-labelled myocytes exposed to NH4CI (lOmM), methylamine (IOITIM) and chloroquine (100^M). Cultures were perfused for 40 min in DMEM, then at time 0 ('In') the perfusate was changed to DMEM plus a weak base for 1 h. At 'Out' normal perfusion in DMEM was resumed for 3 h. Each panel follows the change in pH of an individual myocyte but represents data derived from five to ten cells. Inherent chloroquine fluorescence was subtracted from the fluorescein-dextran emission as described by Ohkuma & Poole (1978). 130 R. S. Decker, M. L. Decker, V. Thomas andjf. W. Fuseler recovery, cultures treated with chloroquine still disclosed markedly depressed rates of proteolysis (Fig. 9). Myocytes in these cultures frequently displayed numerous dilated vacuoles, suggesting that a lysosomal lesion persists for some time after removal of the weak bases and may partially account for the continued depression of protein degradation. Yet another distinction between the apparent mode of action of these lysosomotropic compounds on lysosomal activity was uncovered when intralysosomal pH was measured fluorimetrically. Selected myocytes that had been loaded for 24 h previously with fluorescein dextran were mounted into Rose chambers and perfused in serumfree DMEM with or without amino acid methyl esters or weak bases. Continuous monitoring of these cells revealed that the methylated amino acids elevated lysosomal pH only modestly, from 4 8 to 5 3 over a 10-min interval. This small rise in lysosomal pH appeared not to be simply related to the concentration or variety of amino acid methyl ester (Reeves, 1979) examined or to the changes in lysosomal geometry, for the small rise in pH also occurred when fluorescein dextran loaded myocytes were exposed to the D-amino acid methyl ester analogue (Fig. 10). Returning to a perfusate of serum-free DMEM 1 h later resulted in a prompt decline of the lysosomal pH to 4-9, which remained constant for an addition 2h. Conversely, ammonium chloride, methylamine and chloroquine promptly stimulated a decrease in the fluorescence intensity ratio, demonstrating a marked rise in intralysosomal pH. Within 3-5 min of exposure to NH4CI, methylamine or chloroquine, pH values of 6 3 and 6 - 5, respectively, were observed (Fig. 11). Moreover, the rise in pH preceded any significant vacuolarization of myocytic lysosomes. The elevated lysosomal pH values drifted slightly higher during the 1-h exposure until the perfusate was returned to normal DMEM, whereupon the pH fell dramatically. In the case of ammonium chloride, the pH returned to ambient levels (pH4 - 9) within 3—5 min. However, removal of chloroquine resulted in a more gradual decline in lysosomal pH, with an initial sharp fall to 5 9 after 5 min and to an apparent stabilization of pH in the 5-5 to 5-8 range after 3 h of perfusion in normal medium (Fig. 11). The response of methylamine appeared comparable to that described for NH4CI. Furthermore, lysosomes remained dilated throughout this 'recovery' period, suggesting that the weak base had perturbed the organelle structure in such a fashion that several hours were required to eliminate osmotic water and, presumably, the accumulated weak base. DISCUSSION Acridine Orange is a member of a group of weak bases that accumulate within lysosomes by virtue of the acidic intralysosomal pH and the impermeability of the lysosomal membrane to the protonated form of the dye (De Duve et at. 1974; Poole, 1977;Ohkuma&Poole, 1978, 1981; Rabon, Chang & Sachs, 1978; Poole &Ohkuma, 1981). It is also a fluorescent compound whose emission spectrum is dependent upon its packing density. At low concentrations, AO emits in the green range, while at higher concentrations it emits at a red-orange frequency (Steiner & Beers, 1961; Allison & Young, 1964; Barrett & Dingle, 1968). It is this latter property that makes Lysosomal effects of amino acid methyl esters 131 AO an ideal marker for the study of the permeability of lysosomal membranes in response to amino acid methyl esters in living cells. In the present experiments, exposure of myocytes to L-Leu-OMe or L-Met-OMe altered the spectral properties of acridine orange staining after as little as 5 min, and by 40 min essentially all of the stained lysosomal profiles were dilated, with their emitted fluorescence shifted to a pale green spectrum (Fig. 3). We believe these changes represent a dilution of the acridine orange compartment during vacuolar swelling and/or alteration of its spatial arrangement within the lysosomes. Since methylated amino acids perturb lysosomal pH minimally (Fig. 10), dilation of the lysosome, which accompanies hydrolysis of the methylated amino acid, may favour a leakage of the protonated form of AO from the organelle. Moreover, the swelling of the vacoules may also disrupt the ionic interactions between the dye and matrix glycolipids (Barrett & Dingle, 1969; Dingle & Barrett, 1969), as a massive influx of osmotic water lowers the ionic strength of the lysosome, making conditions for electrostatic interactions unfavourable. Other small molecular probes also diffuse from lysosomes when they are exposed to amino acid methyl esters. Neutral Red and Lucifer Yellow redistribute within 15 min of exposure to L-Leu-OMe (unpublished observations). Since the latter is not a weak base and reaches the lysosome via bulk phase endocytosis, the present observations support the contention that methylated amino acids alter lysosomal membrane permeability, probably through some form of osmotic perturbation of the lipid bilayer. Conversely, lysosomotropic weak bases, which raise intralysosomal pH (Fig. 11) and promote the osmotic 'swelling' of lysosomes, failed to alter the distribution of AO in prelabelled myocytes. Even though each class of compound osmotically dilates secondary lysosomes (DeDuveef al. 1974; Ohkuma&Poole, 1978, 1981;Reeves, 1979;Reeves et al. 1981; Poole & Ohkuma, 1981), it appears that only the amino acid methyl esters provoke enough disruption to induce a redistribution of the dye. The nature of the permeability change in the lysosomal membrane is not known. The simplest interpretation appears to be that the accumulation of the free amino acid within lysosomes leads to osmotic disruption of the lysosomal membrane with the subsequent redistribution of the intralysosomal contents (including, perhaps acid hydrolases). This process is believed to mediate the disruption of isolated liver lysosomes, as reported by Goldman and colleagues (Goldman, 1973, 1976; Goldman & Kaplan, 1973; Goldman & Naider, 1974; Reeves, 1979). Alternatively, there may be more subtle changes in lysosomal properties and/or pH, which lead to a selective redistribution of smaller intralysosomal compounds such as AO. Our observations on how methylated amino acids and the weak bases modulate lysosomal proteinase activity, protein degradation and intralysosomal pH provide direct evidence that these compounds interfere with lysosomal function by different mechanisms. One of the well-recognized features of weak bases is that they inhibit lysosomal enzyme processing (Hasilik & Neufeld, 1980a,fe; Gonzalez-Noriega, Grubb, Talkad & Sly, 1980; Rosenfeld et al. 1982) while stimulating the secretion of incompletely processed acid hydrolases (Hickman, Shapiro & Neufeld, 1974; Kaplan, Fischer, Accord & Sly, 1977; Sando & Neufeld, 1977; Wilcox & Rathray, 1979; Gonzalez-Noriega et al. 1980). Both ammonia and chloroquine have been 132 R. S. Decker, M. L. Decker, V. Thomas andj. W. Fuseler demonstrated to provoke the secretion of pro-cathepsin D in fibroblasts (Hasilik & Neufeld, 1980a) and hepatoma cells (Rosenfeld et al. 1982). Although our experiments present little evidence of augmented secretion of acid proteinase, a more prolonged exposure to a weak base or methylated amino acid may be required, along with a more sensitive assay system (i.e. immunoblotting of cathepsin B or D) to detect subtle changes in enzyme processing. However, the inactivation of cathepsin B by weak bases but not by amino acid methyl esters clearly differentiates the two groups of lysosomally active compounds. Since cathepsin B is believed to become unstable and irreversibly denatured above pH 6 (Wibo & Poole, 1974; Barrett & Heath, 1977; Barrett, 1980), the 80% loss of cathepsin B activity measured in this investigation (Table 1) indirectly supports the contention that methylated amino acids do not 'alkalinize' the lysosome as do chloroquine or ammonium chloride. There are two perspectives in which to interpret the inactivation of cathepsin B. The marked decrease in thiol proteinase activity may have resulted from a direct, drug-induced inactivation of the lysosomal enzyme, for Ohkuma & Poole (1981) have reported that within 30min chloroquine concentrations may reach 50-100 mM within the lysosomes of cells exposed to 100 ya/i weak base. At these levels cathepsin B could be directly inactivated by chloroquine (Wibo & Poole, 1974; Barrett, 1980). Alternatively, the weak base may have stimulated the secretion of cathepsin B where it is inactivated because of the high pH (7 4) of the medium. We tend to support the first hypothesis for several reasons. First, the secretion of other lysosomal enzymes mediated by weak bases requires a significant time interval (i.e. 8—24 h) to reach a level equivalent to that reported here (Wilcox & Rathray, 1979; Gonzalez-Noriega et al. 1980). Secondly, cathepsin B is susceptible to direct inhibition by chloroquine but not ammonia (Wibo & Poole, 1974); nevertheless both weak bases inactivate the myocytic thiol proteinase (Table 1), suggesting that a pH-induced inactivation of the enzyme is probable within the lysosome. The direct demonstration that L-Leu-OMe or L-Met-OMe raised intralysosomal pH only moderately, whereas the weak bases elevated it significantly within 5 min (Figs 10, 11), further strengthens this notion. Lastly, albeit indirect, cathepsin D secretion was not enhanced significantly during exposure to the weak bases, suggesting that little lysosomal secretion had transpired during the period in which cathepsin B was inactivated. More recent experiments from Samarel's laboratory suggest that cardiac myocytes may secrete very small amounts only of lysosomal acid hydrolases, since a 5-h perfusion of young rabbit hearts in the presence of chloroquine provoked little or no immunoprecipitable cathepsin D in the perfusate, even though the processing of the acid proteinase was inhibited (Samarel, Ferguson, Worobec & Lesch, 1985). These observations suggest that the weak bases are acting directly upon cellular lysosomal enzymes and that the inactivation of cathepsin B is mediated by a rise in the intralysosomal pH. Lastly, alterations in the degradation of long-lived myocyte proteins, which are degraded principally within lysosomes (Dean, 1980), further illustrate that the actions of these two compounds are dissimilar. Although both groups inhibit proteolysis (Decker & Fuseler, 1984), amino acid methyl esters appear to be more efficient in this regard and completely reversible in cultured myocytes. Our observations Lysosomal effects of amino acid methyl esters 133 suggest that osmotic dilation seems sufficient to inhibit proteolysis significantly, since acid proteinase activity and lysosomal pH remained essentially unchanged in response to the methylated amino acids. Moreover, since our ultrastructural results indicate that only the secondary lysosome appears to respond to the amino acid methyl esters, this may account for their rapidly reversible action on protein degradation. Conversely, chloroquine inhibits degradation to a lesser extent in our hands but requires many hours for the myocytes to recover from its adverse effects (Dean, 1980). This rather slow recovery may be related to the synthesis of new thiol proteinase (e.g. cathepsin B), which were apparently inactivated by the weak bases (Table 1) and by the relatively high pH retained by myocyte lysosomes after being exposed to chloroquine, for example (Fig. 11). Thus, in summary, the mode of action of methyl esters of amino acids can be distinguished from that of lysosomotropic weak bases by at least five criteria: (1) they alter the permeability of the lysosomal membrane to acridine orange and other small molecules; (2) they appear to alter structurally secondary lysosomes only, (3) they do not inhibit or inactivate lysosomal cathepsin B activity and perhaps other thiol proteinases as well; (4) they do not markedly influence intralysosomal pH; and (5) they appear to be specific, reversible inhibitors of accelerated proteolysis. Although amino acid methyl esters cannot be strictly classified as lysosomotropic compounds (De Duve et al. 1974), under the present circumstances their major pharmacological site of action appears to be lysosomal; as such, the unique properties of this class of compounds make them especially valuable for studying the lysosomal vacuolar apparatus. 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