© 1995 American Heart Association, Inc.
Articles |
Functional Coupling Between Glycolysis and Sarcoplasmic Reticulum Ca2+ Transport
From the Cardiology Division, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Md.
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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.
Key Words: glycolysis • Ca2+ transport • sarcoplasmic reticulum • glycolytic enzymes • ATP
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Introduction |
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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.
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Materials and Methods |
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Experimental 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
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 ß-scintillation counter.
Thapsigargin
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).
Reagents
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.
Experimental Protocols
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.
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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, 3.9x150 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.
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Results |
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SR Preparation
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.
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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.
Glycolytic Enzymes
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
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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.
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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).
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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.
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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.
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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
|
Acknowledgments |
|---|
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.
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Footnotes |
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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.
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References |
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A. D. Karelis, F. Peronnet, and P. F. Gardiner Insulin does not mediate the attenuation of fatigue associated with glucose infusion in rat plantaris muscle J Appl Physiol, July 1, 2003; 95(1): 330 - 335. [Abstract] [Full Text] [PDF]
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 D. L. Trence, J. L. Kelly, and I. B. Hirsch The Rationale and Management of Hyperglycemia for In-Patients with Cardiovascular Disease: Time for Change J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2430 - 2437. [Abstract] [Full Text] [PDF]
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O. J Sichelschmidt, C. Hahnefeld, T. Hohlfeld, F. W Herberg, and K. Schror Trapidil protects ischemic hearts from reperfusion injury by stimulating PKAII activity Cardiovasc Res, June 1, 2003; 58(3): 602 - 610. [Abstract] [Full Text] [PDF]
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L. A Blatter, J. Kockskamper, K. A Sheehan, A. V Zima, J. Huser, and S. L Lipsius Local calcium gradients during excitation-contraction coupling and alternans in atrial myocytes J. Physiol., January 1, 2003; 546(1): 19 - 31. [Abstract] [Full Text] [PDF]
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J. Kockskamper and L. A Blatter Subcellular Ca2+ alternans represents a novel mechanism for the generation of arrhythmogenic Ca2+ waves in cat atrial myocytes J. Physiol., November 15, 2002; 545(1): 65 - 79. [Abstract] [Full Text] [PDF]
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N. Rosenblatt-Velin, C. Montessuit, I. Papageorgiou, J. Terrand, and R. Lerch Postinfarction heart failure in rats is associated with upregulation of GLUT-1 and downregulation of genes of fatty acid metabolism Cardiovasc Res, December 1, 2001; 52(3): 407 - 416. [Abstract] [Full Text] [PDF]
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S. J. Lees, P. D. Franks, E. E. Spangenburg, and J. H. Williams Glycogen and glycogen phosphorylase associated with sarcoplasmic reticulum: effects of fatiguing activity J Appl Physiol, October 1, 2001; 91(4): 1638 - 1644. [Abstract] [Full Text] [PDF]
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J. G. Van Emous, C. L. A. M. Vleggeert-Lankamp, M. G. J. Nederhoff, T. J. C. Ruigrok, and C. J. A. Van Echteld Postischemic Na+-K+-ATPase reactivation is delayed in the absence of glycolytic ATP in isolated rat hearts Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H2189 - H2195. [Abstract] [Full Text] [PDF]
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M. Schafer, D. Bahde, B. Bosche, Y. Ladilov, C. Schafer, H. M. Piper, and T. Noll Modulation of early [Ca2+]i rise in metabolically inhibited endothelial cells by xestospongin C Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1002 - H1010. [Abstract] [Full Text] [PDF]
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D. D. Belke, T. S. Larsen, E. M. Gibbs, and D. L. Severson Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice Am J Physiol Endocrinol Metab, November 1, 2000; 279(5): E1104 - E1113. [Abstract] [Full Text] [PDF]
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N. Ortenblad, P. K. Lunde, K. Levin, J. L. Andersen, and P. K. Pedersen Enhanced sarcoplasmic reticulum Ca2+ release following intermittent sprint training Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2000; 279(1): R152 - R160. [Abstract] [Full Text] [PDF]
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J. Huser, Y. G. Wang, K. A Sheehan, F. Cifuentes, S. L Lipsius, and L. A Blatter Functional coupling between glycolysis and excitation--contraction coupling underlies alternans in cat heart cells J. Physiol., May 1, 2000; 524(3): 795 - 806. [Abstract] [Full Text] [PDF]
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P. H. McNulty, D. Jagasia, G. W. Cline, C. K. Ng, J. M. Whiting, P. Garg, G. I. Shulman, and R. Soufer Persistent Changes in Myocardial Glucose Metabolism In Vivo During Reperfusion of a Limited-Duration Coronary Occlusion Circulation, February 29, 2000; 101(8): 917 - 922. [Abstract] [Full Text] [PDF]
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P. Zhu, L. Lu, Y. Xu, C. Greyson, and G. G. Schwartz Glucose-insulin-potassium preserves systolic and diastolic function in ischemia and reperfusion in pigs Am J Physiol Heart Circ Physiol, February 1, 2000; 278(2): H595 - H603. [Abstract] [Full Text] [PDF]
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T. G. Favero Sarcoplasmic reticulum Ca2+ release and muscle fatigue J Appl Physiol, August 1, 1999; 87(2): 471 - 483. [Abstract] [Full Text] [PDF]
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J. H. James, K. R. Wagner, J.-K. King, R. E. Leffler, R. K. Upputuri, A. Balasubramaniam, L. A. Friend, D. A. Shelly, R. J. Paul, and J. E. Fischer Stimulation of both aerobic glycolysis and Na+-K+-ATPase activity in skeletal muscle by epinephrine or amylin Am J Physiol Endocrinol Metab, July 1, 1999; 277(1): E176 - E186. [Abstract] [Full Text] [PDF]
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D. S. Berger, S. K. Fellner, K. A. Robinson, K. Vlasica, I. E. Godoy, and S. G. Shroff Disparate effects of three types of extracellular acidosis on left ventricular function Am J Physiol Heart Circ Physiol, February 1, 1999; 276(2): H582 - H594. [Abstract] [Full Text] [PDF]
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K. Y. Xu, D. L. Huso, T. M. Dawson, D. S. Bredt, and L. C. Becker Nitric oxide synthase in cardiac sarcoplasmic reticulum PNAS, January 19, 1999; 96(2): 657 - 662. [Abstract] [Full Text] [PDF]
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M. Samaja, S. Allibardi, G. Milano, G. Neri, B. Grassi, L. B. Gladden, and M. C. Hogan Differential depression of myocardial function and metabolism by lactate and H+ Am J Physiol Heart Circ Physiol, January 1, 1999; 276(1): H3 - H8. [Abstract] [Full Text] [PDF]
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C. S. Apstein Glucose-Insulin-Potassium for Acute Myocardial Infarction : Remarkable Results From a New Prospective, Randomized Trial Circulation, November 24, 1998; 98(21): 2223 - 2226. [Full Text] [PDF]
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F. A. Recchia, P. I. McConnell, R. D. Bernstein, T. R. Vogel, X. Xu, and T. H. Hintze Reduced Nitric Oxide Production and Altered Myocardial Metabolism During the Decompensation of Pacing-Induced Heart Failure in the Conscious Dog Circ. Res., November 16, 1998; 83(10): 969 - 979. [Abstract] [Full Text] [PDF]
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W. Chen, R. London, E. Murphy, and C. Steenbergen Regulation of the Ca2+ Gradient Across the Sarcoplasmic Reticulum in Perfused Rabbit Heart : A 19F Nuclear Magnetic Resonance Study Circ. Res., November 2, 1998; 83(9): 898 - 907. [Abstract] [Full Text] [PDF]
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V. A Losito, R. G Tsushima, R. J Diaz, G. J Wilson, and P. H Backx Preferential regulation of rabbit cardiac L-type Ca2+ current by glycolytic derived ATP via a direct allosteric pathway J. Physiol., August 15, 1998; 511(1): 67 - 78. [Abstract] [Full Text] [PDF]
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J. D. Tune, R. T. Mallet, and H. F. Downey Insulin improves contractile function during moderate ischemia in canine left ventricle Am J Physiol Heart Circ Physiol, May 1, 1998; 274(5): H1574 - H1581. [Abstract] [Full Text] [PDF]
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 K. Y. Xu and L. C. Becker Ultrastructural Localization of Glycolytic Enzymes on Sarcoplasmic Reticulum Vesicles J. Histochem. Cytochem., April 1, 1998; 46(4): 419 - 428. [Abstract] [Full Text]
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J. Dizon, D. Burkhoff, J. Tauskela, J. Whang, P. Cannon, and J. Katz Metabolic inhibition in the perfused rat heart: evidence for glycolytic requirement for normal sodium homeostasis Am J Physiol Heart Circ Physiol, April 1, 1998; 274(4): H1082 - H1089. [Abstract] [Full Text] [PDF]
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G. W. Goodwin, F. Ahmad, T. Doenst, and H. Taegtmeyer Energy provision from glycogen, glucose, and fatty acids on adrenergic stimulation of isolated working rat hearts Am J Physiol Heart Circ Physiol, April 1, 1998; 274(4): H1239 - H1247. [Abstract] [Full Text] [PDF]
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M. C. Hogan, E. Ingham, and S. S. Kurdak Contraction duration affects metabolic energy cost and fatigue in skeletal muscle Am J Physiol Endocrinol Metab, March 1, 1998; 274(3): E397 - E402. [Abstract] [Full Text] [PDF]
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B. J. Martin, H. H. Valdivia, R. Bunger, R. D. Lasley, and R. M. Mentzer Jr. Pyruvate augments calcium transients and cell shortening in rat ventricular myocytes Am J Physiol Heart Circ Physiol, January 1, 1998; 274(1): H8 - H17. [Abstract] [Full Text] [PDF]
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C. S. Apstein and H. Taegtmeyer Glucose-Insulin-Potassium in Acute Myocardial Infarction : The Time Has Come for a Large, Prospective Trial Circulation, August 19, 1997; 96(4): 1074 - 1077. [Full Text]
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R. Tian, L. Nascimben, J. S. Ingwall, and B. H. Lorell Failure to Maintain a Low ADP Concentration Impairs Diastolic Function in Hypertrophied Rat Hearts Circulation, August 19, 1997; 96(4): 1313 - 1319. [Abstract] [Full Text]
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J. Booth, M. J. McKenna, P. A. Ruell, T. H. Gwinn, G. M. Davis, M. W. Thompson, A. R. Harmer, S. K. Hunter, and J. R. Sutton Impaired calcium pump function does not slow relaxation in human skeletal muscle after prolonged exercise J Appl Physiol, August 1, 1997; 83(2): 511 - 521. [Abstract] [Full Text] [PDF]
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K.-i. Yabe, Y. Nasa, M. Sato, R. Iijima, and S. Takeo Preconditioning preserves mitochondrial function and glycolytic flux during an early period of reperfusion in perfused rat hearts Cardiovasc Res, March 1, 1997; 33(3): 677 - 685. [Abstract] [PDF]
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K. Y. Xu, J. L. Zweier, and L. C. Becker Hydroxyl Radical Inhibits Sarcoplasmic Reticulum Ca2+-ATPase Function by Direct Attack on the ATP Binding Site Circ. Res., January 1, 1997; 80(1): 76 - 81. [Abstract] [Full Text]
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Z.-P. Gao, H.F. Downey, S. Jie, M.-X. He, and R. T Mallet Adenosine receptor blockade enhances glycolysis in hypoperfused guinea-pig myocardium Cardiovasc Res, January 1, 1997; 33(1): 31 - 44. [Abstract] [Full Text] [PDF]
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S. L. Henning, R. B. Wambolt, B. O. Schonekess, G. D. Lopaschuk, and M. F. Allard Contribution of Glycogen to Aerobic Myocardial Glucose Utilization Circulation, April 15, 1996; 93(8): 1549 - 1555. [Abstract] [Full Text]
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D. R. Meldrum, J. C. Cleveland Jr, B. C. Sheridan, R. T. Rowland, A. Banerjee, and A. H. Harken Cardiac Surgical Implications of Calcium Dyshomeostasis in the Heart Ann. Thorac. Surg., April 1, 1996; 61(4): 1273 - 1280. [Abstract] [Full Text]
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M.-L. Wu, K.-L. Tsai, S.-M. Wang, J.-C. Wu, B.-S. Wang, and Y.-T. Lee Mechanism of Hydrogen Peroxide and Hydroxyl Free Radical–Induced Intracellular Acidification in Cultured Rat Cardiac Myoblasts Circ. Res., April 1, 1996; 78(4): 564 - 572. [Abstract] [Full Text]
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A. Kaasik, V. Veksler, E. Boehm, M. Novotova, A. Minajeva, and R. Ventura-Clapier Energetic Crosstalk Between Organelles: Architectural Integration of Energy Production and Utilization Circ. Res., July 20, 2001; 89(2): 153 - 159. [Abstract] [Full Text] [PDF]
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