Download responses of cultured cardiac myocytes to lysosomotropic

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. Since the lysosomal changes induced by amino acid methyl esters are accompanied by minimal changes in other cell organelles, these rapidly acting, selective probes
should prove useful in the further clarification of secondary lysosomal function in a
variety of cultured cells.
These studies were supported by grants from the National Heart, Lung, and Blood Institute
(HL17669 and HL06296). The authors express their gratitude to Ms Carole Becker for preparing
this manuscript. Dr R. S. Decker is an Established Investigator of the American Heart Association.
REFERENCES
ALLISON, A. C. & YOUNG, M. R. (1964). Uptake of dyes and drugs by living cells in culture. Life
Sci. 3, 1401-1414.
ALLISON, A. C. & YOUNG, M. R. (1969). Vital staining and fluorescence microscopy of lysosomes.
In Lysosomes in Biology and Pathology (ed. J. T. Dingle & H. B. Fell), vol. 3, pp. 600-628.
Amsterdam: North Holland.
BARKA, T. & ANDERSON, P. J. (1962). Histochemical methods for acid phosphatase using
hexazonium pararoszanilin as a coupler. J. Histochem. Cytochem. 10, 453—471.
BARRETT, A. J. (1980). The many forms and functions of cellular proteinases. Fedn Proc. FednAm.
Socs exp. Biol. 39, 9-14.
BARRETT, A. J. & DINGLE, J. T. (1969). A lysosomal component capable of binding cations and
carcinogen. Biochem.J. 105, 20.
BARRETT, A. J. & HEATH, M. F. (1977). Lysosomal enzymes. In Lysosomes: A Laboratory Handbook (ed. J. T. Dingle), p. 119. Amsterdam: North Holland.
CANONICO, P. G. & BIRD, J. W. C. (1969). The use of acridine orange as a lysosomal marker in
rat skeletal muscle. J . Cell Biol. 43, 367-371.
134
R. S. Decker, M. L. Decker, V. Thomas andjf. W. Fuseler
DEAN, R. T . (1980). Protein degradation in cell cultures: general considerations on mechanisms
and regulation. Fedn Proc. FednAm. Socs exp. Biol. 39, 15-19.
DECKER, R. S., DECKER, M. L., CANON, M. K. & THOMAS, V. (1982). Alterations in cathepsin
D and protein degradation in foetal rabbit heart myocytes. J. Cell Biol. 97, 105a.
DECKER, R. S. & FUSELER, J. F. (1984). Methylated amino acids and lysosomal function in cultured
heart cells. Expl Cell Res. 154, 304-309.
D E DUVE, C , D E BORSY, T., POOLE, B., TROUET, A., TULKENS, P. & VAN HOOF, F. (1974).
Lysosomotropic agents. Biochem. Pharmac. 23, 2495-2531.
DINGLE, J. T . & BARRETT, A. J. (1969). Accumulation of various substances within lysosomes.
Biochem. J. 105,22.
FRIEND, D. S. & FARQUHAR, M. G. (1967). Functions of coated vesicles during protein absorption
in the rat vas deferens. J. Cell Biol. 35, 357-376.
GOLDMAN, R. (1973). Dipeptide hydrolysis within intact lysosomes in vitro. FEBS Lett. 33,
208-212.
GOLDMAN, R. (1976). In Lysosomes in Biology and Pathology (ed. J. T . Dingle & R. T . Dean),
vol. 5. pp. 305—336. Amsterdam: North Holland.
GOLDMAN, R. & KAPLAN, A. (1973). Rupture of rat liver lysosomes mediated by L-amino acid
esters. Biochim. biophys. Acta 318, 205-216.
GOLDMAN, R. & NAIDER, F. (1974). Permeation and stereospecificity of hydrolysis of peptide
esters within intact lysosomes in vitro. Biochim. biophys. Acta 388, 224—233.
GONZALEZ-NORIEGA, A., GRUBB, J. H., TALKAD, V. & SLY, W. S. (1980). Chloroquine inhibits
lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor
recycling. J. Cell Biol. 85, 839-852.
HASILIK, A. &NEUFELD, E. F. (1980a). Biosynthesis of lysosomal enzymes in fibroblasts-synthesis
as precursors of high molecular weight. J. biol. Chem. 255, 4937—4945.
HASILIK, A. & NEUFELD, E. F. (19806). Biosynthesis of lysosomal enzymes in fibroblasts—
phoaphorylation of mannose residues. J. biol. Chem. 255, 4946—4950.
HICKMAN, S. L., SHAPIRO, L. J. & NEUFELD, E. F. (1974). A recognition marker for the uptake
of lysosomal enzymes by cultured fibroblasts. Biochem. biophys. Res. Commun. 57, 55-61.
HOLTZMAN, E. (1976). Lysosomes: A Survey, pp. 54—59. Wien, New York: Springer-Verlag.
KAPLAN, A., FISCHER, H. D., ACHORD, D. & SLY, W. (1977). Phosphohexozyl recognition is a
general characteristic of pinocytosis of lysosomal glycosidases by human fibroblasts. J. clin.
Invest. 60, 1088-1093.
KASTAN, F. (1973). Mammalian myocardial cells. In Tissue Culture Methods and Applications (ed.
P. F. Kruse, Jr & M. K. Patterson), pp. 72-81. New York: Academic Press.
LONG, W. H., CHUA, B. H. L., LAUTENSACK, N. & MORGAN, H. E. (1982). Effects of amino
acid methyl esters on cardiac lysosomes and proteolysis. Fedn Proc. Fedn Am. Socs exp. Biol.
41, 881.
LOWRY, O. H., ROSEBROUGH, M. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement
with the Folin phenol reagent. J. biol. Chem. 193, 265-275.
MAXFIELD, F. R. (1982). Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in clutured mouse fibroblasts. J'. Cell Biol. 95, 676-681.
MILLER, O. K., GRIFFITHS, E., LENARD, J. & FIRESTONE, R. A. (1983). Cell killing by
liposomotropic detergents. J. Cell Biol. 97, 1841-1851.
MOSES, R. L. & CLAYCOMB, W. C. (1982). Ultrastructure of terminally differentiated adult rat
cardiac muscle cells in culture. Am.J. Anat. 164, 113—131.
NATH, K., SHAY, J. W. & BOLLAN, A. P. (1978). Relationship between dibutyl cyclic AMP and
microtubule organization in contracting heart muscle cells. Proc. natn. Acad. Sci. U.SA. 75,
319-323.
NOVIKOFF, P. M., NOVIKOFF, A. B., QUINTANA, N. & HAUW, J.-J. (1971). Golgi apparatus,
GERL, and lysosomes of neurons in rat dorsal root ganglia studied by thick and thin section
cytochemistry. J. Cell Biol. 50, 859-886.
OHKUMA, S. & POOLE, B. (1978). Fluorescence probe measurement of the intralysosomal pH in
living cells and perturbation of pH by various agents. Proc. natn. Acad. Sci. U.SA. 75,
3327-3331.
OHKUMA, S. & POOLE, B. (1981). Cytoplasmic vacuolation of mouse peritoneal macrophages and
the uptake into lysosomes of weakly basic substances. J. Cell Biol. 90, 656-664.
Lysosomal effects of amino acid methyl esters
135
POOLE, A.R. (1977). The detection of lysosomes by vital staining with acridine orange. inLysosomes:
A Laboratory Handbook (ed. J. T . Dingle), pp. 313-316. Amsterdam: North Holland.
POOLE, B. & OHKUMA, S. (1981). Effect of weak bases on the intralysosomal pH in mouse
peritoneal macrophages.J. Cell Biol. 90, 665-669.
RABON, E., CHANG, H. H. & SACHS, G. (1978). Quantitation of H + and potential gradients in
gastric plasma membrane vesicles. Biochemistry 17, 750-753.
REEVES , J. P. (1979). Accumulation of amino acids by lysosomes incubated with amino acids methyl
esters. 7. biol. Chem. 254, 8914-8921.
REEVES, J. P., DECKER, R. S., CRIE, J. S. & WILDENTHAL, K. (1981). Intracellular disruption of
rat heart lysosomes by leucine methyl ester: Effects on protein degradation. Proc. natn.Acad. Set.
U.SA. 78, 4426-4429.
ROSENFELD, M. G., KREIBICH, G., POPOV, D., KATO, K. & SABATINI, D. D. (1982). Biosynthesis
of lysosomal hydrolases: Their synthesis in bound polysomes and the role of co - and posttranslational processing in determining their subcellular distribution. J. Cell Biol. 93, 135-143.
SAMAREL, A. M., FERGUSON, A. G., WOROBEC, S. W. & LESCH, M. (1984). Transport and
proteolytic processing of rabbit cardiac cathepsin D. Am. J. Pkysiol. 248, C135-C144.
SANDO, G. N. &NEUFELD, E. F. (1977). Recognition and receptor-mediated uptake of a lysosomal
enzyme, a-L-idouonidase, by cultured human fibroblasts. Cell 12, 619-627.
STEINER, F. & BEERS, R. F. (1961). Polynucleotides, Natural and Synthetic Acids, pp. 179-184.
Amsterdam: Elsevier.
SZASZ, G., BUSCH, E. W. & TAROKS, H. B. (1970). Serum kreatin-kinase. I. Methodiside erfahrungen und normal verte mit einem neuen handelpublischen test. Dt. med. Wschn. 95, 829—835.
TYCKO, B. & MAXFIELD, F. R. (1982). Rapid acidification of endocytic vesicles containing a 2
macroglobulin. Cell 28, 643-651.
WIBO, M. & POOLE, B. (1974). Protein degradation in cultured cells. II. The uptake of chloroquine
by rat fibroblasts and the inhibition of cellular protein degradation and cathepsin B. J. Cell Biol.
63, 430-440.
WILCOX, P. & RATHRAY, R. (1979). Secretion and uptake of /3-7V-acetyl glucosaminidase by
fibroblasts. Biochim. biophys. Ada 586, 442-452.
(Received 13 June 1984-Accepted, in revised form, 17 September 1984)