From the
In isolated rat hepatocytes, several tyrosine protein kinase
inhibitors (tyrphostins) reduced the autophagic sequestration of
electroinjected [
The pathways of autophagy and endocytosis are known to be
subject to regulation by protein phosphorylation. In isolated rat
hepatocytes, autophagy can be inhibited by cyclic AMP, an activator of
the cyclic AMP-dependent protein kinase
(1) , as well as by
okadaic acid
(2) , a protein phosphatase inhibitor that causes a
general hyperphosphorylation of liver cell proteins
(3) . The
effect of okadaic acid on autophagy can be antagonized by a specific
inhibitor of Ca
The endocytic pathway is apparently regulated by
protein phosphorylation at several steps, e.g. receptor
internalization
(4, 5) , receptor recycling
(4) ,
receptor routing
(6) , endosome-endosome
fusion
(7, 8) , endosome binding to
microtubules
(9) , and transport of endosomes along
microtubules
(10) . In addition, several endosomal proteins have
been shown to be phosphorylated
(11) . The present study was
undertaken to investigate the role of tyrosine protein phosphorylation
in regulation of autophagy and endocytosis in freshly isolated
hepatocytes. For this purpose we tested the effects of various tyrosine
protein kinase-inhibitory benzenemalononitrile derivatives, known as
tyrphostins. The tyrphostins were developed as substrate-competitive
inhibitors of the EGF
In cells treated with
okadaic acid (Fig. 7G) or tyrphostin 25
(Fig. 7F), endocytosed 10-nm AOM-gold accumulated
predominantly in multivesicular endosomes (). It thus
seems clear that different tyrphostins can affect different steps in
the endocytic pathway, in accordance with the results obtained by the
sucrose gradient analysis (Fig. 5) and the ligand recycling
studies (Fig. 6).
The effects of the tyrphostins indicate that autophagic
sequestration as well as receptor-mediated endocytosis may be dependent
on tyrosine protein phosphorylation. In contrast, increased
serine/threonine protein phosphorylation, induced by okadaic acid or
other protein phosphatase inhibitors, has been shown to inhibit
autophagy
(2, 24) . It is, therefore, possible that
members of the two classes of protein kinase may regulate autophagy
antagonistically.
All the active tyrphostins had to be given at
relatively high concentrations (100-300 µM) in order
to be effective on isolated hepatocytes. At these levels, some
cytotoxicity was observed with tyrphostins 23 and 63, but not with
tyrphostins 1, 25, 46, or 51. Among the latter, tyrphostin 46 was
uniquely autophagy-specific, whereas the other three inhibited both
autophagy and endocytosis. There was no correlation between the effects
of the individual tyrphostins on
Our results indicate that
tyrphostins have an early effect on
In contrast to their moderate effect on
Like tyrphostins 1 and 51, okadaic acid
caused retention of endocytosed ASGP in a recycling compartment
corresponding to light endosomes (1.10-1.11 g/ml). However, in
contrast to the tyrphostin-treated cells, these endosomes were
identified morphologically as multivesicular rather than as
tubulovesicular. Okadaic acid would thus seem to inhibit endocytosis at
a later step than tyrphostins 1 and 51, possibly by preventing the
detachment of multivesicular endosomes from a continuous endosomal
network
(30) containing tubulovesicular as well as
multivesicular elements of similar low density (1.10-1.11 g/ml).
Tyrphostin 25 would appear to inhibit endocytosis at an even later
step. In the presence of this drug, ASGP accumulated predominantly in
dense (1.14 g/ml), still multivesicular endosomes which had the
properties of a non-recycling compartment, indicating their detachment
from the recycling endosomal network to become ``carrier
vesicles'' (34). Tyrphostin 25 apparently inhibited fusion between
these dense multivesicular endosomes and lysosomes, thereby preventing
the formation of active, light (1.14-1.16 g/ml)
lysosomes
(35, 36) , or
``prelysosomes''
(37) , recognized by their contents of
both acid-soluble and acid-insoluble
Hepatocytes preloaded with
[
Lysosomes were
labeled by injecting rats intravenously with 3 nm of AOM-gold 24 h
before cell isolation. The freshly isolated hepatocytes were incubated
for 30 min at 37 °C with no additions (control) or in the presence
of 50 µM vinblastine, 30 nM okadaic acid, or 100
µM tyrphostin 1, 25, or 63. 10 nm of AOM-gold was then
added and the cells incubated for another 2 h at 37 °C. After the
incubation the cells were rapidly cooled to 0 °C and washed once in
ice-cold perfusion buffer containing 10 mM EGTA to remove
surface-bound ligand. The cells were processed for conventional
electron microscopy as described under ``Experimental
Procedures.'' Ten cell profiles from each treatment group were
examined, and the total number of 10-nm gold particles in all vacuoles
belonging to a certain morphologically defined compartment (endocytic
vesicles/tubules, multivesicular endosomes or lysosomes) was counted
and express as percent of the total number of vacuole-associated gold
particles/cell profile. Each value is the mean ± S.E. of 10 cell
profiles.
We thank Dr. Tor Gjand Prof. Trond Berg for
providing the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H]raffinose by 40-75% at
doses that did not significantly affect cellular ATP levels or plasma
membrane integrity. Tyrphostin 46 specifically inhibited autophagy,
whereas tyrphostins 1, 25 and 51 also suppressed the receptor-mediated
endocytic uptake of
I-tyramine-cellobiose-asialoorosomucoid,
I-TC-AOM, by 20-30% and its degradation by
70-90%. Tyrphostins 1 and 51, and the microtubule inhibitor
vinblastine, inhibited an early endocytic step (endosome
maturation/multivesiculation?), causing accumulation of endocytosed
I-TC-AOM in a recycling compartment that corresponded to
light endosomes (1.10-1.11 g/ml) in sucrose density gradients. In
the electron microscope, these endosomes could be recognized as small,
peripheral endocytic vesicles and tubules accumulating endocytosed
AOM-gold. The serine/threonine protein phosphatase inhibitor okadaic
acid inhibited an intermediate endocytic step (detachment of
multivesicular endosomes from the tubulovesicular network?), causing
accumulation of
I-TC-AOM in a recycling compartment
corresponding to light endosomes (1.10-1.11 g/ml), but with a
multivesicular rather than a tubulovesicular morphology. Tyrphostin 25
inhibited endocytosis at a late step (endosome-lysosome fusion?),
causing accumulation of
I-TC-AOM in a non-recycling
compartment corresponding to dense, multivesicular endosomes (1.14
g/ml) that had probably detached from the light endosomal network.
/calmodulin-dependent protein kinase
II (2), implicating this, or a closely related enzyme, in the negative
regulation of autophagy. On the other hand, several inhibitors of
tyrosine protein kinases suppress autophagy, suggesting that this class
of enzymes may be involved in positive control of the
process
(2) .
(
)
receptor tyrosine kinase
(EGFR)
(12) , and shown to be effective blockers of EGF-dependent
processes in intact cells, e.g. cell
proliferation
(13, 14) . Some of the tyrphostins can
inhibit EGF-independent tyrosine protein kinases as well, such as the
receptors for insulin, platelet-derived growth factor, or nerve growth
factor
(12, 15, 16) . We have measured autophagy
as the sequestration of electroinjected
[
H]raffinose
(17) and receptor-mediated
endocytosis as the uptake, recycling, and degradation of
I-TC-AOM. This probe has the advantage that acid-soluble
degradation products remain inside the vacuoles where they are
generated and can therefore be used to distinguish between proteolytic
and non-proteolytic endocytic vacuoles, separated on sucrose density
gradients
(18) . In addition, endocytic vacuoles have been
characterized ultrastructurally after uptake of AOM-coated gold
particles. The results indicate that tyrphostins can inhibit the
initial sequestration step of autophagy as well as several steps in the
endocytic pathway.
Cell Preparation and Incubation
Hepatocytes were
isolated from 18-h starved male Wistar rats (250-300 g) by
two-step collagenase perfusion
(19) . The cells were washed and
suspended in suspension buffer containing 15 mM pyruvate and
extra Mg (to 2 mM). Two ml of cell
suspension (15-20 mg cellular wet weight) were incubated at 37
°C in 5-cm albumin-coated plastic Petri dishes. For measurement of
endocytosis, the cells were preincubated for 30 min at 37 °C in the
presence of the drugs before addition of
I-TC-AOM, and
the cells were then incubated for a further 2 h at 37 °C. For
isopycnic centrifugation the cells were incubated with drugs for 30 min
at 37 °C, and then for a further 30 or 120 min in the presence of
I-TC-AOM. After the incubation the cells were cooled to 0
°C, transferred to 15-ml plastic centrifugation tubes, and washed
once with perfusion buffer containing 10 mM EGTA, pH 7.5, to
remove receptor-bound
I-TC-AOM from the cell surface. For
uptake and degradation measurements, the cells were washed twice with
10% sucrose (w/v) or once with perfusion buffer.
Measurement of Autophagy
Autophagy was measured as
the sequestration of electroinjected [H]raffinose
into sedimentable vacuoles as described previously
(17) .
Measurement of ATP
For measurement of
intracellular ATP, the incubated cells (2 ml) were precipitated with
0.5 ml of 10% perchloric acid and centrifuged for 15 min at 3700
g. A 1-ml aliquot of the supernatant was transferred
to a separate tube and neutralized with freshly made 2 M KOH.
The amount of ATP in the sample was measured luminometrically by use of
a luciferin/luciferase assay (Pharmacia LKB Biotechnology, Sweden).
Measurement of Protein Synthesis
Protein synthesis
was measured as the incorporation of [C]valine
into the acid-precipitable material of cells incubated in the presence
of amino acids
(20) .
Measurement of Uptake and Degradation of
After washing, the cell pellets were
counted in an LKB-gamma counter to measure the cellular uptake of
I-TC-AOM
I-TC-AOM, i.e. the amount of cell-associated
radioactivity, expressed as the percent of the total acid-insoluble
radioactivity initially added. To each pellet, 1 ml of 10%
trichloroacetic acid was added, the samples were kept on ice for a
minimum of 15 min, and thereafter centrifuged for 15 min at 3700
g. The supernatant, containing the acid-soluble
degradation products of
I-TC-AOM, was transferred to a
separate tube and counted. The amount of
I-TC-AOM
degraded was calculated as a percentage of the total acid-insoluble
radioactivity added to each sample initially.
Measurement of
To
measure the release of endocytosed I-TC-AOM Recycling
I-TC-AOM from
ligand-recycling compartments, hepatocytes were incubated for 30 min at
37 °C in the presence of drugs.
I-TC-AOM was then
added, and the cells incubated for another 60 min. The cells were then
rapidly cooled by addition of ice-cold buffer and washed once at 0
°C with buffer containing 10 mM EGTA to remove
surface-bound ligand. The cells were subsequently incubated at 37
°C in the presence of 2.5 mM EGTA for up to 60 min. In the
presence of the chelator, any AOM that recycles to the cell surface and
is released into the medium will be prevented from rebinding to the
receptor. The amount of recycled
I-TC-AOM was measured
and expressed as percent of the total cell-associated radioactivity.
Cell Fractionation and Isopycnic Sucrose Density Gradient
Centrifugation
All fractionation and gradient work was performed
at 0 °C. Cell samples were resuspended in 0.5 ml of unbuffered 10%
sucrose and pooled before electrodisruption. An equal volume of
buffered sucrose (0.25 M sucrose, 10 mM HEPES, 1
mM EDTA, pH 7.3) was then added to each sample, followed by
10-20 strokes in a Dounce homogenizer with a tight-fitting
pestle. The homogenate was centrifuged at 3000 g
min to obtain a nuclear pellet and an post-nuclear
supernatant. The nuclear pellet was washed once in 4.5 ml of buffered
sucrose, and the two post-nuclear supernatants were combined. A 1.5-ml
sample of post-nuclear supernatant was layered on top of a linear
sucrose density gradient. The gradient was made by layering 5.5 ml of
63% buffered sucrose underneath 5.5 ml of 15% buffered sucrose and
mixing with a Biocomp Gradient Master (Nycomed Pharma, Oslo, Norway) to
obtain densities ranging from 1.04 g/ml in the top fraction to 1.23
g/ml in the bottom fraction. The gradients were centrifuged at 4 °C
in a Beckman L8-70 M ultracentrifuge with an SW40/9E
2800 rotor for 1 h 45 min at 40,000 revolutions/min (3.1
10
g
min). Following centrifugation the gradients were split
into approximately 20 fractions by upward displacement using Maxidenz
as a displacement fluid. The densities of the fractions were calculated
from the refractive indices. Acid phosphatase activity was determined
spectrophotometrically in a Technicon RA-1000 autoanalyzer and
expressed as percentage of the total acid phosphatase in the cell.
Total radioactivity, acid-soluble, and acid-insoluble radioactivity in
each fraction were measured in a gamma counter as described above,
except that albumin was added to a final concentration of 0.1% (w/v)
before acid precipitation (0.4 ml gradient fraction, 0.1 ml of albumin,
and 0.5 ml of 20% trichloroacetic acid). Radioactivities were expressed
as percentage of the total acid-insoluble radioactivity initially added
to the sample.
Electron Microscopy
Colloidal gold particles (3-
and 10-nm diameter) were prepared according to Slot and Geuze
(21) and coated with AOM by adsorption according to established
procedures. Gold sol (250 ml) was complexed with AOM (1.5 mg for 10-nm
gold; 0.75 mg for 3-nm gold) by mixing at room temperature, and the
gold-protein complexes recovered by centrifugation at 47,000
g for 120 min (3-nm) or 45,000
g for 45 min
(10-nm). The gold-AOM probes were dialyzed against 500 ml of 0.9% NaCl
(w/v) overnight. To prelabel hepatocytic lysosomes, 1 ml of 3-nm
AOM-gold in collodial suspension (with an absorbance value of 1.70 at
520 nm after 100-fold dilution) was injected intravenously into rats 24
h before cell isolation. To label endosomes and lysosomes in
vitro, 2 ml of hepatocytes in suspension (15 mg cells/ml, wet
weight) were incubated at 37 °C with 15 µl of 10-nm AOM-gold
(final absorbance of 2.0 at 520 nm) for 120 min. The cells were washed
once in perfusion buffer containing 10 mM EGTA to remove dead
cells and surplus gold-AOM, and twice in ice-cold 0.1% glutaraldehyde,
0.1 M cacodylate buffer, pH 7.4. Pellets were fixed in 2%
glutaraldehyde, 0.1 M cacodylate buffer overnight and
postfixed for 60 min with 2% OsO
containing 1.5% potassium
ferrocyanide, followed by en bloc staining with 1.5% uranyl
acetate. After serial dehydration in ethanol and propylene oxide,
specimens were embedded in Epon, sectioned, and post-stained with 0.2%
lead citrate. Sections were examined in a Phillips CM10 electron
microscope at 80 kV.
Reagents
I-TC-AOM was kindly
provided by dr. Tor Gjand Prof. Trond Berg, University of
Oslo. [
H]Raffinose (5 Ci/mmol, 1 Ci/liter) was
from NEN Du Pont and [
C]valine (260 Ci/mol, 50
mCi/l) from Amersham International, Amersham, Bucks, United Kingdom.
Okadaic acid was from Moana Bioproducts Inc., Hawaii. Metrizamide and
Maxidenz were from Nycomed A/S, Oslo, Norway. Texas red sulfonyl
chloride (T-1905) and rhodamine green succinimidyl ester (R-6112) were
purchased from Molecular Probes Inc., Eugene, OR. Tyrphostins and all
other biochemicals were from Sigma.
Effects of Tyrphostins on Autophagic Sequestration,
Protein Synthesis, Intracellular ATP Levels, and Viability
The
structural formulas of the tyrphostins investigated in this study, and
their potencies against the EGF receptor tyrosine protein kinase
(EGFR), are listed in Fig. 1. All of the tyrphostins tested
inhibited the autophagic sequestration of
[H]raffinose (). Tyrphostins 25, 46,
and 51 inhibited autophagy by 70-76%, tyrphostin 1 by 54%,
without significantly affecting hepatocytic integrity (measured as the
loss of total cellular radioactivity). Tyrphostin 25 was somewhat more
potent than the other two, with maximal effect at 100 µM.
At higher concentrations, cytotoxicity (reduced cellular integrity)
became more prominent. With tyrphostins 23 and 63, cytotoxicity was
evident already at 30 and 150 µM, respectively.
Figure 1:
Molecular structure of the tyrphostins
used in the present study. The data, including the potency of
tyrphostins against EGF-receptor tyrosine kinase activity, have been
adapted from Ref. 12.
Protein
synthesis was inhibited by 15-40% at nontoxic tyrphostin
concentrations. Intracellular ATP levels were not reduced by any of the
tyrphostins tested, even at high concentrations, showing that the drugs
did not interfere with the mitochondrial respiratory chain as has been
reported for some tyrphostins
(22) . The inhibition of autophagy
by tyrphostins would, therefore, be unlikely to be the result of
nonspecific cytotoxicity.
Effects of Tyrphostins on Uptake and Degradation of an
Endocytic Probe
Isolated hepatocytes rapidly endocytose
I-TC-AOM by a receptor-mediated mechanism
(18) :
within 2 h, 80% of the added probe had been taken up by control cells
(Fig. 2A), and approximately 50% of this had become
degraded (Fig. 2B). Two of the tyrphostins (1 and 25)
had moderate effects on
I-TC-AOM uptake
(Fig. 2A) but strong inhibitory effects on
I-TC-AOM degradation (Fig. 2B). Similar
effects were exerted by the microtubule inhibitor, vinblastine, the
serine/threonine protein phosphatase inhibitor, okadaic acid, and,
somewhat paradoxically, by the tyrosine protein phosphatase inhibitor,
vanadate. Fig. 3shows dose-response curves for the effects of
various tyrphostins on uptake and degradation of
I-TC-AOM
during a 3-h incubation period. Tyrphostins 23 (panel A), and
46 (panel B) caused only small alterations in the uptake and
degradation of
I-TC-AOM, even at the highest doses
employed. Tyrphostins 1, 25, 51, and 63 (panels C-F) had
moderate inhibitory effects (30% or less) on
I-TC-AOM
uptake, but inhibited the degradation of endocytosed
I-TC-AOM strongly (70-90%) in the concentration
range 100-300 µM. These tyrphostins would thus seem
to be effective inhibitors of
I-TC-AOM
endocytosis/degradation at some step(s) beyond ligand uptake.
Figure 2:
Effects of tyrphostins, okadaic acid,
vinblastine, and vanadate on accumulation and degradation of
endocytosed I-TC-AOM. Hepatocytes were incubated for 30
min at 37 °C without additions (
), with 50 µM
vinblastine (
), 30 nM okadaic acid (
), 100
µM tyrphostin 1 (
), 100 µM tyrphostin
25 (
), or 10 mM vanadate (
).
I-TC-AOM was then added, and the cells incubated further
at 37 °C for the length of time indicated. The cells were then
cooled to 0 °C, washed, precipitated with ice-cold 10%
trichloroacetic acid, and the total cell-associated radioactivity
(A) as well as the acid-soluble radioactivity (B) was
measured and expressed as percent of the acid-insoluble radioactivity
initially added. The data are from a single
experiment.
Figure 3:
Effects of tyrphostins on endocytic uptake
and degradation of I-TC-AOM. Hepatocytes were incubated
for 30 min at 37 °C, in the presence of tyrphostin at the doses
indicated.
I-TC-AOM was then added, and the incubation
continued for another 2 h at 37 °C. The cells were then cooled to 0
°C, washed, and precipitated with 10% ice-cold trichloroacetic
acid. The total cell-associated radioactivity (uptake,
), as well
as the acid-soluble radioactivity (degradation,
), was measured
and expressed as percent of the acid-insoluble radioactivity initially
added. Each value is the mean ± S.E. of three to six independent
experiments.
Effects of Tyrphostins on Density Distribution of
After 30
min of continuous I-TC-AOM-containing Endocytic Vacuoles
I-TC-AOM uptake, radiolabeled endocytic
vacuoles from control hepatocytes banded in a sucrose density gradient
as a major peak at 1.10 g/ml, with a shoulder at 1.13-1.14 g/ml
(Fig. 4). This distribution was not much changed by any of the
inhibitors applied, although the light peak was somewhat sharper and
the heavy shoulder somewhat less developed after treatment with okadaic
acid, vinblastine, and some of the tyrphostins (1, 51, and 63). At this
time, no radioactive material coincided with the lysosomes, i.e. the major peak of acid phosphatase at 1.18 g/ml. Vinblastine
shifted this peak to a lighter position (1.14 g/ml), whereas the other
treatments had no effect on lysosome distribution.
Figure 4:
Effects of tyrphostins on density
distribution of endocytic vacuoles after 30 min of
I-TC-AOM endocytosis. Hepatocytes were incubated for 30
min with no additions (control), or in the presence of 30 nM
okadaic acid, 50 µM vinblastine, 30 µM
tyrphostin 23, 100 µM tyrphostins 46, 25, or 1, 300
µM tyrphostin 51 or 150 µM tyrphostin 63.
I-TC-AOM was then added and the cells incubated for
another 30 min at 37 °C, then rapidly cooled to 0 °C, and
washed twice with 10% sucrose, electrodisrupted, and homogenized.
Post-nuclear supernatants were prepared and fractionated by isopycnic
sucrose density gradient centrifugation. Radioactivity was measured and
acid phosphatase (ACP) assayed in each fraction, and the
values per fraction are expressed as percent of the total
cell-associated radioactivity (
) or acid phosphatase (
). The
data are from a single experiment.
After 2 h of
I-TC-AOM endocytosis, some of the radioactivity in
control cells was still present in the light gradient peak at 1.10
g/ml, but most of it had been shifted to peaks at 1.14 and 1.18 g/ml,
the latter colocalizing with the lysosomal marker enzyme
(Fig. 5). The radioactivity at 1.18 g/ml was largely
acid-soluble, indicating that
I-TC-AOM had been degraded
by lysosomal enzymes (results not shown). Cells treated with
tyrphostins 23 or 46 (which had negligible effects on
I-TC-AOM endocytosis, cf.Fig. 3
)
displayed the same distribution as control cells.
Figure 5:
Effects of tyrphostins on density
distribution of endocytic vacuoles after 120 min of
I-TC-AOM endocytosis. The experiment and the analyses
were performed as described in the legend to Fig. 4, except that the
hepatocytes were incubated for 120 min after the addition of
I-TC-AOM. The data are from a single
experiment.
In cells treated
with okadaic acid, a major fraction of the endocytosed
I-TC-AOM was still retained at 1.10 g/ml after 120 min,
although some had also reached the lysosomal peak at 1.18 g/ml
(Fig. 5). With tyrphostin 25, most of the radioactivity
accumulated at 1.14 g/ml, less at 1.10 g/ml, and very little at 1.18
g/ml. Tyrphostins 1, 51, and 63 produced yet another pattern,
essentially the same as vinblastine, with radioactivity retained in a
major peak at 1.10 g/ml and a minor peak at 1.14 g/ml, and little or
none at 1.18 g/ml. Unlike the tyrphostins, vinblastine shifted the
lysosome peak to 1.14 g/ml, but no acid-soluble radioactivity was found
in this region in either vinblastine- or tyrphostin-treated cells
(results not shown).
Effects of Tyrphostins on Ligand
Recycling
Endocytosed I-TC-AOM undergoes extensive
recycling from early endosomes, but when the probe reaches late
endosomes it dissociates from the receptor and recycling gradually
decreases
(18) . Fig. 6shows the degree of
I-TC-AOM recycling, measured as the release of
radioactivity from the cells in the presence of EGTA (to cause ligand
detachment at the cell surface), during a 1-h incubation in the
presence of okadaic acid, vinblastine, or various tyrphostins.
Vinblastine increased the extent of recycling 4-fold, and okadaic acid
3-fold, indicating increased retention of ligand in a recycling
compartment (Fig. 6A). Tyrphostins 1, 51, and 63
similarly increased
I-TC-AOM recycling severalfold
(Fig. 6B). Tyrphostins 23 and 46, which had little or no
effect on
I-TC-AOM endocytosis, respectively
(Fig. 3), also had little or no effect on recycling
(Fig. 6A). Tyrphostin 25 caused much less recycling than
the other active tyrphostins (Fig. 6B), suggesting
ligand retention primarily in a non-recycling compartment.
Figure 6:
Effects of tyrphostins on ligand
recycling. Hepatocytes were incubated for 30 min in the presence of
various drugs as indicated. A, , no addition (control);
, 50 µM vinblastine;
, 30 nM okadaic
acid;
, 100 µM tyrphostin 46;
, 30
µM tyrphostin 23. B,
, no addition
(control);
, 100 µM tyrphostin 25;
, 300
µM tyrphostin 51;
, 100 µM tyrphostin
1;
, 150 µM tyrphostin 63.
I-TC-AOM
was then added, and the incubation continued for 1 h at 37 °C. The
cells were then rapidly cooled to 0 °C and washed once in ice-cold
perfusion buffer containing 10 mM EGTA to remove surface-bound
ligand. Finally, the cells were incubated in suspension buffer
containing 2.5 mM EGTA (to prevent rebinding of released
ligand) at 37 °C for the length of time indicated.
I-TC-AOM released to the medium was measured and
expressed as percent of the total radioactivity in the system. Each
value is the mean ± S.E. or range of two to eight
experiments.
Effects of Tyrphostins on the Distribution of Endocytic
Vacuoles Identified by Electron Microscopy
To obtain a
structural identification of the endocytic vacuoles affected by
tyrphostins, hepatocytes were prelabeled with 3 nm of AOM-gold in
vivo overnight (to label lysosomes) and allowed to endocytose 10
nm of AOM-gold during the period of drug treatment (to identify
endosomes). In control cells, three major vacuole types could be
identified: 1) small endocytic vesicles and tubules (50-100 nm
diameter). These were the predominant structures labeled with 10 nm of
AOM-gold after 5 min of endocytosis (Fig. 7A). 2)
Multivesicular endosomes (200-600 nm diameter), the predominant
type of endosome at later times of endocytosis
(Fig. 7B). 3) Lysosomes (300-1500 nm diameter),
recognized as vacuoles containing 3 nm of AOM-gold or visibly degraded
material of cellular origin (Fig. 7C). Both small, dense
lysosomes (Fig. 7C, upper right-hand corner)
and large, electron-lucent lysosomes (Fig. 7C, lower
left-hand corner) could be seen; in addition, the lysosome class
as defined here may include some of the ``prelysosomes'' and
``late endosomes'' characterized by other workers on the
basis of different criteria
(23) .
Figure 7:
Morphology of endocytic vacuoles.
Hepatocytes, isolated from a rat injected with 3-nm AOM-gold 24 h
before sacrifice, were preincubated for 30 min without additions
(control, A-C), 50 µM vinblastine (D),
100 µM tyrphostin 1 (E), 100 µM
tyrphostin 25 (F), or with 30 nM okadaic acid
(G). 10-nm AOM-gold was then added, and the cells incubated
for another 2 h at 37 °C before harvesting and processing for
conventional electron microscopy. 3-nm gold was found in both light and
dense lysosomes (C). Endocytosed 10-nm gold could be found in
early endocytic vesicles/tubules (A, D, and
E), in multivesicular endosomes (B, F, and
G) and in light and dense lysosomes (C). In cells
treated with vinblastine (D) or tyrphostin 1 (E),
10-nm gold was present mainly in early endocytic vesicles/tubules. In
cells treated with tyrphostin 25 (F) or okadaic acid
(G), 10-nm gold was primarily found in multivesicular
endosomes. Bar, 100 nm.
A quantitative morphometric
analysis revealed that after 2 h of endocytosis in control cells, the
bulk of the endocytosed 10-nm AOM-gold was approximately equally
distributed between multivesicular endosomes and lysosomes, only a
small fraction (4%) remaining in endocytic vesicles/tubules
(). A similar distribution was seen in cells treated with
tyrphostin 46, which does not affect endocytosis detectably. However,
vinblastine treatment altered the distribution radically, causing
two-thirds of the endocytosed 10-nm AOM-gold to be retained in
endocytic vesicles/tubules, while hardly anything (2%) reached the
lysosomes (Fig. 7D, ). A similar
distribution was seen in cells treated with tyrphostin 1
(Fig. 7E, ).
I-TC-AOM degradation and
their reported potencies as inhibitors of the EGFR
(12) : two of
the most effective inhibitors in our studies, tyrphostins 1 and 63, are
in fact essentially inactive as EGFR inhibitors (cf.
Fig. 1
). Wijetunge et al.
(25) concluded that the
cellular effects of tyrphostin 1 could not be due to EGFR inhibition,
and other observations likewise indicate that tyrphostin effects are
not necessarily the result of inhibition of tyrosine protein
kinases
(22) . Any implication of these enzymes to explain the
effects of tyrphostins on autophagy or endocytosis should, therefore,
be regarded as tentative. As an additional paradox, a tyrosine protein
phosphatase inhibitor, vanadate, has been found to inhibit
asialoglycoprotein (ASGP) endocytosis
(26) as effectively as do
the tyrosine protein kinase inhibitors.
I-TC-AOM uptake as
well as later effects on intracellular endocytic flux. The ASGPR has a
single cytoplasmic tyrosine residue that might be a substrate for a
receptor-associated tyrosine protein kinase
(27) , although the
functional role of phosphorylation at this tyrosine has been
questioned
(28) . Fallon et al. showed that several
tyrosine kinase inhibitors, among them genistein and a tyrphostin,
inhibited tyrosine phosphorylation of the ASGPR in vitro(5) and internalization of the ASGPR in intact HepG2 cells,
suggesting a causal relationship. In our studies, we found no effect of
genistein at concentrations that strongly affected other hepatocellular
processes
(2) and at most a 30% inhibition of
I-TC-AOM uptake by the tyrphostins. In normal
hepatocytes, at least, tyrosine phosphorylation of the ASGPR would,
therefore, seem to have a modulating rather than an obligatory function
in ASGP uptake.
I-TC-AOM uptake, several tyrphostins strongly reduced the
transfer of
I-TC-AOM to lysosomes and its degradation to
acid-soluble material. Tyrphostins 1 and 51, as well as the
microtubule-disrupting drug vinblastine, exerted their effect at a
relatively early endocytic step, causing accumulation of endocytosed
ASGP in a recycling compartment identified as small (50-100 nm),
light (1.10-1.11 g/ml) endocytic vesicles and tubules. These
tyrphostins may thus inhibit the microtubule-dependent
(29) transfer of ligand from tubulovesicular to multivesicular
endosomes within a continuous endocytic network
(30) , possibly
by interfering with endosome
maturation/multivesiculation
(31, 32) . However, the
tyrphostins did not share the ability of vinblastine to disrupt the
hepatocytic microtubule organization,
(
)
suggesting a different mechanism of action, e.g. interference with the binding of endosomes to
microtubules
(33) .
I-TC-AOM-derived
material. This precluded the subsequent degradation of
I-TC-AOM and the concomitant formation of dense
(1.18-1.19 g/ml) lysosomes containing acid-soluble degradation
products only. A tyrosine protein kinase involvement at a late step in
ASGP endocytosis has previously been suggested
(38) , but on the
basis of inhibition by staurosporine, a broadly nonspecific protein
kinase inhibitor. Hopefully, the use of tyrphostins with different
specificities may help to elucidate the complexity of the endocytic
pathway in greater detail.
Table:
Effects of tyrphostins on autophagic
sequestration, protein synthesis, intracellular ATP levels, and
cellular integrity
H]raffinose were incubated for 3 h at 37 °C
in the presence of tyrphostins at the concentrations indicated.
Autophagy was measured as the net sequestration of
[
H]raffinose, and expressed as percent of the
autophagic rate measured in control cells (3.7%/h). Cellular (plasma
membrane) integrity was measured as the loss of total cellular
radioactivity between 1 and 3 h of incubation, and expressed as percent
of the loss from control cells (3.0%/h). Protein synthesis was measured
as the net incorporation of [
C]valine during a
3-h incubation in the presence of a complete, balanced amino acid
mixture, and expressed as percent of the protein synthesis rate
measured in control cells (0.5%/h). ATP was measured luminometrically
in acid-precipitated cells after 3 h of incubation at 37 °C, and
expressed as percent of the ATP level measured in control cells (2.1
µg/g cellular wet mass). Each value is the mean ± S.E. or
range of the number of experiments given in parentheses.
Table:
Effects of okadaic acid, vinblastine, and
various tyrphostins on endocytosis of 10 nm gold-AOM
I-TC-AOM. The skillful technical assistance
of Mona Birkeland is gratefully acknowledged.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.