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.
- adenosine 5′-triphosphate
- sodium azide
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 thediscussion) (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).
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 ofl-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 −1.04 t+ 0.70e −0.031 t(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).
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 α- or β-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.
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 inB–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.2 A) (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. 2 B). 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. 2 B). 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. 2 B). 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 and2 A). 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.2 E). 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 thediscussion); 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.2 C). 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.2 D), 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.2 D. 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.2 D). 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. 2 D, 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.2 F). 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. 2 F). 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.2 F). After a 2- to 3-h diamide (100 μM) exposure, the proteolytic inhibition was almost irreversible under any perfusate conditions (data not shown).
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; and3) 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).
This work was supported by a grant from the American Heart Association, Ohio Affiliate.
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:).
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