Abstract To investigate whether the energy derived from glycolysis is functionally coupled to Ca2+ active transport in sarcoplasmic reticulum (SR), we determined whether glycolytic enzymes were associated with SR membranes and whether metabolism through these enzymes was capable of supporting 45Ca transport. Sealed right-side-out SR vesicles were isolated by step sucrose gradient from rabbit skeletal and cardiac muscle. Intravesicular 45Ca transport was measured after the addition of glycolytic substrates and cofactors specific for each of the glycolytic reactions being studied or after the addition of exogenous ATP and was expressed as transport sensitive to the specific Ca2+-ATPase inhibitor thapsigargin. We found that the entire chain of glycolytic enzymes from aldolase onward, including aldolase, GAPDH, phosphoglycerate kinase (PGK), phosphoglyceromutase, enolase, and pyruvate kinase (PK), was associated with SR vesicles from both cardiac and skeletal muscle. Iodoacetic acid, an inhibitor of GAPDH, eliminated 45Ca transport supported by fructose-1,6-diphosphate, the substrate for aldolase, but transport was completely restored by phosphoenolpyruvate (the substrate for PK), indicating that both of the ATP-producing glycolytic enzymes, GAPDH/PGK and PK, were associated with the SR and functionally capable of providing ATP for the Ca2+ pump. Addition of a soluble hexokinase ATP trap eliminated 45Ca transport fueled by exogenous ATP but had markedly less effect on 45Ca transport supported by endogenously produced ATP (via glycolysis). Similarly, at very low concentrations of ATP and ADP (10 to 50 nmol/L), ATP that was produced endogenously from ADP and phosphoenolpyruvate supported 15-fold more 45Ca transport than ATP that was supplied exogenously at the same concentration. These results are consistent with functional coupling of glycolytic ATP to Ca2+ transport and support the hypothesis that ATP generated by SR-associated glycolytic enzymes may play an important role in cellular Ca2+ homeostasis by driving the SR Ca2+ pump.
During ischemia, the myocardium becomes critically dependent on ATP produced by anaerobic glycolysis, but after reperfusion, oxidative phosphorylation resumes, and glycolysis is relegated to its normal role as a minor producer of cellular ATP. Under conditions of maximal stimulation, glycolysis is believed to contribute no more than 7% of the total ATP produced by the normal heart.1 Despite this fact, there is evidence that glycolytic ATP production has special importance in the reperfused heart. After ischemia and 24 hours of reperfusion, an enhancement of glycolytic activity in the myocardium has been demonstrated,2 and positron emission tomographic studies have shown increased uptake of the glucose analogue fluorodeoxyglucose, consistent with increased glycolytic metabolism. Mallet et al3 and Bunger et al4 have shown that glycolytic activity during the transition period from anaerobic to aerobic metabolism is critical for functional recovery of the heart and that enhancement of glycolysis through provision of glucose, as opposed to other oxidative substrates, results in better recovery. Our own studies have shown that glycolytic metabolism is critical for functional and metabolic recovery of isolated rabbit hearts during reperfusion after 20 minutes of global ischemia.5 6 When glycolysis was inhibited during reperfusion by the administration of iodoacetic acid (IAA) or 2-deoxyglucose, the recovery of contractile function and high-energy phosphates was markedly impaired despite provision of oxidative substrates in the perfusion medium.5 If glycolytically inhibited hearts were reperfused transiently with perfusate containing low Ca2+, functional recovery was similar to that of hearts without glycolytic inhibition.6 19F nuclear magnetic resonance measurements using the fluorinated Ca2+ indicator 5F-BAPTA demonstrated a marked and persistent elevation of cytosolic free Ca2+ concentration in hearts with glycolytic inhibition during reperfusion.6 These data suggest that glycolytic ATP, as opposed to ATP produced by oxidative phosphorylation, may be essential to achieve Ca2+ homeostasis during periods of increased Ca2+ influx, as occurs during ischemia/reperfusion.
Evidence exists to support the concept of functional compartmentation of ATP in myocytes. Glycolytic ATP appears to preferentially fuel membrane ion pumps, such as the Na+,K+-ATPase and the Ca2+-ATPase, whereas ATP produced by mitochondrial oxidative phosphorylation is used preferentially to support myocyte contractile activity. Glycogenolytic enzymes are known to be associated with the sarcoplasmic reticulum (SR),7 and enzymes of the glycolytic pathway have also been found in SR fractions in rabbit skeletal muscle8 and myocardium.9 Glycolytic ATP appears to be used preferentially over ATP derived from oxidative phosphorylation to close sarcolemmal (SL) ATP-sensitive K+ channels (KATP channels).10 11 Experiments in excised inside-out patches of sarcolemma suggest that the key glycolytic enzymes, phosphoglycerate kinase (PGK), pyruvate kinase, or both, are located in the membrane or adjacent cytoskeleton near the KATP channels, which potentially could account for a preferential use of glycolytic ATP.10 11
The present study was performed to determine whether glycolytic enzymes are associated with SR isolated from both cardiac and skeletal muscle and whether the addition of glycolytic substrates and cofactors to isolated SR (without the addition of exogenous ATP) could lead to sufficient ATP production to support SR Ca2+-ATPase activity and transport of Ca2+ into SR vesicles. We also examined whether the ATP produced endogenously from glycolytic substrates and isolated SR could support greater Ca2+ transport than exogenously added ATP and whether endogenously produced ATP was protected from a soluble ATP trap, thereby supporting the concept of functional compartmentation of glycolytic ATP in SR.
Materials and Methods
SR Vesicle Preparation
Cardiac and skeletal muscle SR was prepared from hearts and white hind-leg skeletal muscle of New Zealand White rabbits by the method of Chu et al,12 with modifications. For cardiac SR, the atria were removed, and the ventricles were trimmed of fat and connective tissue and sliced into small pieces. The initial homogenization was carried out twice in 0.29 mol/L sucrose and 10 mmol/L imidazole/HCl buffer without KCl, pH 6.8, for 15 seconds at 22 000 rpm. The homogenate was centrifuged at 8000 rpm (11 000g) for 15 minutes, and the pellet was discarded. The supernatant was then centrifuged at 30 000 rpm (110 000g) for 90 minutes, and the pellet was harvested, resuspended, and loaded on the top of a sucrose step gradient (45%, 38%, 34%, 32%, 26%, and 20%). Centrifugation was carried out for 16 hours at 20 000 rpm (70 000g). The SR fraction at the interfaces between 32% and 34% gradient steps was collected and sedimented for 90 minutes at 32 000 rpm (125 000g). The final pellet was resuspended in 10 mmol/L imidazole/HCl and 0.29 mol/L sucrose buffer and stored at −70°C. The specific activity for cardiac SR Ca2+-ATPase, was 150 to 300 μmol inorganic orthophosphate (Pi) per milligram per hour, and for skeletal muscle SR Ca2+-ATPase, it was 600 to 1100 μmol Pi per milligram per hour.
Determination of Ca2+-ATPase Activity
Ca2+-ATPase was assayed by modification of the method of Kyte.13 The enzymatic activity was defined as the thapsigargin-sensitive hydrolysis of Mg2+-ATP (2 to 3 mmol/L) in the presence of Ca2+ (10 μmol/L), ouabain (10 μmol/L) to inhibit Na+,K+-ATPases, P1,P5-di(adenosine-5′) pentaphosphate (DPP, 0.4 mmol/L) to inhibit myokinase, and oligomycin (4 μg/mL) and ruthenium red (30 nmol/L) to inhibit F0F1- ATPase (ATP synthetase). The reaction was initiated by adding SR and stopped after 10 to 30 minutes at 37°C by adding 0.75 mL quench solution (0.5% ammonium molybdate plus 0.5N H2SO4) and 0.02 mL developer (25 mg of a mixture of 0.2 g 1-amino-2-naphthol-4-sulfonic acid plus 1.2 g sodium bisulfate plus 1.2 g sodium sulfite dissolved in 1 mL water). The color was allowed to develop for 30 minutes at room temperature, and phosphate was then determined at 700 nm with a spectrophotometer. Over the range used in these experiments, the liberation of phosphate was linear with the amount of SR added and was also linear with time from 0 to 30 minutes. An incubation mixture without enzyme served as a blank to correct for the time-dependent evolution of phosphate in strong acid.
45Ca uptake was initiated by the addition of SR to the reaction mixture for 15 to 20 minutes at 37°C. The mixture contained Ca2+ (10 μmol/L), 45Ca (1 μCi/mL), DPP (0.2 to 0.4 mmol/L), oligomycin (2 to 4 μg/mL), ruthenium red (30 nmol/L) to block mitochondrial Ca2+ uptake, Mg2+ (5 mmol/L), and specific glycolytic substrates and cofactors or exogenous ATP (1 to 2 mmol/L). The reaction was stopped by pelleting the samples at 14 000 rpm (16 000g) for 15 minutes. The pellet was washed three times with 25 mmol/L imidazole/HCl buffer, pH 7.2, and then dissolved in 50 to 300 μL of 10% SDS solution. An aliquot (5 or 10 μL) was taken from each sample, and the radioactivity was determined by a β-scintillation counter.
Thapsigargin (6 to 20 μmol/L), a specific inhibitor of SR Ca2+-ATPase, was used in each experiment. ATPase activity or 45Ca transport in the presence of thapsigargin was considered to represent nonspecific activity. All the results were expressed as thapsigargin-sensitive Ca2+-ATPase activity (micromoles Pi per milligram per hour) and thapsigargin-sensitive 45Ca uptake (micromoles per milligram protein).
45Ca was purchased from Du Pont; glyceraldehyde 3-phosphate (GAP), from ICN Biomedicals, Inc; and oligomycin, CaCl2, MgCl2, DPP, ruthenium red, potassium phosphate dibasic anhydrous, NADH, fructose 1,6-diphosphate (FDP), glucose, thapsigargin, ADP, ATP (vanadium, <1 ppm), hexokinase, sucrose, imidazole, 2-phosphoglycerate (2-PG), 3-phosphoglycerate (3-PG), phosphoenolpyruvate (PEP), ammonium molybdate, 1-amino-2-naphthol-4-sulfonic acid, sodium bisulfate, sodium sulfite, and IAA, from Sigma Chemical Co.
The presence of functional glycolytic enzymes associated with the SR was inferred by adding to isolated SR the appropriate substrates and cofactors (without exogenous ATP) for the specific enzyme being examined and by measuring the resulting Ca2+-ATPase activity and/or 45Ca uptake fueled by endogenously produced ATP (Fig 1⇓). By this approach, the presence of each of the glycolytic enzymes, from pyruvate kinase back to aldolase, was examined in a stepwise manner.
Presence of Glycolytic Enzymes
For pyruvate kinase, both SR Ca2+-ATPase activity and 45Ca uptake were measured after the addition of 1 mmol/L ADP and 2 mmol/L PEP in the presence and absence of thapsigargin. As with each of these experiments, Ca2+-ATPase activity and 45Ca uptake was expressed as thapsigargin-sensitive activity or uptake, and results were compared with those obtained after the addition of 1 mmol/L ATP to the isolated SR.
For enolase, 45Ca uptake was measured after the addition of 1 mmol/L ADP and 4 mmol/L 2-PG. The occurrence of 45Ca uptake in this experiment indicated that PEP was produced in the enolase reaction, which was used in turn by pyruvate kinase to produce ATP.
For phosphoglyceromutase, 45Ca uptake was measured after the addition of 1 mmol/L ADP and 4 mmol/L 3-PG. The presence of 45Ca uptake indicated that 2-PG was produced in the phosphoglyceromutase reaction, which was used by enolase to produce PEP, which in turn was used by pyruvate kinase to produce ATP.
For the coupled ATP-producing enzyme complex GAPDH/PGK, 45Ca uptake was measured after the addition of 6 mmol/L GAP, 1 mmol/L ADP, 4 mmol/L Pi, and 4 mmol/L NAD+. The presence of 45Ca uptake indicated that 1,3-diphosphoglycerate was produced from GAP, NAD+, and Pi, which was then used by PGK, along with ADP, to produce ATP and 3-PG. It is possible that the 3-PG continued through the remainder of the glycolytic chain to yield additional ATP via the pyruvate kinase reaction.
Finally, for aldolase, 45Ca uptake was measured after the addition of 1 mmol/L ADP, 4 mmol/L Pi, 4 mmol/L NAD+, and 10 mmol/L FDP. The presence of 45Ca uptake was indicative of the production of GAP, which in turn was used to produce ATP by GAPDH/PGK and possibly also to produce substrate for the more distal glycolytic reactions and pyruvate kinase.
Demonstration of Pyruvate Kinase on SR
To directly measure the production of ATP from ADP by pyruvate kinase, skeletal muscle SR (0.14 mg/mL) was incubated with or without PEP (1 mmol/L) in the presence and absence of thapsigargin for 1 or 15 minutes at 37°C in a water bath. The final concentrations of ADP, Ca2+, Mg2+, DPP, and ruthenium red were 1 mmol/L, 10 μmol/L, 2 mmol/L, 0.4 mmol/L, and 30 nmol/L, respectively. The reaction mixtures were then centrifuged at 14 000 rpm (16 000g) to remove the membrane-bound proteins. One hundred microliters of each sample solution was injected on to a high-pressure liquid chromatography (HPLC) column (C18 μBondapak, 3.9×150 mm) and eluted with 1 mL/min NH4H2PO4/NH4OH buffer at pH 5.5. The effluent was monitored spectroscopically at 229 nm to detect the appearance of ATP and disappearance of ADP.
To determine whether pyruvate kinase was bound to SR or present free in the solution, SDS-PAGE was performed on the pellets and supernatants from the samples in the experiment described above. Samples were added to SDS and separated by electrophoresis on a 4% to 15% slab gel according to the method of Laemmli.14 The polyacrylamide gel was then stained with Coomassie blue and destained with methanol and acetic acid.
Absence of Creatine Kinase
To determine whether creatine kinase was present on the isolated SR, cardiac and skeletal muscle SR was incubated with ADP (1 mmol/L) and phosphocreatine (2 mmol/L) in the presence of thapsigargin for 15 minutes. Ca2+, Mg2+, DPP, and ruthenium red were present in the concentrations described above. After centrifugation, the supernatant (100 μL) of each sample was injected on to a C18 HPLC column and eluted with 1 mL/min NH4H2PO4/NH4OH buffer at pH 5.5. The effluent was monitored at 229 nm for the appearance of ATP.
In addition, to determine whether phosphocreatine and ADP could support 45Ca uptake (indicating the presence of creatine kinase), cardiac and skeletal muscle SR was incubated with 45Ca (1 μCi) and ADP, phosphocreatine, Ca2+, Mg2+, oligomycin, DPP, and ruthenium red (1 mmol/L, 2 mmol/L, 10 μmol/L, 2 mmol/L, 4 μg/mL, 0.4 mmol/L, and 30 nmol/L). The final volume of the reaction mixture was 0.5 mL. 45Ca uptake by SR was measured as described above.
Effect of Glycolytic Inhibition
To examine the effect of glycolytic inhibition on SR 45Ca uptake, IAA (2 mmol/L), which at low doses produces a selective inhibition of GAPDH,15 was added to the 45Ca uptake reaction medium in the presence of FDP, NAD+, Pi, and ADP. This concentration of IAA had no effect on the enzymatic activity of either Ca2+-ATPase or Na+,K+-ATPase (data not shown). To verify that the Ca2+-ATPase was intact and that the glycolytic block did not involve the entire glycolytic cascade, PEP, the substrate for pyruvate kinase, was added with IAA in some experiments.
Effect of a Soluble ATP Trap
To determine whether the ATP produced by SR-associated glycolytic enzymes was in free exchange with the reaction medium or instead was compartmented with the Ca2+-ATPase for Ca2+ transport, 45Ca uptake experiments were performed in the presence and absence of a soluble ATP trap consisting of hexokinase (5.4, 10.8, or 20 U/mL) and glucose (4 mmol/L). This trap functions as an ATPase in the bulk phase by using ATP to phosphorylate glucose essentially irreversibly with a Km for ATP of ≈200 μmol/L.16 45Ca uptake was compared in the presence or absence of the ATP trap after addition to isolated SR of (1) glycolytic substrates and cofactors (mmol/L: ADP 1, FDP 4, NAD+ 4, and Pi 4) to produce ATP endogenously or (2) exogenous ATP (1 mmol/L).
Comparison of Exogenous ATP With Endogenously Produced ATP
To determine whether endogenously produced ATP could provide more effective support of SR 45Ca uptake than exogenously added ATP (in the same concentration), skeletal muscle SR (0.43 mg/mL) was incubated with various low concentrations of ADP or ATP (10, 50, and 100 nmol/L) in the presence and absence of PEP for 6 minutes at room temperature. The final concentrations of PEP, Ca2+, Mg2+, oligomycin, DPP, and ruthenium red were 2 mmol/L, 10 μmol/L, 4 mmol/L, 10 μg/mL, 0.4 mmol/L, and 20 nmol/L. The 45Ca uptake was stopped by adding 10 μmol/L thapsigargin. Samples were centrifuged, washed, and counted for radioactivity as described above.
Electron micrographs of cardiac and skeletal muscle SR preparations showed that the great majority of vesicles were tightly sealed (Fig 2⇓). Approximately 70% to 75% of cardiac SR and 85% to 90% of skeletal muscle SR had a “right-side-out” orientation based on the amount of increase in Ca2+-ATPase activity observed after the addition of saponin (0.6%) to increase membrane permeability and permit the access of substrates to the inside of the vesicles. Right-side-out SR vesicles have the active side of Ca2+-ATPase (ATP binding site) located on the outside of the vesicles; enzymatic activity is the same with and without saponin. Inside-out vesicles, in contrast, have the active side of Ca2+-ATPase on the inside of the vesicles; enzymatic activity is therefore detected only in the presence of saponin. We found that 0.6% saponin had no inhibitory effect on Ca2+-ATPase activity, and Kyte et al17 have demonstrated that there is no inhibitory effect on Na+,K+-ATPase activity.
Although the great majority of membrane vesicles obtained were SR vesicles, there was some contamination by SL. To estimate the extent of SL contamination, Na+,K+-ATPase was used as a marker enzyme, and Mg2+-ATP, Ca2+, Na+, and K+ were included in the incubation medium to reflect total ATPase activity (Ca2+-ATPase plus Na+,K+-ATPase). The addition of ouabain (10 μmol/L), a specific inhibitor of the Na+,K+-ATPase, reduced activity by ≈10% in skeletal muscle SR and 15% to 20% in cardiac SR. However, in the Ca2+-ATPase and 45Ca uptake assays reported below, the effects of SL contamination were minimized, since Na+ and K+ were excluded from the reaction medium and all results were expressed as thapsigargin-sensitive activity (which represented ≈90% of total Ca2+-ATPase activity or 45Ca uptake). In addition, ruthenium red and oligomycin were added to block Ca2+ uptake into any mitochondrial vesicles that may have been present,18 and a myokinase inhibitor (DPP) and an inhibitor of F0F1-ATPase (2 μg/mL oligomycin or 2 mmol/L sodium azide) were used to prevent formation of ATP from sources other than glycolysis.
The key ATP-producing enzyme pyruvate kinase was associated with both skeletal muscle and cardiac SR. When the substrates for pyruvate kinase, PEP and ADP, were added to isolated SR, sufficient ATP was produced to support Ca2+-ATPase activity (Fig 3⇓). With 1 mmol/L ADP, Ca2+-ATPase activity averaged 24 μmol Pi per milligram per minute for skeletal muscle SR and 3.2 μmol Pi per milligram per minute for cardiac SR (mean of three experiments). When 1 mmol/L ATP was added instead of PEP and ADP, Ca2+-ATPase activity averaged 27 and 5.1 μmol Pi per milligram per minute for skeletal muscle SR and cardiac SR, respectively. The absence of substrates or ATP was associated with no measurable Ca2+-ATPase activity. Similarly, PEP and ADP added to isolated SR produced sufficient ATP to fuel 45Ca uptake by the SR (Fig 4⇓). With 1 mmol/L ADP, 45Ca uptake averaged 13 nmol/mg protein for skeletal muscle SR and 0.2 nmol/mg protein for cardiac SR (average of three experiments). When 1 mmol/L ATP was added to the SR instead of PEP and ADP, 45Ca uptake averaged 17 and 0.25 nmol/mg protein for skeletal muscle SR and cardiac SR, respectively. The greater Ca2+-ATPase activity and 45Ca uptake in skeletal muscle SR compared with cardiac SR in these and other experiments appears to reflect the greater number of Ca2+ pumps existing in SR in fast-twitch muscle.19
The coupled enzyme GAPDH/PGK, which serves as a second source of glycolytically produced ATP, was also associated with skeletal muscle and cardiac SR. The addition of substrates and cofactors for these enzymes (GAP, NAD+, Pi, and ADP) resulted in the generation of sufficient ATP by SR to support 45Ca uptake. For skeletal muscle SR, 45Ca uptake averaged 4.16 nmol/mg protein when glycolytic substrates and 1 mmol/L ADP were added compared with 2.81 nmol/mg protein after the addition of 1 mmol/L ATP (average of three experiments). For cardiac SR, 45Ca uptake averaged 0.18 and 0.24 nmol/mg protein with endogenous ATP production and exogenously added ATP, respectively.
Further experiments examining 45Ca uptake by SR in the presence of substrates for enolase, phosphoglyceromutase, and aldolase demonstrated that each of these glycolytic enzymes was also associated with skeletal muscle and cardiac SR. The amount of SR 45Ca uptake resulting from the addition of various combinations of glycolytic substrates and cofactors, chosen to demonstrate the presence of specific SR-associated glycolytic enzymes, is presented in the Table⇓. The amount of 45Ca uptake occurring with substrates and 1 mmol/L ADP is expressed as a percentage of the uptake observed when 1 mmol/L ATP was added without ADP, substrates, or cofactors. Uptake resulting from endogenous glycolytic ATP production was less than that produced by the addition of exogenous 1 mmol/L ATP in both cardiac and skeletal muscle SR for pyruvate kinase, enolase, and phosphoglyceromutase but not for GAPDH/PGK or aldolase. The amount of 45Ca uptake was lowest for both types of SR when enolase and phosphoglyceromutase were required for ATP production. It is unknown whether this finding was due to lower intrinsic levels of these two enzymes in SR or to greater loss of activity during SR isolation.
Pyruvate Kinase Activity on SR
The addition of PEP and ADP to skeletal muscle SR resulted in a rapid and essentially complete conversion of ADP to ATP (Fig 5⇓). The enzymatic production of ATP by pyruvate kinase was revealed only when thapsigargin was added to the reaction mixture to block the hydrolysis of ATP by the SR Ca2+-ATPase. Without thapsigargin (Fig 5C⇓), only small amounts of ATP could be detected in the supernatant because of its immediate conversion back to ADP. SDS-PAGE of the pellets and supernatants from this experiment demonstrated that pyruvate kinase was found only in the pellets of each sample, consistent with tight association of this enzyme with SR membranes (Fig 5⇓).
Absence of Creatine Kinase
Creatine kinase could not be detected on either cardiac or skeletal muscle SR. The addition of ADP and phosphocreatine to SR resulted in no measurable ATP in the supernatant when HPLC was used (data not shown). Similarly, the addition of ADP and phosphocreatine to both types of SR failed to support significant 45Ca uptake (data not shown). For cardiac SR, no 45Ca uptake occurred, whereas for skeletal muscle SR, uptake was <4% of that seen after the addition of ADP and PEP.
Effect of Glycolytic Inhibition
The addition of IAA to the reaction medium containing FDP, NAD+, Pi, and ADP completely inhibited 45Ca uptake by both cardiac and skeletal muscle SR (Fig 6⇓). To prove that this finding was not due to a nonspecific inhibitory effect of IAA on the entire glycolytic enzyme chain or on the Ca2+-ATPase, PEP was added with IAA in some experiments. PEP completely restored 45Ca uptake (Fig 6⇓). These results are consistent with the notion that IAA can inhibit SR-associated glycolytic ATP production at a specific step, most likely at the GAPDH enzyme15 but that this block in glycolytic ATP production can be circumvented by provision of a distal glycolytic substrate.
Effect of a Soluble ATP Trap
45Ca was measured in cardiac and skeletal muscle SR after the addition of either (1) ADP (1 mmol/L), Pi, NAD+, and FDP to produce ATP endogenously via GAPDH/PGK (and potentially pyruvate kinase, as well, if the glycolytic cascade proceeded to the end) or (2) exogenous ATP (1 mmol/L). Hexokinase was added to trap available ATP by essentially irreversible phosphorylation of glucose. This was possible because hexokinase has a Km for ATP of ≈200 μmol/L,16 whereas the Km of the SR Ca2+-ATPase is higher (0.4 to 3 mmol/L).20 In skeletal muscle SR, hexokinase (5.4 U/mL) reduced 45Ca transport by 60% when ATP had been added exogenously but had no effect on 45Ca transport supported by endogenous ATP production (Fig 7⇓). More inhibition was produced by higher concentrations of hexokinase with both endogenous and exogenous ATP, but the trapping of exogenous ATP was consistently greater at each hexokinase concentration. For cardiac SR, the results were even more striking: hexokinase (5.4 U/mL) produced complete inhibition of 45Ca transport supported by exogenous ATP but had no effect on transport fueled by endogenous ATP. These results suggest that hexokinase did not have the same access to ATP produced endogenously by GAPDH/PGK as it did to exogenous ATP, consistent with the existence of compartmentation of ATP produced by SR-associated GAPDH/PGK.
Comparison of Exogenous ATP With Endogenously Produced ATP
When skeletal muscle SR and very low concentrations of ATP and ADP were used, ATP was found to be markedly less effective than ADP (provided in the same concentration) plus PEP in supporting 45Ca uptake (Fig 8⇓). As the concentration of ATP or ADP increased from 10 to 100 nmol/L, the uptake of 45Ca increased by a factor of ≈2, but uptake with ADP and PEP was consistently higher than with the same concentration of ATP. With 10 and 50 nmol/L ADP, 45Ca uptake was 15-fold greater than with the same concentrations of exogenously supplied ATP. With 100 nmol/L ADP, there was a 9-fold difference. These results indicate that ATP formed by SR-associated pyruvate kinase from ADP is used more efficiently by the Ca2+-ATPase than exogenous ATP and is consistent with functional coupling between pyruvate kinase and the Ca2+-ATPase.
The experiments reported in the present study were designed to determine which glycolytic enzymes are associated with SR from skeletal and cardiac muscle and whether the ATP generated from these glycolytic reactions can support the SR Ca2+-ATPase and 45Ca pumping into the SR. Our results indicate that the entire chain of glycolytic enzymes from aldolase onward, including aldolase, GAPDH, PGK, phosphoglyceromutase, enolase, and pyruvate kinase are bound to SR membranes in cardiac and skeletal muscle as evidenced by the ability of glycolytic substrates and cofactors (without ATP) to support 45Ca transport. IAA, an inhibitor of GAPDH, eliminated SR 45Ca transport supported by FDP (the substrate for aldolase), but transport was completely restored by PEP, indicating that both of the ATP-producing glycolytic enzymes, GAPDH/PGK and pyruvate kinase, were associated with the SR and functionally capable of providing ATP for the Ca2+ pump. Addition of a soluble hexokinase ATP trap eliminated 45Ca transport fueled by exogenous ATP but had a markedly reduced effect on 45Ca transport supported by endogenously produced ATP. Similarly, at very low concentrations of ATP and ADP, ATP produced endogenously from ADP and PEP supported 15-fold more 45Ca transport than ATP supplied exogenously at the same concentration. These results suggest that ATP generated by SR-associated glycolytic enzymes is transferred to the Ca2+ pump in a protected microenvironment and is functionally coupled to Ca2+ transport.
Our SR vesicles were contaminated to some degree by SL membranes (10% for skeletal muscle and 15% to 20% for cardiac muscle). Initial attempts were made to obtain highly purified SR by including 600 mmol/L KCl in the isolation buffer, but this resulted in loss of GAPDH/PGK and pyruvate kinase enzyme activities that was probably due to dissociation of the enzymes from the SR membranes.9 Activity was fully retained at physiological levels of KCl in the isolation buffer (150 to 200 mmol/L) or when KCl was absent. Consequently, we decided to eliminate KCl from the isolation medium. SL is known to have Ca2+ pumps, although far fewer than SR.21 To eliminate possible confounding of our results by Ca2+ uptake into SL or mitochondrial vesicles, all assays were performed in the presence of ruthenium red, an inhibitor of mitochondrial Ca2+ transport, and in the presence and absence of thapsigargin, a plant-derived sesquiterpene lactone, which is a potent and specific inhibitor of all of the isoenzymes in the SR or endoplasmic reticulum Ca2+-ATPase family22 but without effect (in the concentrations used) on the plasma membrane Ca2+-ATPase.22 23 All results were then expressed as thapsigargin-sensitive 45Ca uptake by the SR. SL membranes are believed by some investigators to contain glycolytic enzymes bound to the cytoplasmic side.10 To the extent that inside-out SL vesicles were present in the preparations and did in fact contain these enzymes, the addition of glycolytic substrates and cofactors could have resulted in ATP production by the SL, with release into the reaction medium, diffusion to the SR, and support of the Ca2+ pumps. However, this effect should have been minor, because of the relatively small amount of SL contamination present. Furthermore, the ATP trap experiment demonstrated that the ATP generated by glycolysis and used to support SR Ca uptake was “protected” and was not in free equilibrium with soluble ATP in the reaction medium.
The association of glycolytic enzymes with SR from skeletal and cardiac muscle is unlikely to represent an artifact of our system and procedures. The only sources of ATP after the addition of glycolytic substrates to SR were the glycolytic reactions at GAPDH/PGK and pyruvate kinase; inhibitors were given to block the myokinase and mitochondrial F0F1-ATPase reactions. No significant creatine kinase activity was detectable in the SR preparation. Controls with SR but without glycolytic substrate failed to exhibit Ca2+-ATPase activity or 45Ca uptake. SDS gels demonstrated that the key glycolytic enzyme, pyruvate kinase, was bound to the SR and was not free in the supernatant, making nonspecific “sticking” of the enzyme to the SR membranes unlikely. Therefore, it seems probable that glycolytic enzymes are, in fact, associated with SR in the intact myocyte.
It should be noted that the concentrations of ADP, NAD+, PEP, and other glycolytic intermediates used in our experiments were probably higher than the concentrations that occur in vivo. In cardiac ischemia/reperfusion, transition levels of GAP are in the micromolar range3 rather than the millimolar concentrations used here. The same is true for free ADP in vivo.24 On the other hand, it is unknown whether the concentrations of the various SR-associated enzymes present in our preparation are similar to the concentrations occurring in vivo. Enzyme concentrations could be higher in vivo if significant dissociation of enzymes occurred during the isolation procedure. Furthermore, the activities of these enzymes in vivo are dependent on local concentrations of specific substrates and coenzymes, which could be much higher than average cellular concentrations. To facilitate measurement of SR-associated glycolytic enzyme activities in our experiments, relatively high concentrations of substrates and coenzymes were chosen to saturate the active site of the enzymes. Given these uncertainties, our data demonstrate the potential for glycolytic ATP to support the SR Ca2+ pump but do not prove that this actually occurs in vivo.
The nature of how glycolytic enzymes are associated with SR membranes of cardiac and skeletal muscle is unknown. Our observation that glycolytic enzyme activity was lost when isolation buffer containing 600 mmol/L KCl was used is consistent with the report of Pierce and Philipson,9 who found that GAPDH and PGK activities were lost from a rabbit cardiac particulate fraction containing SL and SR vesicles in the presence of a high concentration of NaCl. However, the activities were fully recoverable in the supernatant, indicating that the enzymes had been solubilized from the membranes. Dimethonium, an organic divalent cation that screens membrane surface charge, removed >90% of exogenously added GAPDH that had bound to purified SL vesicles. These results suggest that the binding of at least GAPDH and PGK to SR and SL membranes is electrostatic in nature and potentially reversible. Electrostatic interactions have also been invoked to explain the binding of hexokinase to mitochondrial membranes in brain and tumor cells25 26 and of hemoglobin to band 3 in erythrocyte membranes.27
Paul and colleagues28 29 have reported that plasma membrane vesicles (obtained from pig stomach antrum smooth muscle) contain a full complement of glycolytic enzymes, including LDH, pyruvate kinase, enolase, phosphoglyceromutase, PGK, GAPDH, aldolase, and hexokinase. Addition of FDP, NAD+, ADP, and Pi to these membrane vesicles supported 45Ca transport and production of NADH and lactate. Han et al8 recently reported that triads from rabbit skeletal muscle can synthesize ATP from GAP or FDP and that this ATP is compartmentalized and not in equilibrium with bulk ATP. In nonmyocyte cell types, substrates for GAPDH and PGK have been shown to stimulate Na+ transport into vesicles from human erythrocyte membranes without the need for added ATP.30 Coupling of glycolysis and Na+,K+-ATPase activity was also demonstrated to exist in cultured MDCK cells derived from renal epithelium.31 32 Other than our own studies, there are no reports available concerning the possible association of glycolytic enzymes with cardiac SR or support of cardiac SR function by glycolytic substrates and cofactors in the absence of exogenous ATP.
Whether functional compartmentation of ATP production exists in myocytes has been controversial. Weiss and colleagues10 11 33 have reported that glycolytic ATP is used preferentially over ATP derived from oxidative phosphorylation to close sarcolemmal KATP channels. Studies using patch clamping of permeabilized ventricular myocytes demonstrated that glycolysis was more effective than oxidative phosphorylation in preventing the opening of K+ channels. Experiments in excised inside-out patches suggested that ATP-producing glycolytic enzymes were located in the membrane near the channels, potentially accounting for their preference for glycolytic ATP.10 11 Glycolysis also appears to be linked to Na+-K+ transport across the cell membrane in vascular smooth muscle; oxygen consumption is related more to isometric force.34
However, the physical basis for intracellular compartmentation of ATP has been questioned previously.35 Although intracellular diffusion of ATP from the mitochondrion appears to be unhindered and should be sufficient to support SR Ca2+-ATPase function under normal circumstances, intracellular gradients of ATP have been shown to occur during conditions of limited ATP supply (eg, hypoxia), whereby enzymes located further from the mitochondria experience a more dramatic decrease in ATP concentration than enzymes located more closely.36 37 Functional intracellular compartmentation based on spatial localization of glycolytic enzymes could provide efficient spatial translocation of ATP to sites of usage, such as the SR Ca2+ pump. It should be pointed out, however, that we have no direct data pointing to the existence of microcompartmentation in the intact myocyte. The ATP concentrations that we used in vitro were lower than those reported to exist in vivo. The diffusion gradient for bulk ATP may therefore be higher in vivo, and intracellular ATP compartmentation may be of significance only under conditions of marked ATP depletion. In addition, a phosphocreatine/creatine “energy shuttle” may transport ATP from the mitochondria to the SR to a certain extent in vivo, provided that creatine kinase is located on the SR near the Ca2+-ATPase. However, we could find no evidence of creatine kinase activity in our SR preparations.
Kusuoka and Marban38 recently reported that glycolytic inhibition produced by glycogen depletion and IAA or 2-deoxyglucose resulted in increased end-diastolic left ventricular pressure and intracellular Ca2+ concentration in isolated perfused ferret hearts. However, intracellular Ca2+ overload occurred only when glucose was included in the perfusate, leading to accumulation of sugar phosphates. The authors suggested that the deleterious effects of glycolytic inhibition may have been related to the accumulation of toxic intermediates or to “phosphate trapping” rather than to a reduction in glycolytically derived ATP. These results, however, do not exclude intracellular compartmentation of ATP with depletion at specific intracellular locations (eg, the SR Ca2+ pumps) nor do they speak to the role of glycolytic ATP in the setting of myocardial ischemia/reperfusion with concomitant intracellular ATP depletion. Our own results using IAA in SR vesicles supported by glycolytic metabolism are most consistent with IAA producing a decrease in Ca2+ transport through a block in ATP production. If Ca2+ uptake were inhibited by sugar phosphates accumulating proximal to the IAA-induced glycolytic block, it should not have been so readily restored by the addition of PEP, a downstream glycolytic substrate, to the reaction medium (Fig 4⇑).
Although ATP derived from oxidative phosphorylation is sufficient to support SR Ca2+ transport and preserve cytosolic Ca2+ homeostasis under normal conditions, as evidenced by the lack of functional deterioration when perfused hearts or isolated myocytes undergo glycolytic inhibition, the same may not be true under conditions of increased Ca2+ “stress.” Perfused hearts subjected to ischemia/reperfusion in order to increase cytosolic Ca2+ concentration or given isoproterenol to stimulate cellular Ca2+ entry developed marked functional and metabolic deterioration when glycolysis was inhibited that was apparently due to an inability to restore Ca2+ homeostasis.5 6 39 The capacity to reduce cytosolic Ca2+ concentration promptly during periods of increased Ca2+ stress may be essential for avoiding cellular injury related to Ca2+-activated proteases and lipases and may be critically dependent on glycolytic ATP generated by SR-associated enzymes.
In conclusion, the entire chain of glycolytic enzymes from aldolase onward, including the two key ATP-producing enzymes, GAPDH/PGK and pyruvate kinase, is bound to both cardiac and skeletal muscle SR. Ca2+-ATPase activity and SR Ca2+ uptake can be supported solely by provision of glycolytic substrates and cofactors without the addition of exogenous ATP. Since glycolytic ATP is more efficient at supporting SR Ca2+ uptake and is protected from an ATP trap, the glycolytic enzymes and the Ca2+ pump appear to be functionally coupled, with the ATP produced by glycolysis directly channeled to the Ca2+ pump rather than in free equilibrium with bulk phase ATP. These observations are consistent with a preferential role of glycolytic ATP in supporting SR Ca2+ transport and cellular Ca2+ homeostasis and may provide a physiological basis for the critical role of glycolytic metabolism in the functional recovery of the reperfused heart.
This study was supported by US Public Health Service grant HL-33360 from the National Heart, Lung, and Blood Institute, Bethesda, Md. We thank Drs Peter L. Pedersen, Richard G. Hansford, James L. Ellis, David L. Huso, and Jeffrey P. Froehlich for their help and useful discussions and Christine Borkowski for expert typing of the manuscript.
Reprint requests to Dr Lewis C. Becker, The Johns Hopkins Hospital, 600 N Wolfe St, Halsted 500, Baltimore, MD 21287.
- Received August 8, 1994.
- Accepted March 17, 1995.
- © 1995 American Heart Association, Inc.
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