Vol. 276, Issue 5, E945-E954, May 1999
Redox-dependent and redox-independent subcomponents of protein
degradation in perfused myocardium
Thomas D.
Lockwood
Department of Pharmacology and Toxicology, School of Medicine,
Wright State University, Dayton, Ohio 45435
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ABSTRACT |
The integration of proteolytic pathways with
metabolism was investigated in perfused rat myocardium. After a 10-min
incorporation period, the minute-to-minute release of
[3H]leucine from
myocardial proteins was measured in nonrecirculating effluent
perfusate. The nontoxic pro-oxidant probe diamide (100 µM) or a
supraphysiological concentration of the endogenous oxidative metabolite
dehydroascorbic acid (200 µM) reversibly inhibited 75% of myocardial
proteolysis consisting of several known subcomponents (redox
dependent); however, 25% of proteolysis was diamide insensitive (redox
independent). Decrease in extracellular glucose concentration from 10 to 0.1 mM strongly increased the potencies of minimally effective
concentrations of diamide (10 µM) or dehydroascorbic acid (15 µM)
by ~10-fold to the respective potencies maximally inhibiting
proteolysis. The reversal of diamide action was also strongly dependent
on the perfusate glucose concentration observed at 0.1, 0.2, 1.0 and 10 mM glucose. Proteolytic inhibition caused by diamide (100 µM) was not
accompanied by change in basal tissue ATP content of 5 µmol/g wet wt.
Conversely, nearly lethal 60% ATP depletion caused by sodium azide
(0.4 mM) was not accompanied by change in total
[3H]leucine release.
Results indicate that a large proteolytic subcomponent (75%) is
maintained by redox chains fed by glucose; however, there is no
apparent linkage of this proteolysis to short-term ATP fluctuations. A
distinct major proteolytic subcomponent (25%) does not vary in
response to experimental intervention in either ATP content or redox chains.
proteolysis; oxidation-reduction; adenosine 5'-triphosphate; diamide; sodium azide; glucose
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INTRODUCTION |
CELL PROTEINS ARE degraded by alternative major
pathways. Subcomponents of cell proteolysis are incompletely identified
and characterized with regard to such features as subcellular location, compartmental control, substrate designation reactions, proteases involved, substrate translocation, integration with cell metabolism, neuroendocrine and postreceptor controls, and roles in cell function (2, 23, 27).
Although peptide bond hydrolysis is exergonic, an unexplained energy
requirement exists for at least some intracellular proteolysis. Exposure of cultured cells to metabolic poisons can decrease averaged cell proteolysis before cell death; however, proteolysis is
proportional to ATP depletion down to 5% of normal ATP content (12).
Some proteolytic systems are directly activated by ATP, and some
protein degradation might be influenced by the ATP requirement of the ubiquitin conjugation reactions (2, 23, 27). Other conceivable ATP
requirements for proteolysis include proton or other transport processes, cytoplasmic motility driving vesicular fusions or fluid motion, and substrate translocation. However, it is not known how much
of total cell protein degradation has a direct ATP requirement, or
whether any such energy requirement is ever limiting under the range of
nonlethal ATP fluctuations. Second, severe experimental ATP depletion
can be associated with many additional covariables that might alter
proteolysis indirectly.
Energy from glucose can be transferred to various cell processes by two
major networks serving distinct cell functions; however, no studies
have determined whether any amount of proteolysis is responsive to
selective fluctuations of either network. The glycolytic pathway and
Krebs cycle provide high-energy phosphate bond energy serving such
processes as transport, macromolecular synthesis, and mechanical
contraction. The hexose monophosphate (HMP) pathway transfers reductive
energy from glucose to the cell redox chains serving various
bioreductive processes. Among cell reductive processes is the reduction
of enzymes and nonenzymatic proteins by several protein reductase
systems deriving energy from glucose via NADPH or glutathione (see the
DISCUSSION) (9, 14, 15, 26). The
separation of the high-energy phosphate system and reducing system
permits separate control of distinct energetic functions; however,
interconversion pathways provide some degree of metabolic transfer
between these networks. Attempts at selective experimental interventions in either the glycolytic branch or HMP pathway can be
imperfect and subject to interpretive reservations depending on the
outcome. Nonetheless, several experimental interventions can be
employed to determine whether a particular phenomenon is a metabolic
parameter of selective changes in either high-energy phosphates or the
cell redox chains (described below) (18, 35).
We have distinguished four subcomponents of total myocardial protein
degradation thus far, three of which are inhibited by either the redox
probe diamide (19, 28), or the oxidative metabolite dehydroascorbic
acid (DHA) (20), or almost entirely by the sulfhydryl protease active
site inhibitor E-64 (see Ref. 28). A fourth proteolytic process is
unresponsive to redox probes or sulfhydryl protease inhibitor. We
suggested that some cell proteolytic pathways might be integrated with
cell redox metabolism, whereas other proteolysis is independent. Among
probable redox-related proteolytic mechanisms is the reductive
activation of sulfhydryl proteases (20, 26), although other mechanisms
are also conceivable.
It is presently reported that total myocardial protein degradation is
unlinked to nearly lethal ATP depletion, or vice versa; however, three
of four pathways are strongly responsive to metabolic intervention with
noninjurious redox probes without ATP depletion. The onset and reversal
of proteolytic inhibition under redox probes was strongly dependent on
the extracellular glucose concentration. The dependence of
intracellular protein degradation upon cell-reducing chains has not
recently been investigated due to several independent prior reports
suggesting no relationship; however, those studies did not address the
questions described below (5, 8, and reviewed therein).
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METHODS |
Perfusion of hearts.
Hearts were perfused as Langendorff preparations by modification of
methods previously described (28). A constant flow rate of 7.0 ml/min
sustained a low perfusion pressure of 40 mmHg for 6 h. The
preparation remained minimally loaded, as evidenced by palpation of a
limp flaccid myocardium. The nonrecirculating perfusate (95%
O2-5%
CO2) routinely contained
Krebs-Henseleit salts, glucose (10 mM), and physiological
concentrations of citrate (0.1 mM), pyruvate (0.1 mM), lactate (0.3 mM), complete amino acids, and BSA (0.2%), all adjusted to pH
7.42 after addition.
Measurement of tissue ATP content.
Midventricular biopsies of 100-150 mg were placed in previously
weighed containers containing cold perchloric acid (0.2 M, 
10°C) within 4 s of excision and were then weighed again.
The ATP content per gram of tissue weight was determined in homogenates using HPLC by methods previously established for myocardium (1). ATP
calibration standards were from Sigma.
Measurement and interpretation of contractile rhythm in a nonloaded
preparation.
A catheter was inserted into the right ventricle through the pulmonary
artery, and the chamber was closed by ligating the artery around the
catheter. In the nonloaded preparation, the ventricular fluid volume is
small, resulting in zero diastolic pressure. Right ventricular
contractile pressure is shown in units of millimeters of mercury;
however, the wall tension developed in a nonloaded ventricle of small
volume is a small fraction of the in vivo wall tension. For present
purposes, it is the contractile rhythm that is compared with ATP
content and protein degradation, and not the peak systolic pressure
(see below).
Measurement of protein degradation in myocardium.
Proteins were biosynthetically labeled by infusion of
L-4,5-[3H]leucine
(40-60 Ci/mmol, 4.5 µCi/ml) for 10 min, then nonradioactive leucine (1.5 mM) was routinely added to prevent reincorporation of
label except as indicated below (28). After labeling, a 20-min preliminary period preceded measurements of
[3H]leucine release,
providing ~40 half times of intracellular-extracellular leucine
exchange before the designated zero time points as previously described
(28). The nonrecirculated effluent perfusate was collected at 2-min
intervals in a fraction collector, and TCA-soluble radioactivity was
determined. It has previously been determined that the percent metabolism of
[3H]leucine to other
forms of radioactivity is below the limits of detection of several
percent (19). A two-component equation describing the progress of
macromolecular
[3H]leucine remaining
in myocardial protein over 5 h has been described: Y = 0.30e
1.04t + 0.70e0.031t
(28). The present data illustrate the rate of
[3H]leucine release
per minute, or the differential of this equation. Under constant
intracellular degradative conditions, the rate of total
[3H]leucine release
declines continually in proportion to change in the declining amount of
undegraded proteins remaining. The declining curvilinear control
baseline of
[3H]leucine release
represents the total degradation of diverse proteins with heterogeneous
half-lives labeled in proportion to their turnover rates. The percent
inhibition of protein degradation is proportional to the percent
downward displacement of the baseline rate of
[3H]leucine release
following the transition time to a new steady state. Control values of
protein degradation are presently illustrated as either the declining
baseline of
[3H]leucine release
or, alternatively, as the normalized 100% value of the declining
baseline. The percent downward displacement of the baseline is
identical using either method of presentation; however, representation
of data as percent inhibition enhances visualization of the time course
of transition to steady-state inhibition.
During the first 3 h after labeling, the steep decline and curvature of
[3H]leucine release
from rapidly degraded proteins resulted in large experimental
variability in statistical extrapolation of the baseline. Therefore,
the progress of proteolytic inhibition during the first 3 h after
labeling was determined by comparison of the inhibited [3H]leucine release to
the measured mean control rate of
[3H]leucine release
from four separate identically labeled parallel preparations. However,
after elimination of rapidly degraded proteins at 3 h, each preparation
can be accurately employed as its own internal experimental control by
extrapolation of the fitted baseline of
[3H]leucine release
from 3 to 4 h over the following period of degradative inhibition from
4 to 6 h as previously described (28).
Drugs or variable nutrients, indicated below, were infused into flowing
perfusate from 100-1,000 concentrated solutions. Materials were as
previously described (28).
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RESULTS |
Subcomponents of total
[3H]leucine release from
myocardial proteins.
Immediately after a 10-min incorporation period, the declining rate of
myocardial [3H]leucine
release describes a rapidly declining subcomponent approaching a second
subcomponent of slower decline by 3 h postlabeling (Figs. 1-3).
Beginning at 3 h postlabeling, the total rate of
[3H]leucine release
can be divided into three additional subcomponents as previously
described (28). Either insulin or chloroquine nonadditively inhibit
40-45% of
[3H]leucine release
(3-6 h postlabeling), corresponding to lysosomal vesicular
degradation. After 45% inhibition with chloroquine, the simultaneous
infusion of 
- or 
-adrenergic agonists inhibits an additional 30%
of total proteolysis, although not further characterized in present
studies. The pro-oxidant redox probe diamide (100 µM) inhibits the
rapid turnover subcomponent from 0 to 3 h postlabeling, and diamide
also causes a 75% proteolytic inhibition at any time from 3 to 6 h,
including all of the above lysosomal and adrenergic-responsive pathways
(Fig. 1 and Ref. 28). Thus three of four myocardial subcomponents
are diamide inhibitable; however, 25% of total proteolysis is
uninhibited by diamide indefinitely.
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Fig. 1.
Simultaneous effects of diamide and sodium azide on myocardial protein
degradation and ventricular contractile function. In
left panels, mean release of
[3H]leucine from 4 untreated control preparations is shown ±SD ( ). Total
[3H]leucine release
was measured in effluent perfusate at 2-min intervals, and each point
shown at 20-min intervals represents mean of 10 separate measurements.
Initial rates of
[3H]leucine release
were >104 counts/min (cpm);
however, they were normalized to 100% of
104 cpm/min. Control values from
200 to 300 min are statistically extrapolated using measured values
from 140 to 200 min (see METHODS).
Myocardial contractile function was measured as right intraventricular
pressure (right panels) simultaneous
with measurement of
[3H]leucine release
(left panels). Contractile function
is illustrated from representative individual preparations shown in
left panels ( ). Letters in
left panels indicate times of
measurement of contractile function in individual preparations
corresponding to letters in right
panels. Release of
[3H]leucine from
individual experiments () is included in means of parallel replicate
experiments ( ). Arrows in right
panels are described in text. In
experiment 1 (EXP. 1) of
left panels ( ±SD), agents were
infused beginning at 20 min. In experiment
2 (EXP. 2) of left
panels ( ), agents were infused after a 3-h
preliminary perfusion period. The 100% mean values of cpm/min before
infusions were superimposed on means of separate group of untreated
control experiments. Diamide concentration was 100 µM, and azide
infusion is described in text.
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Fig. 2.
A and
B: effects of diamide
(A) and sodium azide
(B) on myocardial protein
degradation and simultaneous ATP content. All preparations of
A-F
were perfused with routine perfusate composition (see
METHODS) except as indicated.
Normalized 100% values of
[3H]leucine release
shown in
A-F
correspond to 100% control values of curvilinear baselines illustrated
in Fig. 1. Normalized 100% values of
[3H]leucine release of
separate preparations were superimposed at time
0, resulting in definition of no experimental
variability at this point (see
METHODS). Time
0 in A and
B corresponds to 40 min postlabeling;
therefore, results from 0 to 60 min represent largely rapid turnover
proteolysis. ATP contents of midventricular biopsies were measured
immediately before and 20 min after drug infusions in 4 separate
treated preparations in A and 4 in
B, using identical averaged controls
for each group. Action of diamide on
[3H]leucine release is
illustrated from a single preparation, as also shown statistically in
Fig. 1. In B, a separate group of 4 preparations was perfused preliminarily for 3 h to eliminate rapid
turnover proteins as shown in Fig. 1; therefore, results at 140 min
represent 3 remaining subcomponents described in text. Constant
proteolysis and decreased ATP levels under sodium azide at 4 h (not
shown) have previously been described under a different perfusate
composition (19). C and
D: effect of glucose concentration on
proteolytic inhibitory action of submaximal diamide and dehydroascorbic
acid (DHA). Experiments were preceded by a 3-h preliminary degradation
period before time 0 as shown in Fig.
1. Previously reported maximal actions of diamide (100 µM) and DHA
(200 µM) under routine perfusate are shown by dashed traces in
C and
D. Actions of minimally effective
diamide (10 µM) and DHA (15 µM) are shown in single continuous
representative experiments with means ± SD at 80 min in
C and 40 min in
D () with control measurements
superimposed (, 40 to 0 min). A single experiment is shown in
D, in which 15 µM DHA caused no
proteolytic inhibition until glucose was terminated () (see text).
Effects of glucoprivation in C and
D are representative of 2 experiments
each (). Absence of any immediate effect of glucose termination in
absence of diamide or DHA is shown statistically in Fig. 3
(P < 0.01).
E: effect of glucose concentration on
protein degradation. Three separate groups of preparations were labeled
and perfused over a 3-h preliminary period inroutine perfusate containing 10 mM glucose. Control measurements
(60 to 9 min) are superimposed. Variability of each individual
group before time 0 was not greater
than individual groups shown after time
0. A glucose concentration of 0.1 mM did not support
acceptable viability of preparation past 60 min. Percent control
[3H]leucine release
under 0.2 mM glucose is shown ±SD at 180 min.
F: effect of glucose concentration on
reversal of proteolytic inhibitory action of diamide. Four separate
groups of 3 or 4 preparations were labeled and perfused for 3 h before
time 0 in routine perfusate containing
10 mM glucose. Glucose (10 mM) infusions were changed to indicated
glucose concentrations with diamide infusions. A single representative
experiment from group changed to 0.1 mM glucose is illustrated
continuously () and also shown statistically at 120 min. Reversal of
action of diamide (100 µM) under 0.1, 0.2, 1.0, and 10 mM glucose
after a 20-min diamide exposure is shown as means ± SD in each
separate group of individual preparations with cpm/min superimposed so
as to coincide at measured percent inhibitions at 50 min. Dashed trace
shows action of continuous 100 µM diamide in unchanged routine
perfusate as shown in A and Fig. 1.
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Fig. 3.
Short-term effects of acute glucoprivation on myocardial contractile
function and protein degradation. Time
0 was preceded by a 2.75-h preliminary degradation
period eliminating rapidly degraded proteins as in Fig. 1. After first
2 h of preliminary perfusion, routine perfusate was changed to one
without amino acids, using cycloheximide (20 µM) to prevent
reincorporation of label in place of 1 mM nonradioactive leucine.
Routine concentrations of glucose, citrate, pyruvate, and lactate (see
METHODS) were delivered by infusion
from a concentrated solution from 0 to 2.75 h. At time
0, infusion of all exogenous nutrients was terminated
except 0.1 mM glucose, leaving a perfusate consisting of only
Krebs-Henseleit salts, BSA (0.2%), and 0.1 mM glucose. A single
experiment is illustrated with means ± SD
(n = 3) shown at 32 min. Control
ventricular contractile function is shown at time
0, and ventricular contractile dysfunction is
illustrated at 19-20 min from 1 of 3 replicate experiments (see
text). In 2 separate experiments, change to 0 mM glucose also caused no
significant change from control
[3H]leucine release
(both experiments illustrated at 23 min, ). In 2 separate
experiments, change to 0 mM glucose with addition of 2-deoxyglucose
(2.5 mM) also caused no significant change in
[3H]leucine release
(both experiments illustrated at 20 min,  ).
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Inhibition of proteolytic pathways by a pro-oxidant redox probe
without depletion of cell ATP, and depletion of cell ATP by an
inhibitor of oxidative phosphorylation without inhibition of protein
degradation.
Deficiency of ATP causes impairment of contractile function, and
deficiency of reducing energy permits eventual oxidation of reduced
cell constituents after depletion of redox chain intermediates. The net
levels of ATP and redox chain intermediates are the results of the
relative rates of their production from the glucose supply vs. their
utilization by the cell demand for their respective functions. With the
use of glucose as sole substrate, glycolysis or the HMP pathway can be
altered either by experimental change in the glucose supply (see below)
or by use of metabolically active experimental agents of known action
(schematized in Fig. 1). A decrease in ATP can be imposed by an
uncoupler of oxidative phosphorylation, such as sodium azide, and
depletion of redox chains can be imposed by pro-oxidant redox probes
such as diamide (reviewed below). The approach presently developed
permitted the observation that a major proteolytic subcomponent can be
a minute-to-minute covariable of the cell redox status under some
conditions; however, none of observable cell proteolysis is linked to
severe ATP fluctuations, or vice versa.
The progress of protein degradation was compared with simultaneous
changes in measured tissue ATP content, as well as contractile evidence
of ATP deficiency caused by sodium azide. The hallmark of ATP
deficiency in myocardium is the impaired ability of contractile filaments to relax when ATP content declines to ~50% of normal. More
severe contractile impairment results when ATP declines to 40% of the
basal level. In the present preparation, the onset of impaired
relaxation under ATP deficiency can be readily observed qualitatively
as irregularities in the downslope of the systolic ventricular
pressure pulse (Figs. 1 and 3). Irregular relaxation began with slight
change in the rate of fall in ventricular pressure and progressed
through more severe stages of the inability to relax (Fig. 1,
bottom right, arrows in
B-D). The right
ventricle of this preparation is closed by ligation around the pressure catheter inserted through the pulmonary artery into the chamber. Although there is no venous return, a small amount of fluid can enter
or leave the chamber from the muscle wall by interstitial flow and
small vessels opening into the chamber. The fluid content and volume of
the nonloaded preparation without venous return are normally maintained
very small by ventricular contraction; therefore, the diastolic
pressure is essentially zero. However, when the ventricular contraction
is severely weakened by ATP deficiency, the chamber eventually fails to
expel the fluid that enters from the muscle wall, and the diastolic
pressure is elevated above zero (Fig. 3). Such ventricular dysfunction
is well characterized and also presently calibrated in terms of severe
ATP deficiency; therefore, this minute-to-minute functional indicator
of sublethal energy deficiency can be compared with simultaneous
minute-to-minute changes in proteolytic pathways.
Basal ATP of the preparation was 5 µmol/g tissue wet wt, which is
identical to the reported in vivo myocardial ATP content (Fig.
2A) (1). To observe proteolysis
under prolonged severe ATP depletion, the concentration of sodium azide
was gradually increased until the beginning of contractile dysrhythmia
was observed at 15-20 min, then maintained at that submaximal
azide level of ~0.275-0.325 mM. By adjusting the submaximal
sodium azide concentration under observation of rhythm, the
characteristic abnormality in contraction-relaxation could be sustained
for >1.5 h of severe ATP deficiency, during which time total release
of [3H]leucine was
simultaneously unchanged (Fig. 1). To observe proteolysis under the
onset of prelethal ATP deficiency, a higher sodium azide concentration
of 0.4 mM was infused (Fig. 2B). The
preparation can usually recover from 0.4 mM azide if exposure is
terminated in ~20 min. A measured prelethal 60% ATP depletion from 5 to 2 µmol/g at 20 min was not associated with any change in total
[3H]leucine release at
1 h postlabeling (Fig. 2B). A second
20-min exposure of the same preparations to 0.4 mM sodium azide at 80 min or separate preparations at 180 min also caused no change in total
protein degradation (Fig. 2B).
Therefore, the total of all proteolytic subcomponents is unchanged by
severe ATP deficiency at any time from 0 to 4 h postlabeling.
Maximally effective diamide concentrations (100 µM) inhibited
proteolysis without causing change in contractile function or decrease
in measured basal ATP content (Figs. 1 and
2A). A 20-min diamide exposure time
was selected to permit comparison of ATP levels with the maximal
practical prelethal exposure time to sodium azide in above parallel
experiments. The contractile rhythm of the basal preparation exhibits
occasional irregularities as does the in vivo organ; however, rhythm is
maintained in a largely regular pattern for several hours. Premature
ventricular contractions followed by a strong beat occur at a frequency
comparable to the in vivo heart, although not quantitatively
characterized (Fig. 1, top right,
arrow in A). Diamide (100 µM)
caused a slight slowing of heart rate similar to this concentration of
many agents that dissolve in cell membranes (Fig. 1,
top,
B); however, diamide inhibited 75%
of proteolysis without causing contractile dysfunction for much more
than 1.5 h. In separate experiments, diamide caused no contractile
irregularities for 
3 h at a 40 µM concentration that
caused more than half-maximal sustained proteolytic inhibition (data
not shown). Although tissue ATP content has not been measured under
such prolonged diamide exposure times, the maintenance of unchanged
contractile function for several hours suggests that effective
antiproteolytic diamide concentrations do not appreciably decrease ATP
over this time.
Prompt and delayed effects of glucoprivation on total myocardial
proteolysis.
If a proteolytic pathway hypothetically requires reductive energy from
glucose, then the pathway should decline under severe sustained
glucoprivation when glucose is the only metabolic substrate provided.
Glucose, as sole metabolic substrate, simultaneously feeds both the
glycolytic pathway and the HMP pathway; therefore, glucoprivation leads
to eventual depletion of both ATP and redox chain intermediates with
respective time courses dependent on their initial amounts and the
rates at which the cell consumes them. Under acute nutrient deficiency,
ATP is depleted more rapidly than redox chain intermediates, and the
myocardium becomes ATP deficient before injury from redox imbalance.
Indeed, the cell redox system is buffered against moderate short-term
supply-demand imbalance, and moderate depletion is not necessarily
injurious (7, 35). Upon glucoprivation or mitochondrial inhibition, it
is known that high-energy phosphate pools are significantly depleted
within 10 min by cellular demand. However, in the absence of exogenous
oxidative agents, various cell constituents can be oxidized with
greatly differing time courses, depending on their individual
tendencies to spontaneously oxidize, or react, with endogenous
oxidants. In the absence of exogenous pro-oxidants, cell redox chain
components are not immediately oxidized upon glucose deficiency but
rather decline only after the cell reservoir of reducing energy is
depleted. Upon acute glucoprivation, the cell can die from ATP
depletion before depletion of the redox chains. Accordingly, it is not
expected that a hypothetical redox-dependent process should decline
immediately upon glucose insufficiency in the absence of an exogenous
experimental pro-oxidant.
Various approaches were employed to observe proteolysis under
short-term glucose deficiency with identical findings. Reincorporation of [3H]leucine
degradation product can be prevented competitively with supraphysiological nonradioactive leucine (1 mM or greater) or by
inhibiting protein synthesis with cycloheximide (20 µM) with indistinguishable results, as previously described (19). Under the
present experimental design, results are not complicated by the routine
presence of 1.5 mM nonradioactive leucine because all comparisons
involve percent changes in the identical parallel conditions except for
the indicated experimental variables. However, by substitution of
cycloheximide (20 µM) for supraphysiological nonradioactive leucine
(1.5 mM), it was previously determined that responsiveness of major
subcomponents was indistinguishable with or without supraphysiological
leucine (see Refs. 19, 28). Therefore, any possible effect of 1.5 mM
leucine is below the limits of present detection. Cycloheximide (20 µM) is toxic after 2 h and cannot be routinely employed.
To eliminate all exogenous sources of metabolic energy except glucose,
radiolabeled hearts were preliminarily perfused for 30 min with 10 mM
glucose in only Krebs buffer (i.e., without added amino acids, citrate,
pyruvate, or lactate) using cycloheximide (20 µM) to prevent
reincorporation of label in place of 1.5 mM nonradioactive leucine (see
Fig. 3 legend). These conditions support basal contractile function for
more than 1 h. Subsequent acute decrease from 10 to 0.1 mM
glucose as sole exogenous nutrient caused severe contractile
dysfunction at 20 min without changing total
[3H]leucine release at
30 min (Fig. 3). Contractile dysfunction included elevated diastolic
pressure from weakened contraction, as well as irregular relaxations as
described above (Fig. 3, inset). Upon acute total deprivation of all perfusate glucose in the absence of
all other nutrients, the contractile dysfunction of the nonloaded preparation can be sustained for 10 min while cytoplasmic metabolites and endogenous glycogen stores are depleted. After 10-15 min of substrate-free perfusion, the onset of irregular relaxations signaled a
severe depletion of ATP (contractile function not shown). At the time
of nearly lethal contractile dysfunction after 24 min of completely
substrate-free perfusion, total
[3H]leucine release
was unchanged in two experiments (Fig. 3). When glucose is restored
after 20 min of such complete glucoprivation, contractile function
typically returns in ~10 min. In two separate experiments (not
shown), repeated alternating 20-min periods of complete glucoprivation
followed by glucose restoration (10 mM) caused no appreciable sustained
change in [3H]leucine
release over 1-2 h in the absence of all other metabolites. Infusion of the glucose analog 2-deoxyglucose (2.5 mM) inhibits glucose
utilization. When perfusate with only 10 mM glucose was changed to 0 mM
glucose and 2.5 mM 2-deoxyglucose, contractile dysfunction ensued
before 10 min; however, total
[3H]leucine release
was unchanged at 19 min in two preparations (Fig. 3). Thus ATP
deficiency can be induced either by various impositions of an
insufficient cell glucose supply or an uncoupler of mitochondrial
oxidative phosphorylation without immediately changing total protein
degradation in the absence of exogenous pro-oxidant.
To study the effect of prolonged glucoprivation on protein degradation,
the preparation must be kept alive under severe prolonged metabolic
deficit. It was determined that 0.2 mM glucose in otherwise routine
perfusate (physiological citrate, pyruvate, lactate, and complete amino
acids) can sustain the preparation for several hours, although 0.1 mM
glucose in routine perfusate was lethal in ~1-2 h. Because the
preparation performs almost no mechanical contractile work, these
conditions cannot be directly compared with the loaded in vivo organ.
When perfused with 0.2 mM glucose in otherwise routine perfusate, the
total [3H]leucine
release remained unchanged for 40 min and then began a gradual
decline, reaching a submaximal inhibition of ~50% at 3 h (Fig.
2E). Thus, in the absence of
exogenous pro-oxidants, protein degradation is not an immediate
covariable of the glucose supply, but rather a delayed variable under
present conditions.
Effect of glucoprivation on the proteolytic inhibitory potencies of
the exogenous pro-oxidants diamide and DHA.
The rate limitation over the transfer of glucose to cell redox chains
is attributable to the first enzyme of the HMP pathway, glucose-6-phosphate dehydrogenase (G-6-PD). In the presence of low
amounts of exogenous pro-oxidants, the cell can increase the transfer
of reductive energy through redox chains so as to meet the increased
demand of the exogenous oxidant. However, in the presence of excess
amounts of exogenous pro-oxidants, the consumption, diversion, or
disruption of reducing energy exceeds the capacity of G-6-PD to
transfer reductive energy into the chains (35). When the exogenous
demand for reductive energy exceeds the ability of the redox chains to
supply it, the cell exists in a state of oxidative imbalance. Moderate
depletion of cell-reducing chains is not necessarily injurious and
might be part of normal cell function (7) (see the
DISCUSSION); however, severe
prolonged depletion causes an undefined syndrome called oxidative stress.
Under perfusion with routine perfusate (10 mM glucose), protein
degradation was unchanged under 1 µM diamide, 1-10% inhibited under 10 µM diamide, and maximally inhibited by 75% under 100 µM
diamide (Figs. 1 and 2 and data not shown). To determine the interaction of experimental redox probes with glucose supply, a
minimally effective concentration of diamide (10 µM) was first infused to cause slight partial inhibition of proteolysis, then the
perfusate glucose infusion was terminated. Acute termination of glucose
promptly converted the slight submaximal proteolytic inhibitory action
of diamide (10 µM) to the maximal action observed at 10-fold higher
concentrations of 100 µM (Fig.
2C). Therefore, the presence of
glucose retarded the proteolytic inhibitory action of submaximal
diamide, and the deprivation of glucose greatly increased the potency
of submaximal diamide by nearly 10-fold.
DHA is a particularly interesting endogenous pro-oxidant for present
studies, although its exact intracellular concentration and roles in
cell function are unknown (further reviewed and discussed in Ref. 20).
Extracellular DHA is readily taken up and reduced to ascorbic acid by
several known reductase systems deriving reducing energy from glucose,
including some of the same reductases that reduce cell proteins (22,
30) (see the DISCUSSION). A
supraphysiological DHA concentration of 200 µM maximally inhibited
diamide-sensitive proteolysis within 2 h (Fig.
2D), and 500 µM DHA maximally
inhibited within 45 min (20). Physiological perfusate DHA (5 µM)
caused no appreciable proteolytic inhibition, presumably because the cell-reducing systems can accommodate the exogenous oxidant taken up at
this exposure. Slightly supraphysiological DHA (15 µM) caused a mean
proteolytic inhibition of several percent; however, some preparations
were uninhibited by 15 µM DHA as shown in Fig.
2D. A 6 mM glucose concentration
was substituted for routine 10 mM glucose to decrease the time
necessary for depletion of presumably lesser intracellular glucose
pools. After a slight submaximal mean proteolytic inhibition with
supraphysiological DHA (15 µM) in routine perfusate except with 6 mM
glucose, the subsequent termination of 6 mM glucose infusion caused the
prompt onset of a much greater potency of DHA action corresponding to
the maximal 200 µM DHA concentration (Fig.
2D). Thus cell processes fed by glucose can oppose the proteolytic inhibitory actions of an
experimental redox probe and an endogenous pro-oxidative metabolite.
Possible effects of glucose on the uptake of DHA or diamide are unknown.
The combined proteolytic inhibitions caused by DHA (500 µM) and
diamide (100 µM) did not exceed the 75% proteolytic inhibition caused by either agent alone as previously described statistically (Fig. 2D, Ref. 20).
Glucose dependence of the reversal of diamide antiproteolytic
action.
Diamide is a unique metabolic probe with a redox potential conveniently
poised for gentle reversible reactivity with reduced sulfhydryls, and
other sites, under noninjurious conditions (3, 18). Diamide does not
denature or form covalent bonds with its targets. Unlike harsh
irreversible oxidants, diamide action is weakly concentration
dependent. Cells can withstand diamide concentrations of several
millimolar that are >10-fold above present maximally effective
concentrations of 75-100 µM. Diamide does not react equally with
all cell sulfhydryls, but preferentially with the more reactive or
acidic sites that tend to donate protons readily. Because diamide does
not denature or form permanent bonds with its targets, the altered
redox ratios of various cell constituents can be restored to the normal
reduced levels upon diamide washout. It is well established that
reductive restoration of oxidized diamide targets can be prevented if
the glucose supply is deficient. Thus it is generally accepted that the
demonstration of a glucose dependence of the reversal of a particular
diamide action implies a role of the cell redox chains in maintaining
the inhibited process under observation (3, 18).
In routine perfusate (10 mM glucose), the 75% proteolytic inhibitory
action of diamide (100 µM) is largely, but not entirely, reversed
beginning within several minutes of diamide discontinuation, and
reaching ~80% reversal by 1 h (Fig.
2F). Under a decreased glucose
concentration of 0.1 mM in otherwise routine perfusate, the proteolytic
inhibitory action of diamide (100 µM) was essentially irreversible
(Fig. 2F). As observed over periods
of 1 h after diamide termination, the reversal of diamide action was
proportional to increasing perfusate glucose concentrations of 0.2, 1, and 10 mM added to other nutrients of the routine perfusate (Fig. 2F). After a 2- to 3-h diamide (100 µM) exposure, the proteolytic inhibition was almost irreversible
under any perfusate conditions (data not shown).
| |
DISCUSSION |
Possible mechanisms of redox-dependent proteolytic changes.
Known oxidative mechanisms inactivating sulfhydryls include oxygenation
to SO, SO2, or
SO3; metal ion binding; formation
of protein-mixed disulfides or thiolation reactions; intramolecular disulfide formation; and adduction with various metabolites.
Cell-reducing systems serve to prevent or reverse oxidative changes
caused by metabolic demand or some spurious oxidative reactions (4, 9, 10, 29, 31, 33, 34). Several of the major nonspecific sulfhydryl
proteases are spontaneously inactivated (26). Assay of redox-sensitive
proteases reveals little activity in the absence of dithiol reductant
and metal chelator to remove copurified endogenous metals such as
Zn2+,
Cu2+, and
Fe2+ (17). Although much of the
present redox dependence of proteolysis appears to involve the
inactivation-reactivation of sulfhydryl proteases (20, 26), additional
redox-sensitive mechanisms are likely.
A second mechanism of reductive stimulation of proteolysis is the
well-known increase in substrate protein susceptibility to proteolysis
upon reduction of intramolecular disulfide bonds (24). Substrate
unfolding is believed to be a requirement for proteolysis by the
proteasome (2, 27); however, unfolding can also increase the
susceptibility of a native protein to nonspecific attack by most
proteases by 10- to 100-fold. A third conceivable mechanism of
activation of proteolysis is the removal of inhibitory metal ions from
the surface of substrate proteins. The surface of most proteins
contains abundant associated Zn2+,
Cu2+,
Fe2+ or other ions that can be
attracted by weak electrostatic interactions or higher affinity binding
sites. Because sulfhydryl proteases can be completely inhibited by
endogenous levels of Zn2+,
Cu2+, or
Fe2+, substrate-associated metals
can obviously deliver an inhibitor to the active site of an attacking
protease. Various endogenous disulfide mechanisms might contribute to
dissociation of substrate-bound inhibitory metals. Vicinal disulfides
strongly bind metals, similar to the
Zn2+-dithiothreitol
(DTT) complex with stability constant of
1011 (6). Interestingly, substrate
reduction, removal of inhibitory metals from protease and/or substrate,
and protease reduction might all be simultaneously related actions of
disulfide reductive enzymes. A fourth mechanism of reversible
inactivation of sulfhydryl enzymes is reaction with a wide variety of
endogenous pro-oxidative metabolites. For example, the vicinal keto
oxygens of the ring multiketone DHA are very similar to the classic
thiol oxidant alloxan. Both DHA and alloxan can form reversible adducts
with sulfhydryl enzyme active sites (20). Various other endogenous metabolites can also bind to or react with sulfhydryls including aldehydes, as well as keto oxygens. Fifth, diamide promotes reversible disulfide formation, including protein-mixed disulfides such as protein-S-S-glutathione
(11, 16). Reversible inactivation of some sulfhydryl enzyme active
sites by spontaneous formation of mixed disulfides is well known to
occur (10, 34), although this phenomenon has not been characterized in
sulfhydryl protease active sites. Although many cardiac proteins can be
thiolated by glutathione under diamide (11), the effect of thiolation on substrate proteolytic susceptibility is completely unknown. Sixth, a
variety of reactive intermediates or radical species can oxygenate
sulfhydryls to -SO, -SO2, or
-SO3 (26); however, reversal of
protein oxygenation is not well characterized. Seventh, the enzymatic
conjugation of ubiquitin and some other substrate designation reactions
requires an intermediate thiol-ester bond that can be inactivated by
thiol reactive agents (23). Finally, motion of the
cytoskeletal-vacuolar system has been suggested to require reducing
energy, which might serve to promote cytoplasmic motion or vesicular
fusions (32). Despite these many conceivable possibilities, the
predominant mechanism(s) underlying redox inactivation-reactivation of
proteolysis awaits further investigations.
Possible reducing chains and branches transferring energy from
glucose to the reduction of enzymatic and nonenzymatic proteins.
It was previously assumed that reductive activation of sulfhydryl
proteases is nonenzymatically coupled to the oxidation of glutathione
to its disulfide. In association with recent advances in the
cell-reducing network, it has been suggested that oxidatively inactivated sulfhydryl proteases are enzymatically reactivated by the
cell redox chains similar to some other enzymes (7, 10, 26, 34).
Enzymes and other cell proteins are reduced by three types of protein
reductases, sharing a similar vicinal disulfide mechanism typified by
thioredoxin (14, 15). Thioredoxin and glutaredoxin are small redox
shuttle proteins with a disulfide reductase mechanism (14, 15). A
thioredoxin domain is also found in a third class of larger protein
oxidoreductases serving similar functions (21). A growing number of
cell proteins have been found to contain thioredoxin domains (21). The
thioredoxin reductive mechanism has been compared with DTT, and some
functions of mutationally deleted thioredoxin can be replaced by
exogenous synthetic dithiol agents in bacteria. After myocardial
proteolytic inhibition with either diamide or DHA, the simultaneous
infusion of excess DTT reverses the proteolytic inhibition without cell injury under concurrent exposures to pro-oxidants and dithiol reductant
(21). The intracellular content of thioredoxin disulfides is estimated
at 30 µM, and an exogenous perfusate DTT concentration of only 100 µM can appreciably accelerate the reversal of diamide action after
diamide discontinuation (Ref. 28, and data not shown).
Although thioredoxin has been implicated in reductive reactivation of
sulfhydryl proteases (26), present glucose effects might be explained
by additional metabolic pathways. Thus far, three branches of
reducing chains are known to transfer the energy of glucose to cell
protein reduction: 1) glucose
NADPH thioredoxin protein;
2) glucose NADPH glutathione glutaredoxin protein; and
3) glucose NADPH various protein oxidoreductases protein (24). The
enzymes mediating these transfers (not diagrammed) have been reviewed,
e.g., NADPH:thioredoxin reductase (14, 15, 24, 33). The known
nonspecificity and redundancy of the three types of protein reductase
systems reviewed above (24) suggest that all three might be involved in
redox-responsive proteolysis. Pending further advances, interpretation
of present results cannot presume a complete understanding of the
mechanisms of protein oxidation-reduction or proteolytic
inactivation-reactivation. Nonetheless, the cell redox system is
emerging as a potential integrated signaling network (7).
Several studies suggest that markedly increased myocardial functional
demand is associated with an increased demand on the cell redox system.
Prolonged functional stimulation with isoproterenol leads to an
induction of G-6-PD, the rate-limiting enzyme of the HMP pathway (35).
Extreme stimulation of myocardial contractile function can lead to a
shift toward oxidized pyridine nucleotides (13, 25). Extreme exercise
can lead to a change in myocardial glutathione status (4, 29). If
verified, such studies suggest that extreme functional demand might
cause an increased oxidative demand on redox chains and a decrease in
protein degradation contributory to a net gain in protein, i.e.,
myocardial hypertrophy. However, it is not yet known whether decreases
in redox chain components such as NADPH or glutathione can become
metabolically limiting to various processes under any physiological
conditions. Despite remaining uncertainties, separate studies clearly
indicate that mammalian cell protein degradation can be experimentally
inhibited under the noninjurious range of redox imbalance in vivo. The
actions of the entire class of pro-oxidant antimalarial drugs are
associated with their intervention in host cell redox status at
therapeutic levels. Therapeutic concentrations of primaquine and
quinine inhibit much of redox-responsive proteolysis (unpublished data).
| |
ACKNOWLEDGEMENTS |
This work was supported by a grant from the American Heart
Association, Ohio Affiliate.
| |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. D. Lockwood,
Dept. of Pharmacology and Toxicology, School of Medicine, Wright State
University, Dayton, OH 45435 (E-Mail:
thomas.lockwood{at}wright.edu).
Received 29 July 1998; accepted in final form 14 January 1999.
| |
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Am J Physiol Endocrinol Metab 276(5):E945-E954
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ABSTRACT
INTRODUCTION
