ATP from glycolysis is required for normal sodium homeostasis in resting fast-twitch rodent skeletal muscle

Ken Okamoto1,2, Weiyang Wang2, Jan Rounds1, Elizabeth A. Chambers1, and Danny O. Jacobs2

1 Laboratories for Surgical Metabolism and Nutrition, Department of Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115; and 2 Laboratories for Surgical Research, Department of Surgery, Creighton University, Omaha, Nebraska 68131


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Myocellular sodium homeostasis is commonly disrupted during critical illness for unknown reasons. Recent data suggest that changes in intracellular sodium content and the amount of ATP provided by glycolysis are closely related. The role of glycolysis and oxidative phosphorylation in providing fuel to the Na+-K+ pump was investigated in resting rat extensor digitorum longus muscles incubated at 30°C for 1 h. Oxidative inhibition with carbonyl cyanide m-chlorophenylhydrazone, known as CCCP (0.2 µM), or by hypooxygenation did not alter myocellular sodium or potassium content ([Na+]i, [K+]i, respectively), whereas treatment with iodoacetic acid (0.3 mM), which effectively blocked glycolysis, dramatically increased [Na+]i and the [Na+]i/[K+]i ratio. Experiments using ouabain and measurements of myocellular high-energy phosphates indicate that Na+-K+-ATPase activity is only impaired when glycolysis is inhibited. The data suggest that normal glycolysis is required to regulate intracellular sodium in fast-twitch skeletal muscles, because it is the predominant source of the fuel for the Na+-K+-ATPase.

oxidative phosphorylation; sodium-potassium-adenosine triphosphatase; high-energy phosphate; metabolic inhibition


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A NORMAL TRANSMEMBRANE Na+ gradient is required for normal myocellular excitability and contractility. However, changes in Na+ gradient also modulate nutrient transport, energy metabolism, intracellular calcium, pH, volume, and osmolarity (15, 39, 41). An alteration in myocellular Na+ homeostasis manifested as a decrease in membrane potential and an accumulation of intracellular Na+ may occur during ischemia, hypoxia, and septic shock (39). These changes are associated with changes in myocellular energetics. Indeed, a progressive failure of cellular energy metabolism is a common finding in septic patients who do not respond to therapeutic intervention (40). The precise mechanism by which Na+ homeostasis is altered during critical illness is unknown. However, because extrusion of intracellular Na+ normally occurs primarily via the Na+-K+-ATPase, it has been hypothesized that alterations in cellular ATP production or consumption are responsible for changes in intracellular Na+. The relationship between ATP supply and changes in cation transport is important to understand. Previous studies have suggested that glycolysis plays an important role in energy production in skeletal muscle during metabolic inhibition (10, 18), and several investigators have determined that glycolytically derived ATP fuels a number of sarcolemmal ion transporters, including the Na+-K+-ATPase (4, 9, 14), the ATP-sensitive K+ channel (45), and the Ca2+ ATPase (32) in vascular and cardiac muscle. To our knowledge, however, the preferred source of energy for normal Na+ homeostasis in fast-twitch skeletal muscle has not been determined. Therefore, we examined the relationship between energy supply and intracellular Na+ content in ex vivo resting fast-twitch skeletal muscle isolated from infant rats. We used interventions designed to selectively inhibit glycolysis or oxidative phosphorylation to test the hypothesis that ATP derived from glycolysis preferentially fuels the Na+-K+-ATPase. Pharmacological inhibition of Na+/K+ transport was used to assess the relative contribution of glycolysis and oxidative phosphorylation to alterations in myocellular Na+ homeostasis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Treatment of animals. Male, specific-pathogen-free Wistar rats (Harlan Sprague Dawley, Indianapolis, IN), weighing 35-46 g, were acclimatized to 12:12-h periods of light and dark exposure, housed at a constant temperature of 22°C with 50-70% humidity, and were allowed standard rodent chow (Ralston Purina, Louis, MO) and water ad libitum for 3 days before experimentation. After acclimatization, the rats were weighed (~50-70 g) and anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). The hindlimb extensor digitorum longus (EDL) muscle, which contains primarily fast-twitch glycolytic (~45%) and oxidative-glycolytic (~45%) fibers (26), was carefully dissected, with the supporting tendons left intact. Infant rats have thin EDL muscles weighing 20-30 mg that permit adequate quantities of oxygen and nutrients to diffuse freely during incubation. The experiments described herein adhered to the guidelines of the National Institutes of Health for the use of experimental animals, and the Animal Care and Use Committee of Harvard Medical School approved the study protocols that were employed.

Muscle incubation. Immediately after dissection, each muscle was mounted on a solid support at resting length, placed in a tightly stoppered 25-ml flask (1 muscle per flask) containing 3 ml of Krebs-Henseleit buffer (KHB; in mM: 118 NaCl, 25.3 NaHCO3, 4.6 KCl, 1.16 KH2PO4, 1.16 MgSO4, 2.5 CaCl2, and 10 D-glucose, pH 7.4), and preincubated in a shaking (70 Hz) water bath for 30 min to reduce blood contamination and to allow equilibration of extracellular fluid with KHB (16). In separate experiments, muscles were preincubated for 15 min in KHB as described above, subsequently transferred to another flask containing glucose-free KHB with 2 mg/l glucagon, and incubated for a second 15-min period to deplete myocellular glycogen (9). The KHB solution used for preincubation and incubation was treated by continuous bubbling of 95% O2-5% CO2 gas for 30 min before it was pipetted into the flasks. Hypoxic KHB was created by exposing KHB to 95% N2-5% CO2 gas before pipetting. This procedure decreased PO2 to 35 mmHg in the hypoxic KHB compared with a partial pressure of 560 mmHg in the oxygenated KHB, as measured by a blood gas analyzer (Stat Profile Ultra, Nova Biochemical, Waltham, MA).

After preincubation, the muscles were blotted, weighed, and randomly transferred into another flask containing 3 ml of the following media: normal KHB (control); glucose-free KHB containing acetate (5 mM) as substrate for oxidative metabolism with or without varying concentrations (0.1, 0.3, 1.0, or 2.0 mM) of iodoacetate (IAA), which inhibits glyceraldehyde-3-phosphate dehydrogenase (43); normal KHB with varying concentrations (0.02, 0.2, 2.0, or 20 µM) of carbonylcyanide m-chlorophenylhydrazone (CCCP), which uncouples oxidative phosphorylation (27); or hypoxic KHB (9). In experiments to assess Na+-K+-ATPase activity, ouabain (1 mM), a specific inhibitor of Na+-K+-ATPase, was added to the incubation media. Muscles that weighed >30 mg were not studied, because previous investigations have shown that nutrient diffusion during ex vivo incubation is inadequate in samples of this mass (2). After these interventions, the muscle samples were returned to a water bath and incubated for 1 h. The incubation temperature for all experiments was set at 30°C, rather than 37°C, to slow the rate of metabolism and further ensure adequate O2 delivery by diffusion into the muscles. Immediately after incubation, muscle samples were either assayed for intracellular Na+ and K+ content ([Na+]i and [K+]i, respectively) or frozen with metal tongs cooled in liquid N2 and stored at -80°C until analyzed for other metabolites. Aliquots of the media from the incubation flasks were also stored separately at -80°C.

[Na+]i and [K+]i. [Na+]i and [K+]i in the EDL muscles were determined as described by Everts and Clausen (12). Briefly, after a 1-h incubation period, muscles were washed for 4 × 15 min in 3 ml of ice-cold Tris-sucrose buffer (Tris-HCl 10 mM, sucrose 263 mM, pH 7.4) in a shaking water bath. The Tris-sucrose buffer was oxygenated by continuous bubbling of 95% O2-5% CO2 at 0°C for 30 min before pipetting into 25-ml flasks. Each flask was then individually gassed with the same mixture and tightly stoppered. Because some loss of [Na+]i occurs during washout (12), the correction coefficient to account for sodium loss was determined in a separate experiment. A correction coefficient of 1.41 was obtained and used to calculate [Na+]i. After washout, the muscles were blotted and homogenized in 2 ml of 0.38 M trichloroacetic acid with a sonicator (W-380; Heat Systems-Ultrasonics, New York, NY) and centrifuged at 2,000 g for 10 min. The Na+ and K+ contents of these extracts were determined using a flame photometer (IL643; Instrumentation Laboratory, Lexington, MA), with cesium as the internal standard. The values of [Na+]i and [K+]i in the EDL are expressed in micromoles per gram wet weight.

ATP, ADP, AMP, creatine phosphate, and glycogen content of the muscle. Frozen muscle specimens were ground to a fine powder in liquid N2 and dissected free of connective tissue. The muscle powder was weighed and mixed with 1.0 ml of 0.02 N HCl. A 0.1-ml portion was removed and heated to 100°C for 10 min for glycogen determination by use of amyloglucosidase hydrolysis (25). Perchloric acid (112.5 µl of 3.0 N) was added to the remaining sample. Extraction was performed using gentle agitation for 20 min at 4°C followed by vortex mixing. The precipitate was separated by centrifugation. The supernatant was collected, neutralized with 225 µl of 2.5 M KHCO3, and centrifuged again to remove the neutralized KClO4. The resulting supernatants were used to measure myocellular ATP, ADP, AMP, and phosphocreatine (PCr) by HPLC (47). A reversed-phase HPLC Binary Gradient System (model 338; Beckman Instruments, San Ramon, CA) consisting of two model 110B solvent delivery modules, a 330 organizer with 210A sample injection valve and dynamic mixer, a model 167 programmable scanning detector, a model 406 analog interface, and an IBM PC equipped with Beckman System Gold Software was used to measure the energy metabolites. Separation was performed on a reversed-phase C2/C18 silica column, Mino RPC S5/20 (5 µm, 4.6 × 200 mm) at 25°C. The values for each sample were determined by averaging triplicate samples.

Lactate assay. Lactate production from the muscle was determined by serially measuring lactate concentration in the incubation media. Lactate was assayed as previously described (18), by a standard microfluorometric enzymatic procedure involving reduction of NAD by lactate dehydrogenase (LDH) to produce NADH. Briefly, 50 µl of incubation medium or lactate standards in triplicate were pipetted into each well of a 96-well plate. Next, 200 µl of LDH and NAD in hydrazine-glycine buffer (pH 9.1) were added to each sample. The mixture was agitated for 5 min and then left at room temperature for 45 min. Fluorescence of each well was then detected (excitation: 360 nm, emission: 530 nm) using a microplate reader (CytoFluor II; PerSeptive Biosystems, Foster City, CA). Each plate was read three times over a period of <2 min, and the average of three readings for triplicate samples was adopted as the fluorescence for each sample.

Assay of LDH and creatine kinase. To evaluate myocellular viability and alterations in membrane permeability, LDH and creatine kinase (CK) released from the muscle samples during the 1-h incubation period were determined. LDH and CK activities in the incubation media were measured with a spectrophotometer (model U-2000; Hitachi Instrument, San Jose, CA) at 465 and 430 nm, respectively, by use of commercially available kits (Sigma Chemical, St. Louis, MO).

Statistical analysis. Statistical analysis was performed by analysis of variance (ANOVA) using STATISTICA for Windows Release 4.3 (StatSoft, Tulsa, OK). Post hoc pair-wise group comparisons were performed using Tukey's test when appropriate. Probability values of <0.05 were considered significant. Results in the text, tables, and figures are expressed as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of glycolytic inhibition on myocellular Na+ and K+, lactate production, and LDH release. Isolated EDL muscles were incubated for 1 h in glucose-free media containing varying concentrations of IAA to examine the effects of glycolytic inhibition on myocellular Na+. The efficiency of glycolytic inhibition was assessed by measuring lactate production during incubation. LDH released into the media was also quantified. The amount of CK released into the media was not measured in the muscles treated with IAA, because this inhibitor alters hexokinase activity (35) in the assay used to measure CK activity.

The results are shown in Fig. 1. In control muscles incubated with normal KHB, the average values of [Na+]i and the [Na+]i/[K+]i ratio were 10.8 ± 1.0 and 0.14 ± 0.01 µmol/g wet wt, respectively. No differences in [Na+]i, the [Na+]i/[K+]i ratio, lactate production, or the amount of LDH released were detected in muscle incubated in glucose-free KHB relative to controls. In contrast, addition of 0.3 mM IAA to glucose-free KHB dramatically increased [Na+]i to a maximum mean value of 42.6 ± 0.7 µmol/g wet wt. IAA treatment also increased [Na+]i/[K+]i ratios in a dose-dependent manner. A >16-fold increase (2.32 ± 0.25) in [Na+]i/[K+]i was detected at the highest concentration (2.0 mM) of IAA tested. Lactate production was significantly decreased, even at the lowest concentration (0.1 mM) of IAA administered. Higher concentrations (1.0 and 2.0 mM) of IAA reduced lactate production to near zero but also significantly increased LDH release. Thus the addition of IAA at 0.1 or 0.3 mM concentration to glucose-free media significantly altered myocellular Na+ homeostasis and efficiently blocked glycolysis without altering myocellular viability.


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Fig. 1.   Effects of glycolytic inhibition on intracellular sodium concentration ([Na+]i) (A), the [Na+]i/[K+]i ratio (B), lactate production (C), and lactate dehydrogenase (LDH) release (D) in resting isolated extensor digitorum longus (EDL) muscles. The muscles were incubated for 1 h at 30°C in Krebs-Henseleit buffer (KHB, D-glucose 10 mM), Control; in glucose-free KHB containing acetate (5 mM) as substrate for oxidative metabolism [G(-)]; or in glucose-free KHB with a varying concentration (0.1, 0.3, 1.0, or 2.0 mM) of iodoacetate (IAA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase. Data are means ± SE for 6-8 muscles in each group. *P < 0.05 vs. Control by one-way ANOVA and Tukey's test. Glycolysis inhibition by IAA at 0.1 or 0.3 mM was highly effective and significantly altered myocellular sodium homeostasis without altering myocellular viability.

Effects of inhibition of oxidative phosphorylation on myocellular Na+ and K+, lactate production, LDH, and CK release. The muscles were incubated for 1 h and then hypooxygenated or treated with varying concentrations of CCCP to examine the effects of oxidative phosphorylation inhibition on myocellular Na+ homeostasis, glycolysis, and myocellular viability. Exposure to hypoxia for 1 h significantly increased lactate production but did not alter [Na+]i, the [Na+]i/[K+]i ratio, or the release of LDH and CK (Figs. 2 and 3). Treatment with CCCP significantly increased lactate production at all concentrations tested. Furthermore, although higher concentrations of CCCP (2.0 and 20 µM) increased [Na+]i, the [Na+]i/[K+]i ratio, and LDH and CK release compared with control incubation, lower concentrations (0.02 and 0.2 µM) did not significantly alter these enzyme levels. Thus inhibition of oxidative phosphorylation by a 1-h exposure to hypoxia or treatment with 0.02-0.2 µM CCCP did not significantly alter [Na+]i or myocellular viability.


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Fig. 2.   Effects of oxidative inhibition on [Na+]i (A), the [Na+]i/[K+]i ratio (B), and lactate production (C) in resting isolated EDL muscles. The muscles were incubated for 1 h at 30°C in normal KHB (PO2 560 mmHg), Control; in hypooxygenated KHB (PO2 35 mmHg), Hypoxia; or in normal KHB with varying concentrations (0.02, 0.2, 2.0, or 20.0 µM) of carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation. Data are means ± SE for 6-8 muscles in each group. *P < 0.05 vs. Control by one-way ANOVA and Tukey's test. Lactate production was significantly increased in all treatment groups. Hypoxia and lower concentrations (0.02 and 0.2 µM) of CCCP did not alter [Na+]i or the [Na+]i/[K+]i ratio, whereas higher doses (2.0 and 20 µM) of CCCP significantly increased both indexes.



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Fig. 3.   Effects of oxidative inhibition on release of LDH (A) and creatine kinase (CK, B) from resting isolated EDL muscles. These specific interventions in each group are as described in Fig. 2. Data are means ± SE for 6-8 muscles in each group. *P < 0.05 vs. Control by one-way ANOVA and Tukey's test. Higher concentrations (2.0 and 20 µM) of CCCP significantly increased LDH and CK release, which suggests that membrane damage had occurred.

Effects of selective metabolic inhibition on myocellular Na+ and K+, lactate production, and LDH and CK release in the absence or presence of ouabain. To determine whether changes in Na+-K+-ATPase activity were associated with alterations in myocellular Na+ homeostasis, muscles were incubated for 1 h with IAA (0.3 mM) to inhibit glycolysis, or were hypooxygenated to inhibit oxidative phosphorylation, in the absence or presence of ouabain (1 mM). These doses were selected because they did not significantly alter cell viability as determined by LDH release (Table 1).

                              
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Table 1.   Release of LDH and CK from EDL muscles during 1-h exposure to interventions to selectively inhibit glycolysis (IAA) or oxidative metabolism (hypoxia) in the absence or presence of ouabain

In control EDL muscles, as well as in muscles that were hypooxygenated, the addition of ouabain significantly increased [Na+]i and the [Na+]i/[K+]i ratio compared with samples that were not treated with this metabolic inhibitor (Fig. 4). Ouabain treatment also significantly decreased lactate production in these two groups. In contrast, ouabain treatment had no effect on myocellular [Na+]i, the [Na+]i/[K+]i ratio, or lactate production in muscles incubated with IAA. Finally, the amount of LDH and CK released from incubated muscles was statistically indistinguishable among the three groups irrespective of treatment with ouabain (Table 1). These observations are consistent with altered Na+-K+-ATPase activity when glycolysis is inhibited by IAA.


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Fig. 4.   Effects of ouabain on [Na+]i (A), the [Na+]i/[K+]i ratio (B), and lactate production (C) in resting isolated EDL muscles after selective inhibition of glycolysis or oxidative metabolism. In the absence or presence of ouabain (1 mM), an inhibitor of Na+-K+-ATPase, the muscles were incubated for 1 h at 30°C in normal KHB, Control; glucose-free KHB with IAA (0.3 mM); or hypoxic KHB (Hypoxia). Data are means ± SE for 6-8 samples in each group. *P < 0.05 vs. corresponding group in the absence of ouabain by one-way ANOVA and Tukey's test. Oxidative inhibition by hypooxygenation significantly increased [Na+]i and the [Na+]i/[K+]i ratio in the presence of ouabain, whereas no further ionic changes were observed with ouabain treatment after glycolysis was inhibited by IAA, which is consistent with impairment of ouabain-sensitive Na+-K+-ATPase activity in the latter group.

Effects of selective metabolic inhibition on ATP, ADP, AMP, PCr, and glycogen levels. To determine how inhibition of glycolysis or oxidative phosphorylation affects myocellular energy metabolites, ATP, ADP, AMP, PCr, and glycogen were measured after 1-h incubation and selective metabolic inhibition (Fig. 5 and Table 2). Myocellular ATP levels were reduced and ADP and AMP levels were elevated in muscles treated with 0.3 mM IAA, although incubation in glucose-free KHB did not significantly affect the levels of any of these metabolites.


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Fig. 5.   Myocellular contents of ATP, ADP, and AMP after 1-h exposure to interventions designed to inhibit glycolysis or oxidative metabolism selectively. EDL muscles were incubated in normal KHB, Control; glucose-free KHB [G(-)]; glucose-free KHB with IAA (0.3 mM); hypooxygenated KHB, Hypoxia; or normal KHB with CCCP (0.2 µM). Data are means ± SE for 6-8 samples in each group. *P < 0.05 vs. Control by one-way ANOVA and Tukey's test.


                              
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Table 2.   Myocellular content of PCr and glycogen after 1-h incubation and treatments to selectively inhibited glycolysis or oxidative metabolism

Inhibition of oxidative phosphorylation by hypoxia and CCCP caused ATP levels to increase (P < 0.05). After glycolytic and oxidative inhibition, myocellular PCr content decreased to ~50-70% of control levels. Glycogen levels were also reduced during both glycolytic and oxidative inhibition. The reduction of glycogen with glucose-free KHB was extenuated by 22% in the presence of IAA, which is consistent with a prior study (35).

Effects of glycogen depletion on alterations in myocellular Na+ and K+. To determine whether an accumulation of glycogenolytically derived intermediates induced by IAA might have altered [Na+]i, muscle glycogen stores were depleted before incubation in separate experiments. Preincubation with 2 mg/l glucagon for 15 min reduced myocellular glycogen contents to almost zero (0.9 ± 0.5 µmol glucosyl units/g wet wt, n = 4) compared with the values of 21.0 ± 6.4 µmol glucosyl units/g wet wt in control muscles after 1-h incubation. Figure 6 shows the effects of glycogen depletion with glucagon on [Na+]i and the [Na+]i/[K+]i ratio in the muscles incubated with control media or IAA. In both groups of control and IAA, glycogen depletion before incubation did not significantly change myocellular Na+ levels. These results suggest that the accumulation of glycolytic intermediates derived from glycogen were not responsible for the increase in [Na+]i observed in muscles incubated with IAA.


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Fig. 6.   Effects of predepletion of myocellular glycogen on [Na+]i (A) and the [Na+]i/[K+]i ratio (B) during glycolytic inhibition. EDL muscles were preincubated with and without glucagon (2 mg/l) for 15 min and then incubated in normal KHB (Control) or glucose-free KHB with IAA (0.3 mM) for 1 h. Predepletion of myocellular glycogen did not significantly affect the measured levels of [Na+]i or [Na+]i/[K+]i in either group. This suggests that accumulation of glycogenolytically derived intermediates did not play an important role in mediating the observed changes in sodium homeostasis.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

After Weiss and Hiltbrand (44) suggested that energy production and utilization were compartmentalized such that membrane functions near or within the cell membrane were modulated independently of the bulk energy of the cytoplasm, other investigators endeavored to determine whether this model applied to the control of glycolysis and intracellular Na+ in various tissues (9, 14). Campbell and Paul (4) verified that membrane-bound enzymes of the glycolytic pathway provide the ATP used by the Na+-K+-ATPase in vascular smooth muscle. These studies are consistent with the existence of a PCr-based energy shuttle, which tranports high-energy phosphates from mitochondria to the cytoplasm and primarily fuels the contractile apparatus and other intracellular functions. The prevailing localization of mitochondria near the contractile proteins, which has been shown for cardiac ventricular cells (21), supports this theory. Corresponding morphological data are not yet available for skeletal muscle. However, these observations support the hypothesis that ATP derived from oxidative and glycolytic metabolism provides energy for different cellular processes.

Other investigations have suggested that glycolysis and Na+-K+-ATPase pump activity are linked. Inhibition of oxidative phosphorylation does not significantly alter active Na+/K+ exchange in the skeletal muscle of the frog (10). Lactate production by rat diaphragmatic muscle is reduced by treatment with ouabain or by incubation in K+-free medium to inhibit Na+-K+- ATPase activity (7, 8). James et al. (18) showed that epinephrine or amylin increased glycolysis and Na+-K+-ATPase activity. Although these data suggest that energy production and utilization are compartmentalized, we endeavored to verify this concept by directly comparing the effects of selective suppression of glycolytic or oxidative metabolism on alterations in intracellular sodium in intact resting fast-twitch skeletal muscle.

Results of the present study confirm that the ATP used to maintain normal Na+ gradients in fast-twitch skeletal muscles is preferentially provided by glycolysis. When glycolysis is inhibited, the energy produced by oxidative phosphorylation or PCr breakdown is insufficient to maintain normal [Na+]i in resting skeletal muscles. Our data also indicate that glycolysis and the activity of Na+-K+-ATPase are closely linked and suggest that the predominant role that glycolysis plays in fueling the Na+/K+ pump is due to functional compartmentalization. Glycolysis provides only a small portion of the total energy produced by a muscle cell. However, our data suggest that the maintenance of normal myocellular glucose/glycogen stores is critically important for normal Na+ homeostasis.

IAA was used to suppress glycolysis. IAA is a widely used, nonspecific, sulfhydryl-alkylating agent that affects the cysteine residues on many cellular enzymes, thereby impairing their activity (43). Glyceraldehyde-3-phosphate dehydrogenase is particularly sensitive to IAA and is irreversibly and noncompetitively inhibited at concentrations that do not affect other contractile proteins or the mechanism by which they are activated (34). In our experiments using IAA, glucose was omitted from the incubation media to prevent the accumulation of toxic glycolytic intermediates within the myocyte (24, 34). The lowest concentration (0.1 mM) of IAA that was utilized significantly reduced lactate production, indicating that glycolysis was effectively inhibited. A clear dose-response relationship was found such that, as the IAA dose escalated, greater increases in the [Na+]i/[K+]i ratios were evident. Thus the degree of glycolytic inhibition induced was paralleled by progressive increases in intracellular Na+ and the [Na+]i/[K+]i ratios. However, higher concentrations (1.0 and 2.0 mM) of IAA also significantly increased LDH release, which indicates that membrane permeability was changed, presumably due to irreversible damage. There are several mechanisms by which this damage could occur: 1) at high concentrations, IAA may induce progressive and irreversible sarcolemmal contracture (44) and may interfere with the creatine phosphoryltransfer reaction (6); 2) complete inhibition of glycolysis may critically reduce ATP concentrations below a necessary maintenance level; and 3) the rapid accumulation of glycolytic intermediates derived from myocellular glycogen might cause cellular injury. Because we wanted to evaluate functional alterations in myocellular Na+ transport systems involving the Na+-K+-ATPase, we performed detailed studies using 0.3 mM IAA to selectively suppress glycolysis.

Although IAA has been characterized as a relatively specific inhibitor of glyceraldehyde-3-phosphate dehydrogenase in the glycolytic pathway, IAA could directly inhibit Na+-K+-ATPase activity via its nonspecific binding and alkylation (20). Because we did not measure the activity of Na+-K+-ATPase in the presence of IAA, it is not clear whether IAA at concentrations of 0.1 or 0.3 mM would affect this enzyme function. However, a previous study has shown that 0.1 mM of IAA inhibits anaerobic glycolysis and causes sugar phosphate accumulation but does not inhibit plasma or mitochondrial membrane-bound ATPases in yeast (23). Weiss and Hiltbrand (44) also compared the effects of five different interventions designed to selectively inhibit glycolysis on extracellular [K+] in isolated arterially perfused rabbit hearts. Two of those interventions were 0.1 or 1 mM of IAA. The effects of the five interventions were equivalent, which suggests that extracellular [K+] was increased via inhibition of glycolysis and not through direct effects of IAA on Na+-K+-ATPase. Therefore, we believe that IAA at lower concentrations of 0.1 or 0.3 mM primarily inhibits glycolysis and does not significantly compromise Na+-K+-ATPase activity in isolated, resting, fast-twitch skeletal muscle.

We used two approaches to inhibit oxidative phosphorylation: a mitochondrial uncoupling agent, CCCP (27), and exposure to hypoxia (44). Higher concentrations (2 and 20 µM) of CCCP significantly increased [Na+]i and the [Na+]i/[K+]i ratio but also markedly increased LDH and CK release from the muscles. Thus, in these instances, it is impossible to determine the effect of inhibition of oxidative phosphorylation by CCCP on myocellular Na+/K+ transport because of the observed changes in membrane permeability. The exact mechanism by which these changes occurred is unknown. Uncoupling agents like CCCP can directly affect cellular cation metabolism (22, 27). CCCP directly increases the passive permeability of the cell membrane to proteins and exerts a number of indirect effects on electrically excitable membranes that are apparently caused by the release of Ca2+ from uncoupled mitochondria (27). High concentrations of CCCP dramatically decrease intracellular pH and increase intracellular free Ca2+ levels that can cause muscle membrane depolarization and contraction (22). Thus these cationic changes and the resulting abrupt muscle contraction could significantly alter intracellular Na+ and myocyte availability.

In contrast, lower concentrations (0.02 and 0.2 µM) of CCCP did not affect intracellular [Na+]i and [K+]i levels or any measured index of cellular viability, but they significantly increased lactate production. The increased lactate production is consistent with a compensatory increase in glycolysis. These results are also consistent with the hypothesis that suppression of oxidative phosphorylation does not significantly alter myocellular Na+/K+ transport. Our experiments could not determine whether oxidative phosphorylation was sufficiently inhibited at lower concentrations, although CCCP at concentrations >1 × 10-8 M functions efficiently as a proton ionophore for mitochondria (27). Thus, to verify the effects observed when oxidative inhibition was induced by CCCP, the effects of hypoxia were also studied. The changes induced by a 1-h exposure to hypoxia were indistinguishable from those caused by the lower concentrations of CCCP.

2,4-Dinitrophenol (DNP) has also been extensively characterized as an uncoupler of oxidative phosphorylation in mitochondria. CCCP is a representative uncoupler of the A- class, whereas DNP is an HA<UP><SUB>2</SUB><SUP>−</SUP></UP> class uncoupler (27). Both CCCP and DNP are thought to act specifically to dissipate the proton gradient across the mitochondrial inner membrane and to prevent mitochondrial oxidative phosphorylation. At lower concentrations (~10 µM), DNP binds to the mitochondrial inner membrane, where it acts as a protonophore (42); at higher concentrations, DNP directly affects the activity of a number of membrane-bound ATPases, including the FoF1 ATPase of mitochondria (5), the Mg2+ ATPase of erythrocytes (1), and the glutathione transport ATPase of erythrocytes (46). On the basis of available literature, we would predict that the effects of lower concentrations of CCCP and DNP on myocellular Na+ homeostasis would be equivalent.

Our results also suggest that impairment of ouabain-sensitive Na+-K+-ATPase activity might contribute to alterations in [Na+]i and the [Na+]i/[K+]i ratio observed in incubated fast-twitch skeletal muscle preparations after treatment with 0.3 mM IAA to inhibit glycolysis. Our data are consistent with data obtained from studies of vascular smooth muscle (31), perfused heart (9), cardiac myocytes (38), and Purkinje cells (14). A close relationship between aerobic glycolysis, [Na+]i, and the Na+-K+-ATPase has been found in other cell types, including erythrocytes, neurons, and glia (11, 30), which suggests that coupling of glycolysis to energy utilization by membrane transport processes is a general phenomenon. In our experiments, ouabain treatment significantly reduced lactate production in control and hypooxygenated muscle. This finding is consistent with observations by James and colleagues (17, 18), who demonstrated that lactate generation in resting skeletal muscles after stimulation of glycolysis and glycogenolysis is closely linked to Na+-K+-ATPase activity. There are a number of other Na+ transport mechanisms involving ion channels, exchangers, and cotransporters. The role of these agents remains controversial. Investigations of perfused hearts have implicated altered Na+/H+ exchange (33) and/or voltage-gated Na+ channel activity (3) during ischemia, as well as altered Na+/Ca2+ exchange during beta -adrenergic stimulation (28), as mechanisms that contribute to the accumulation of myocellular Na+. In contrast, more recent studies have shown that altered exchanges of either Na+/Ca2+ or Na+/H+ cannot explain the observed increase in myocellular Na+ detected after glycolysis is inhibited in perfused hearts (9). Furthermore, alteration of voltage-gated Na+ channel activity during metabolic inhibition of isolated myocytes is also not primarily responsible for increasing intracellular Na+ (38). Although we cannot rule out the possibility that those ionic transporters affected Na+ homeostasis, our data suggest a significant dependence of Na+-K+-ATPase activity, and therefore [Na+]i, on glycolytically derived ATP.

When oxidative phosphorylation was inhibited by hypoxia or treatment with CCCP, ATP concentrations in these two groups were higher than those of the control. This may have been secondary to accelerated glycolysis induced by inhibition of oxidative phosphorylation (37) when there is a reduced need for energy produced via this pathway in resting, hypothermic muscle tissue. PCr and glycogen contents were also reduced when oxidative phosphorylation was inhibited, which suggests that glycogenolysis and PCr breakdown were also stimulated or augmented.

Incubating the EDL muscles in glucose-free media where acetate was provided as a substrate for oxidative phosphorylation did not significantly alter myocellular ATP, [Na+]i, or the [Na+]i/[K+]i ratio compared with muscles in the control group. These findings, along with the marked reduction in glycogen stores but unaltered lactate production, are also consistent with a preferential utilization of glycolytically derived ATP to fuel the Na+-K+-ATPase and maintain normal myocellular Na+ levels. Our results suggest that myocellular glycogenolysis occurred under the glucose-free condition presumably to provide ATP for the Na+-K+-ATPase, as has been noted previously (35). Thus there were no significant differences in myocellular [Na+]i and ATP between muscles incubated in glucose-free conditions compared with normal controls. The presence of similar lactate levels in these two groups suggests that the amounts of ATP produced to fuel the Na+-K+-ATPase were equivalent as well.

Our data demonstrate the importance of glycogen metabolism for myocellular Na+ homeostasis. Inhibition of glycolytic and glycogenolytic ATP production with IAA decreased myocellular ATP content, whereas [Na+]i and the [Na+]i/[K+]i ratio were greatly increased. The significant decrease in PCr stores in this group argues that there may be compensatory mechanisms that can provide fuel for the Na+-K+-ATPase when glycolysis is inhibited. However, our data suggest that neither oxidative phosphorylation nor PCr breakdown could provide sufficient energy to maintain normal [Na+]i in our experimental conditions. Further investigation is needed to verify this hypothesis and fully understand its implications.

The reduction in myocellular glycogen induced by exposure to IAA implies that the levels of glycolytic intermediates such as fructose 1,2-diphosphate and glyceraldehyde 3-phosphate levels were increased. Thus the accumulation of glycolytic intermediates could be responsible for the observed changes by directly disrupting cell membrane function or integrity (24). However, because the ATP content of muscles incubated with 0.3 mM IAA was reduced but not depleted, and because predepletion of myocellular glycogen stores with glucagon did not further modify myocellular Na+ homeostasis, we conclude that any effects of glycolytic intermediates were inconsequential in our study.

It has been suggested that glycolysis is tightly coupled to phosphorylation because of the simple and well documented fact that, for each mole of lactate that is formed, one mole of Pi and ADP must be transformed to ATP. Thus the ATPase could be the rate-limiting step in glycolysis. Data presented by Racker (36) further indicate that, in Ehrlich ascites cells and several other tumors, a rate of high aerobic glycolysis is maintained by the generation of Pi and ADP due to defective operation of the plasma membrane Na+-K+-ATPase. Racker's data suggest that the plasma membrane Na+-K+-ATPase is responsible for the ATP hydrolysis that is required for lactate production in tumor cells (36). It has also been generally recognized that there is a direct relationship between the rate of aerobic glycolysis and the activity of Na+-K+-ATPase in other cell types. However, whether there is a "cause and effect" relationship between glycolysis and Na+-K+-ATPase activity is still controversial. Although our data strongly suggest that glycolysis is essential for the normal function of the Na+-K+-ATPase in nondiseased resting skeletal muscle, we cannot specify what role the Na+-K+-ATPase plays in regulating glycolysis. Furthermore, the present study could not determine whether disordered glycolysis, dysfunction of Na+-K+-ATPase, or both mechanisms contribute to alter Na+ homeostasis under pathological conditions. Recent observations indicate that stimulation by insulin or epinephrine of Na+-K+-ATPase is responsible for increased lactate production in skeletal muscle, which suggests that the activity of Na+-K+-ATPase plays an important role in regulating glycolysis (19, 29). Thus the "Warburg effect" (i.e., a higher aerobic glycolysis in malignant tumors) might apply to muscle cells under normal or pathological conditions. Further studies are certainly required to determine the role of the Na+-K+-ATPase in modulating myocellular Na+ homeostasis through the regulation of aerobic glycolysis under normal or pathological conditions.

In well oxygenated muscles, glycolysis provides <5% of the total ATP produced (13). However, our experiments suggest that, during normoxia, basic ion and energy homeostasis may be jeopardized if glycolysis or glycogenolysis is inhibited. Similar problems could occur in patients who are subjected to ischemia and reperfusion, hemorrhage, and resuscitation or who are otherwise recovering from critical illness even in well oxygenated conditions. Therefore, our data suggest that glucose metabolism in skeletal muscle may play a more fundamental and important role during critical illness in preventing myocellular damage or dysfunction than believed, despite the relatively small amount of ATP derived from glycolysis. Our data may also help explain why disorders of glycolysis, which are frequently observed in sick patients, are associated with a poor prognosis and emphasize the importance of supporting glucose and glycogen utilization during critical illness.


    ACKNOWLEDGEMENTS

We thank Dr. J. Howard James, Department of Surgery, University of Cincinnati, for assistance in the lactate assay.


    FOOTNOTES

This study was supported in part by National Institute of General Medical Sciences Grant P50-GM-52585.

Address for reprint requests and other correspondence: D. O. Jacobs, Dept. of Surgery, Creighton Univ./St. Joseph Hospital, 601 N. 30th St., Suite 3520, Omaha, NE 68131 (E-mail: djacobs{at}creighton.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3 January 2001; accepted in final form 26 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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