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Cardiovascular Research 1997 33(1):31-44; doi:10.1016/S0008-6363(96)00182-4
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Adenosine receptor blockade enhances glycolysis in hypoperfused guinea-pig myocardium

Zhi-Ping Gao1, H.Fred Downey, Sun Jie, Miao-Xiang He and Robert T Mallet*

Department of Integrative Physiology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699, USA

Received 7 December 1995; accepted 12 August 1996


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: This study tested the hypothesis that endogenous adenosine depresses anaerobic glycolysis in preischaemic and moderately ischaemic myocardium. Methods: Isolated, working guinea-pig hearts, perfused with glucose-fortified Krebs-Henseleit buffer, were subjected to 15 min mild hypoperfusion (coronary flow 60% of baseline) followed by 10 min ischaemia (coronary flow 20% of baseline). Adenosine A1 receptors were blocked with 8-p-sulfophenyl theophylline (8-SPT; 20 µM). Glucose oxidation and lactate production from exogenous glucose were assessed from 14CO2 and [14C]lactate formation, respectively, from [U-14C]glucose. Energy metabolites, glycolytic intermediates and glycogen were measured in extracts of stop-frozen preischaemic, mildly hypoperfused and ischaemic myocardium. Results: Adenosine receptor blockade did not affect left ventricular function assessed from heart rate x pressure product and pressure x volume work although coronary flow was slightly reduced. Adenosine receptor blockade increased glucose uptake (P < 0.05) by 100% during preischaemia and by 74% during mild hypoperfusion, and increased lactate production from exogenous glucose (P < 0.05) by 89% during preischaemia and fourfold during mild hypoperfusion, but did not stimulate glucose oxidation under any condition. Glycogen degradation was not increased by adenosine receptor blockade during ischaemia. Crossover plots of glycolytic intermediates revealed that phosphofructokinase was activated by adenosine receptor blockade at all three levels of perfusion. Conclusion: Endogenous adenosine attenuates anaerobic glycolysis in normally perfused, hypoperfused and ischaemic myocardium by blunting phosphofructokinase activity; this effect is mediated by adenosine A1 receptors.

KEYWORDS ANOVA = analysis of variance; Cr = creatine; CrP = creatine phosphate; DAP = dihydroxyacetone phosphate; dpm = disintegrations per min; F6P = fructose 6-phosphate; FDP = fructose 1,6-bisphosphate; G6P = glucose 6-phosphate; GAP = glyceraldehyde 3-phosphate; KCK, KMK = equilibrium constants of creatine kinase and myokinase; LAC = lactate; Mgf = cytosolic free magnesium; NMR = nuclear magnetic resonance spectroscopy; PEP = phosphoenolpyruvate; pHi = intracellular pH; PYR = pyruvate; 2PG = 2-phosphoglycerate; 3PG = 3-phosphoglycerate; Pa = aortic pressure; Pv = left atrial filling pressure; PIA = N6-(L-2-phenylisopropyl)adenosine; PKA = cyclic-AMP-dependent protein kinase; SA = specific radioactivity; 8-SPT = 8-p-sulfophenyl theophylline


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Adenosine, formed in large amounts in ischaemic myocardium as a result of cytosolic ATP degradation, has been implicated as a mediator of several cardioprotective phenomena [1], including bradycardia, coronary vasodilatation, adrenergic antagonism, and ischaemic preconditioning. Recent evidence from our laboratory [2] indicates that endogenous adenosine could also mediate acute metabolic down-regulation (i.e., hibernation of moderately ischaemic myocardium). In experiments in isolated, perfused guinea-pig hearts performing external work, adenosine receptor blockade with 8-p-sulfophenyl theophylline (8-SPT) during 60 min of moderately severe ischaemia prevented the recovery of cytosolic energetics that is considered a hallmark of hibernating myocardium [3, 4]. Adenosine receptor blockade also stimulated lactate formation in preischaemic and ischaemic myocardium; this finding raised the possibility that the energetic effects of interstitial adenosine were due, at least in part, to inhibition of anaerobic glycolysis during ischaemia. However, the mechanisms for glycolytic inhibition by endogenous adenosine were unclear.

Considerable disagreement exists in the literature regarding the effects of adenosine on myocardial glucose metabolism. Several investigators have reported that exogenous adenosine inhibits glycolysis during myocardial ischaemia [5–9], whereas others have found that exogenous adenosine stimulates glycolysis in ischaemic and normoxic conditions [10–12]. In ischaemic myocardium, ATP production by oxidative phosphorylation is impaired, and glycolysis could be a crucial source of ATP to support membrane ion transport [13–15] and minimise ischaemic injury [15–17]. Therefore, it would seem essential to delineate the effects of endogenous adenosine on glycolysis and lactate formation in normally perfused and, especially, ischaemic myocardium.

This investigation examined the effect of adenosine receptor blockade with 8-SPT on glucose metabolism in preischaemic, mildly hypoperfused, and ischaemic guinea-pig myocardium. Adenosine A1 receptors were selectively antagonised with 8-SPT before and during ischaemia. The effects of ischaemia and adenosine receptor blockade on glycolysis and glucose oxidation were assessed by measuring [14C]lactate and 14CO2 production, respectively, from [U-14C]glucose. To define the specific glycolytic enzyme activities modulated by adenosine receptor blockade, measured intracellular contents of glycolytic intermediates in preischaemic, hypoperfused and ischaemic myocardium were subjected to crossover plot analyses.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Isolated working hearts
Animal experimentation was approved by the Animal Care and Use committee of the University of North Texas Health Science Center and conformed to the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1985). Hearts (n = 77), beating at intrinsic sinus rhythm, were isolated from methoxyflurane-anaesthetised Hartley guinea-pigs (400–600 g body mass), and perfused as working hearts [2]. Non-recirculating perfusion medium was a modified Krebs-Henseleit bicarbonate buffer (38°C, pH 7.4) fortified with 10 mM glucose and 5 U/1 bovine insulin (Sigma, St. Louis, MO, USA) and aerated with 95% O2/5% CO2 [2]. [U-14C]glucose and 8-p-sulfophenyl theophylline (8-SPT) were purchased from DuPont-NEN (Boston, MA, USA) and Research Biochemicals (Natick, MA, USA), respectively. Spontaneous heart rate, aortic pressure (i.e., afterload: Pa), and left atrial filling pressure (i.e., preload: Pv) were continuously monitored with a multi-channel polygraph (Beckman Model R611). Coronary and aortic flows were measured by timed collections; cardiac output was taken as the sum of coronary and aortic flows. Stroke work was computed as pressure x volume work per beat—i.e., stroke volume x (Pa – Pv). Left ventricular performance was assessed from rate x pressure product (i.e., heart rate x (Pa – Pv)) and from left ventricular power (i.e., stroke work x heart rate).

2.2. Experimental protocols
2.2.1. (I) Hypoperfusion/ischaemia protocol
Experiments were designed to examine the effects of adenosine receptor blockade on glucose metabolism in working guinea-pig hearts following abrupt changes in coronary flow. The experimental protocol is summarised inFig. GR1. Hearts metabolised 10 mM [U-14C]glucose (specific activity: 2 mCi/mol). 8-SPT (20 µM) was continuously infused beginning at 20 min preischaemic baseline perfusion. We recently demonstrated [2] that this 8-SPT concentration was sufficient to completely block the A1-receptor-mediated bradycardia resulting from a bolus injection of 150 µg adenosine, but did not attenuate the hyperaemia mediated by A2 receptors. After 30 min preischaemia, hypoperfusion was produced by decreasing Pa (i.e., coronary perfusion pressure) in two discrete steps, from 90 to 45 cmH2O for 15 min (mild hypoperfusion), then to 22.5 cmH2O for 10 min (ischaemia). Pv was maintained at 12 cmH2O throughout the protocol. Experiments were terminated by freeze-stopping the hearts with Wollenberger tongs precooled to constant temperature in liquid N2. Hearts were stop-frozen at 30 min preischaemia, 15 min mild hypoperfusion, or at 10 min ischaemia, i.e. during the period of maximum anaerobic glycolysis in these ischaemic hearts [2].


Figure 1
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  		Fig. GR1 Hypoperfusion/ischaemia protocol. Coronary hypoperfusion was produced by lowering aortic pressure (Pa) from 90 (preischaemia) to 45 cmH2O for 15 min (mild hypoperfusion), then to 22.5 cmH2O for 10 min (ischaemia). Left atrial filling pressure (Pv) was held at 12 cmH2O throughout the protocol. Perfusion with 8-p-sulfophenyl theophylline (8-SPT) was begun at 20 min preischaemia and continued for the remainder of the experiment. Hearts were stop-frozen at 30 min preischaemia, 15 min mild hypoperfusion, or 10 min ischaemia. Sucrose, a marker of the cardiac extracellular space, was infused for 5 min prior to freeze-stop (hatched bar).

 
2.2.2. (II) Graded coronary perfusion protocol
The moderate reductions in coronary flow produced by 8-SPT in each phase of the experimental protocol raised the possibility that metabolic effects of adenosine receptor blockade could have resulted from ischaemia secondary to the reduction in coronary flow. A separate series of experiments was performed to test this hypothesis. In 8-SPT (20 µM) treated (n = 8) and untreated control (n = 8) hearts, Pa was held sequentially at 90, 100, 80, 60, 40, 30, and 20 cmH2O for 15 min at each level to produce incremental changes in coronary flow over a broad range of aortic pressures. Pv was maintained at 12 cmH2O throughout the protocol. Coronary effluent was sampled during the last 2 min of each phase, and lactate in these samples was measured by enzymatic assay [18] at 337 nm wavelength in a Perkin Elmer Lambda-2 spectrophotometer.

2.3. Oxidation of exogenous [14C]glucose
To quantitatively examine cardiac metabolism of exogenous glucose, hearts were perfused with [U-14C]glucose (specific activity: 2 mCi/mol). Aliquots (2–8 ml) of coronary venous effluent and arterial perfusion medium were collected in 25 ml Warburg flasks and acidified (pH = 1.2–1.5) with 1N HCl to convert HCO3– to CO2. 14CO2 was trapped in 0.4 ml benzethonium hydroxide (Sigma) in a polypropylene center well (Kontes Glassware, Vineland, NJ) without exposure to air. Samples were incubated overnight with gentle shaking to ensure complete trapping of 14CO2. 14CO2 was measured by liquid scintillation counting; disintegrations per min (dpm) were derived from counts per min by use of a standard quench curve. The rate of glucose oxidation (µmol · min–1 · g–1) was computed as 14CO2 production (dpm · min–1 · g–1) divided by the measured specific activity of [14C]glucose (dpm · µmol–1) in the arterial perfusion medium. Rates of 14CO2 release stabilised after about 20 min perfusion with [U-14C]glucose (cf. Fig. GR3 B), indicating that an isotopic steady state had been attained. To test for the presence of volatile radioactive impurities in the left atrial inflow, aliquots of arterial perfusion medium containing [U-14C]glucose were analyzed in parallel with the venous effluent samples [19]. No volatile contaminants were detected by this procedure.


Figure 3
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  		Fig. GR3 Effects of adenosine receptor blockade on metabolism of exogenous glucose. Means ± s.e. from 6–8 experiments. Glucose uptake (panel A), glucose oxidation (panel B) and [14C]lactate release (panel C) were determined as described in Section 2. Perfusion with 8-SPT was begun at 20 min. * P < 0.05 vs. 30 min; {ddagger}P < 0.05 vs. 45 min; {dagger}P < 0.05 vs. untreated.

 
To determine efficiency of 14CO2 trapping, 2–10 ml aliquots of perfusion medium containing 26 mM [14C]HCO3 (specific activity: 2 mCi/mol; Amersham, Arlington Heights, IL, USA) in Warburg flasks were acidified with 1N HCl and incubated overnight to effect 14CO2 trapping in benzethonium hydroxide. 14CO2 trapping efficiency (i.e., dpm 14C in the center well divided by the original dpm 14C in the perfusion medium) equalled 110–115% and did not vary with the volume of medium in the flask. Less than 0.01% of the original 14C remained in the acidified medium following incubation. Thus, it could be assumed that all of the 14CO2 in venous effluent collected in Warburg flasks was captured in the center well following acidification of the venous effluent samples.

2.4. [14C]Lactate formation from exogenous glucose
14C-labeled compounds (glucose, lactate, pyruvate) in coronary venous effluent were separated by anion-exchange chromatography [20, 21] on freshly prepared 2 ml columns (Dowex 1 resin, 1 x 8, formate form, 200–400 mesh) [19]. The elution profile was characterised by chromatographing 5 ml of Krebs-Henseleit buffer containing known amounts of unlabeled glucose, lactate, and pyruvate and measuring each of these compounds in the eluent by spectrophotometric assays [18]. Compounds were separated by a formic acid step gradient: glucose was completely eluted with 20 ml H2O, lactate with 20 ml 0.15N formic acid, and pyruvate with 20 ml 2.0N formic acid. After 5 ml fractions of venous effluent were loaded on the column, [14C]glucose, [14C]lactate and [14C]pyruvate were separated and measured by liquid scintillation counting. [U-14C]Glucose in the perfusion medium contained small amounts of 14C that co-eluted with the lactate and pyruvate fractions. These radioactive contaminants were measured in each experiment by chromatographing left atrial inflow samples collected at 10, 30, and 50 min perfusion. [14C]Lactate and [14C]pyruvate measured in venous effluent were corrected by subtracting the measured co-eluting contaminants. With these corrections, [14C]pyruvate in the coronary effluent consistently fell below the detection limit, and [14C]lactate and 14CO2 were the only metabolites of 14Cglucose detectable in the coronary effluent. [14C]lactate release (dpm · min–1 · g–1) equalled the product of [14C]lactate radioactivity in coronary effluent (dpm · ml–1) times coronary flow (ml · g–1). Total lactate in coronary effluent was measured by enzymatic assay [18]; specific activity of [14C]lactate was expressed as dpm per micromole. Glucose concentrations in left atrial inflow and venous effluent samples were also enzymatically determined[18]. Glucose uptake was taken as the product of the arteriovenous glucose concentration difference times coronary flow per gram tissue wet mass. The rate of metabolism of exogenous glucose to lactate equalled [14C]lactate release divided by the SA of [14C]glucose in the left atrial inflow.

2.5. Assessment of interstitial fluid purines
Concentrations of adenosine, inosine, and other adenylate degradatives in the cardiac interstitium were assessed from measured concentrations in epicardial transudate [22]. A length of absorbent tissue was carefully draped across the cardiac base and great vessel stumps to minimise contamination of epicardial transudate with non-interstitial leakage. Epicardial transudate (0.5–1.0 ml) was collected from the cardiac apex into 1.5 ml Eppendorf vials which contained 20 µl 10 M HCl to denature and precipitate adenosine deaminase and other enzymes. Samples were immediately mixed, neutralised with NaOH, and stored at –90°C prior to analysis. Purines were measured by reverse-phase HPLC (Shimadzu Instruments, Columbia, MD, USA) on a C-18 column. Compounds were eluted by a nonlinear bisolvent gradient as recently described [2] and quantified by absorbances measured at 254 nm wavelength.

2.6. Myocardial metabolites
Creatine kinase reactants (ATP, creatine phosphate (CrP), creatine (Cr)), glycolytic intermediates (glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate), pyruvate, lactate, citrate, glycogen and sucrose were extracted from frozen ventricular myocardium[2, 23] and measured by standard enzymatic assays[2, 18]. Intracellular pH (pHi) was estimated from coronary venous PCO2 as previously described [24]. Extracellular water (ml · g–1) was taken as the sucrose distribution space [25]. Isotonic, filtered (0.45 µM pore diameter) sucrose solution was infused into the left atrial inflow to a concentration of 2 mM for 5 min prior to freeze-stop. The volume of sucrose distribution was determined by dividing total myocardial sucrose content by the extracellular sucrose concentration, which was taken as the mean of the left atrial inflow and coronary effluent concentrations. Intracellular water equalled total myocardial water minus sucrose distribution space [2].

Intracellular ATP concentration ([ATP]ic) was computed by dividing ATP content (µmol · g wet mass–1) by intracellular space (ml · g wet mass–1). Cytosolic free ADP concentration ([ADP]f) was estimated from the mass action ratio of creatine kinase: thus, [ADP]f = ([ATP]ic · [Cr] · KCK)/([CrP] · [H+]), where KCK is the creatine kinase equilibrium constant. The cytosolic free AMP concentration ([AMP]f) was calculated from the myokinase equilibrium; thus, [AMP]f = KMK ([ADP]f2/[ATP]ic), where KMK is the equilibrium constant of myokinase.

The equilibria of the cytosolic creatine kinase and myokinase are Mg-dependent [26]. Accordingly, cytosolic free magnesium concentration (Mgf) was estimated by 31P nuclear magnetic resonance spectroscopy, where Mgf was estimated from the chemical shift difference between the {alpha}- and β-phosphate resonances of ATP in the 31P NMR spectrum [27]. 31P NMR spectra were acquired at 121.5 MHz in a Nicolet model NT300 7.05 tesla spectrometer, using a 20 mm broad-band probe. After stabilisation and shimming, hearts were subjected to experimental protocol I described above. Free Mg2+ was 0.30 ± 0.02 mM during preischaemia, did not change during mild hypoperfusion (0.31 ± 0.03 mM) and increased modestly to 0.44 ± 0.04 mM at 10 min ischaemia (P < 0.02 vs. preischaemia; n = 8). The values of KCK and KMK were adjusted for these estimated Mgf levels [28], based on published relationships between free Mg2+ and KCK [26] and KMK [26]. Thus, at 0.3 mM Mgf, KCK equalled 9.7 · 10–10 M and KMK equalled 0.82; at 0.44 mM Mgf, KCK equalled 8.5 · 10–10 M, and KMK equalled 0.92.

2.7. Enzymes
Aldolase (EC 4.1.2.13 [EC] ), creatine kinase (EC 2.7.3.2 [EC] ), glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.9 [EC] ), {alpha}-glycerophosphate dehydrogenase (EC 1.1.1.8 [EC] ), glycogen phosphorylase (EC 2.4.1.1 [EC] ), hexokinase (EC 2.7.1.1 [EC] ), lactate dehydrogenase (EC 1.1.1.27 [EC] ), myokinase (EC 2.7.4.3 [EC] ), phosphofructokinase (EC 2.7.1.11 [EC] ), phosphoglucoisomerase (EC 5.3.1.9 [EC] ), phosphoglycerate kinase (EC 2.7.2.3 [EC] ), (cyclic-AMP-dependent) protein kinase (EC 2.7.1.37 [EC] ), pyruvate kinase (EC 2.7.1.4 [EC] 0).

2.8. Statistics
Data are presented as mean ± s.e. Single comparisons of means were performed using two-tailed unpaired Student's t-test. Repeated measurements were compared by one-way analysis of variance (ANOVA) with Student-Neumann-Keuls test for multiple comparisons. These analyses were performed with SigmaStat statistical software (Jandel; San Rafael, CA, USA). P-values < 0.05 were taken to indicate statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Left ventricular function during adenosine receptor blockade in preischaemic, hypoperfused and ischaemic myocardium
Isolated working guinea-pig hearts were perfused at 90 cmH2O aortic pressure (Pa) for 30 min (preischaemia baseline), then subjected to graded coronary hypoperfusion by lowering Pa to 45 cmH2O for 15 min (mild hypoperfusion) then further to 22.5 cmH2O for 10 min (ischaemia). Adenosine receptors were blocked with 20 µM 8-p-sulfophenyl theophylline (8-SPT), beginning at 20 min preischaemia. In preischaemic hearts, adenosine receptor blockade slightly lowered coronary flow by 17%, from 6.0 ± 0.3 to 5.0 ± 0.2 ml · min–1 · g–1 (P < 0.05), but did not significantly alter left ventricular function, assessed from heart rate x pressure product (Fig. GR2 B) and left ventricular power (Fig. GR2 C). When Pa was lowered to 45 and then to 22.5 cmH2O, coronary flow and left ventricular function fell in parallel with aortic pressure. Coronary flow (Fig. GR2 A), heart rate x pressure product (Fig. GR2 B), and power (Fig. GR2 C), stabilised at 64, 40 and 55% of the respective baseline levels when aortic pressure was decreased from 90 to 45 cmH2O; these variables fell further to 33, 12 and 16% of baseline when aortic pressure was further decreased to 22.5 cmH2O. 8-SPT significantly lowered coronary flow by 19% in mild hypoperfusion (P < 0.05), but it did not appreciably lower coronary flow during ischaemia. Neither heart rate x pressure product nor power was altered by 8-SPT. Thus, adenosine receptor blockade did not affect left ventricular function in any phase of the protocol, but did moderately increase coronary resistance during preischaemia and mild hypoperfusion.


Figure 2
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  		Fig. GR2 Effects of adenosine receptor blockade on coronary flow and left ventricular function in preischaemic, mildly hypoperfused, and ischaemic myocardium. Means ± s.e. from 6–8 experiments. Left ventricular function was assessed from pressure development per min (heart rate x developed pressure, panel B) and stroke work (panel C). * P < 0.05 vs. 30 min; {ddagger}P < 0.05 vs. 45 min; {dagger}P < 0.05 vs. untreated.

 
3.2. Effects of adenosine receptor antagonism on glucose metabolism in ischaemic myocardium
Glucose uptake in untreated hearts did not change significantly with altered coronary perfusion (Fig. GR3 A; Table 1). 8-SPT increased glucose uptake by 100% prior to ischaemia (P < 0.05), and by 74% during mild hypoperfusion (P < 0.05), but did not significantly increase glucose uptake during ischaemia, relative to untreated control hearts. Glucose oxidation (Fig. GR3 B) fell in lockstep with aortic pressure. 8-SPT did not appreciably alter glucose oxidation at any point in the experiment. In the untreated group, [14C]lactate release did not change markedly until aortic pressure was lowered to 22.5 cmH2O (Fig. GR3 C). At 30 min preischaemia and during mild hypoperfusion, 8-SPT treatment increased [14C]lactate release 1.9- and 4-fold, respectively, relative to untreated hearts (P < 0.05), but did not increase [14C]lactate release significantly during ischaemia. These results indicate that adenosine receptor blockade tended to increase glycolysis, at least in part by enhancing uptake and metabolism of exogenous glucose. Total lactate release was significantly increased by adenosine receptor blockade at all three aortic pressures (P < 0.05; Table 1). Release of [14C]pyruvate in coronary effluent was not detectable in either 8-SPT treated or untreated hearts at any point in the protocol.


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  		Table 1 Effects of adenosine receptor blockade on glucose utilisation during preischaemia, hypoperfusion and ischaemia

 
Table 1 summarises the effects of 8-SPT on glucose metabolism during preischaemia, mild hypoperfusion, and ischaemia. Metabolism of exogenous glucose was assessed with [U-14C]glucose. In untreated preischaemic hearts, 46% of exogenous glucose taken up by the hearts was metabolised to CO2 and 10% to lactate. 8-SPT doubled the rate of glucose uptake and tended to increase lactate formation from exogenous glucose, but did not alter the rate of glucose oxidation; in these preischaemic hearts, 23% of glucose was oxidised to CO2 and 9% metabolised to lactate. In mild hypoperfusion of untreated hearts, oxidation of exogenous glucose fell to 34%, while metabolism of exogenous glucose to lactate remained low at 8%. The respective values in hypoperfused 8-SPT-treated hearts were 20 and 18%. Thus, in mild hypoperfusion, 8-SPT increased glucose uptake by 74% and quadrupled lactate formation from exogenous glucose, but did not alter the rate of glucose oxidation. In ischaemic myocardium, only 17% of glucose was oxidised, but, as expected, lactate formation from exogenous glucose increased fivefold, and accounted for 40% of the glucose taken up. Glucose oxidation fell further to 10% during adenosine receptor blockade; lactate formation accounted for 33% of exogenous glucose metabolised. In all cases, lactate formation from exogenous glucose accounted for most or all of total myocardial lactate release.

3.3. Effects of adenosine receptor antagonism on lactate formation during graded reductions in coronary perfusion
The enhancement of glycolysis by adenosine receptor blockade observed during the hypoperfusion/ischaemia protocol could have resulted from ischaemia due to the 8-SPT-induced decrease in coronary flow. To test this hypothesis, lactate release was measured in untreated and 8-SPT-treated hearts as coronary flow was modified by graded reductions in Pa (i.e., coronary perfusion pressure). Coronary flow, contractile function, and lactate release in the coronary venous effluent were measured at each Pa. 8-SPT moderately lowered coronary flow by 10–20% (P < 0.05) at 80–100 cmH2O Pa (Fig. GR4 A). At lower perfusion pressures, 8-SPT did not exert a significant effect on coronary flow. Despite the differences in coronary flow at higher Pa, cardiac function assessed from stroke work did not differ between the two groups at any Pa (Fig. GR4 B), indicating that adenosine receptor blockade did not induce ischaemia sufficient to impair contractile function at higher Pa, nor exacerbate ischaemia at low Pa. 8-SPT markedly increased lactate release at all but the lowest Pa, where lactate release was sharply elevated in the control condition (Fig. GR4 C).


Figure 4
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  		Fig. GR4 Effects of adenosine receptor blockade on coronary flow, stroke work, and lactate release during graded adjustments in aortic pressure. Means±s.e. from 8 experiments in each group. Hearts were subjected to the graded coronary perfusion protocol detailed in Section 2. Open symbols: treatment with 20 µM 8-p-sulfophenyl theophylline; closed symbols: untreated controls. Coronary flow (panel A), stroke work (panel B) and lactate release (panel C) were determined as described in Section 2. * P < 0.05 vs. untreated.

 
In Fig. GR5, lactate release in these hearts is plotted as a function of coronary flow. 8-SPT increased lactate release at coronary flows above 2 ml · min–1 · g–1. In the hypoperfusion/ischaemia protocol, coronary flows of 8-SPT-treated hearts varied between 4 and 5.5 ml · min–1 · g–1 during preischemia, between 2.25 and 4 ml · min–1 · g–1 in mild hypoperfusion, and between 1 and 2 ml · min–1 · g–1 in ischaemia. Lactate release data from hearts subjected to the graded coronary perfusion protocol (Fig. GR5) were grouped within these flow ranges and the effects of 8-SPT were examined. For each range, the mean coronary flow during 8-SPT treatment did not differ significantly from that of the untreated controls (P > 0.2). At the two higher flow ranges, 8-SPT increased lactate release 2.5- to 3-fold (P < 0.001). As expected, lactate release increased in both groups when coronary flow was lowered to the extent that the hearts became severely ischaemic; in this protocol, 8-SPT did not further increase lactate release at the lowest coronary flows. These results indicate that anaerobic glycolysis was enhanced by 8-SPT independent of coronary flow, and, therefore, did not result from ischaemia induced by adenosine receptor blockade.


Figure 5
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  		Fig. GR5 Myocardial lactate release as influenced by coronary flow and adenosine receptor blockade. Measurements were made in the presence (triangles) or absence (circles) of 20 µM 8-SPT. Open symbols represent individual measurements. The filled symbols represent means±s.e. of data points (n = 8–15) that fell within the three coronary flow ranges of 4–5.5, 2.25–4, or 1–2 ml·min–1·g heart mass–1; these are the respective ranges of coronary flows in 8-SPT-treated hearts during the preischaemia, mild hypoperfusion, and ischaemia phases of the hypoperfusion/ischaemia protocol of Fig. GR1. * P < 0.001 vs. untreated.

 
3.4. Effects of adenosine receptor blockade and ischaemia on glycolytic intermediates
Glycolytic intermediates and cytosolic redox metabolites were measured in preischaemic, mildly hypoperfused, and ischaemic myocardium in the absence and presence of adenosine receptor blockade with 8-SPT. Fig. GR6 presents these data as crossover plots, in which metabolite contents in the experimental condition are expressed as a proportion of the respective contents in the control condition, and plotted in the glycolytic sequence. In preischaemic myocardium, 8-SPT significantly increased levels of fructose 1,6-bisphosphate and phosphoenopyruvate, but lowered dihydroxyacetone phosphate content (Fig. GR6 A); thus, a significant crossover at the level of aldolase was evident. Intracellular lactate content nearly tripled in 8-SPT-treated preischaemic myocardium, while pyruvate content fell 50%. Thus, the intracellular lactate/pyruvate ratio, an index of cytosolic NADH/NAD+ ratio as prescribed by the lactate dehydrogenase equilibrium [29], increased nearly fourfold during 8-SPT perfusion (Table 2), indicating that pronounced cytosolic reduction had occurred as a consequence of adenosine receptor blockade in preischaemic myocardium.


Figure 6
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  		Fig. GR6 Crossover plots of glycolytic intermediates and cytosolic redox metabolites in preischaemic, hypoperfused, and ischaemic myocardium. Metabolites were measured in myocardium stop-frozen at 30 min preischaemia, 15 min mild hypoperfusion, and 10 min ischaemia according to the protocol described in Fig. GR1. G6P = glucose 6-phosphate; F6P = fructose 6-phosphate; FDP = fructose 1,6-bisphosphate; DAP = dihydroxyacetone phosphate; GAP = glyceraldehyde 3-phosphate; 3PG = 3-phosphoglycerate; 2PG = 2-phosphoglycerate; PEP = phosphoenolpyruvate; PYR = pyruvate; LAC = lactate. Means±s.e. from 6–8 experiments. * P < 0.05 vs. reference condition in each panel.

 

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  		Table 2 Effects of adenosine receptor blockade on glycolytic intermediate ratios

 
In moderately hypoperfused myocardium, adenosine receptor blockade, which increased lactate release sevenfold (Table 1), increased fructose 1,6-bisphosphate content nearly 60% (P < 0.05; Fig. GR6 B). Myocardial lactate content also rose significantly. Interestingly, adenosine receptor blockade increased pyruvate content 2.7-fold, while phosphoenolpyruvate did not increase and even tended to fall; these effects of 8-SPT contrasted with its effects on these glycolytic intermediates in preischaemic myocardium.

In the absence of adenosine receptor blockade, ischaemia significantly increased fructose 1,6-bisphosphate and phosphoenolpyruvate (Fig. GR6 C). As expected, intracellular lactate was sharply increased in ischaemic myocardium, and the elevated lactate/pyruvate ratio (Table 2) revealed appreciable cytosolic reduction (i.e., increased cytosolic NADH/NAD+). In ischaemic myocardium, 8-SPT produced further increases in fructose 1,6-bisphophate and lactate content (Fig. GR6 D), but did not increase the lactate/pyruvate ratio above that of untreated ischaemic hearts (Table 2). Thus, the cytosolic reductions induced by adenosine receptor blockade and ischaemia were not cumulative.

To further define the effects of ischaemia and adenosine receptor blockade on glycolytic enzymes, the mass action ratios of phosphoglucoisomerase (glucose 6-phosphate/fructose 6-phosphate), phosphofructokinase (fructose 1,6-bisphosphate/fructose 6-phosphate), and the glyceraldehyde 3-phosphate dehydrogenase/phosphoglycerate kinase couple (3-phosphoglycerate/ dihydroxyacetone phosphate) were determined in untreated and 8-SPT-treated hearts in preischaemia, mild hypoperfusion, and ischaemia (Table 2). In preischaemic hearts, 8-SPT increased the ratio of glucose 6-phosphate/fructose 6-phosphate (P < 0.05), indicating that glucose 6-phosphate formation was increased relative to the rate of its conversion to fructose 6-phosphate by phosphoglucoisomerase. Because fructose 6-phosphate content was not decreased by 8-SPT, the increase in this ratio could only have resulted from increased hexose monophosphate formation, due to increased glucose uptake and phosphorylation by hexokinase, and/or to increased glycogen degradation by glycogen phosphorylase. 8-SPT also increased the ratios of fructose 1,6-bisphosphate/fructose 6-phosphate and 3-phosphoglycerate/dihydroxyacetone phosphate (P < 0.05), indicating that phosphofructokinase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase couple were activated by adenosine receptor blockade in preischaemic myocardium.

Mild coronary hypoperfusion sharply lowered the fructose 1,6-bisphosphate/fructose 6-phosphate ratio in untreated and 8-SPT-treated myocardium. This result indicated that the rate of fructose 1,6-bisphosphate formation by phosphofructokinase was decreased relative to the rate of hexose monophosphate formation when coronary perfusion was moderately lowered. The other ratios were not altered by moderate reductions in coronary perfusion. 8-SPT did not appreciably affect any of the ratios during mild hypoperfusion.

The fructose 1,6-bisphosphate/fructose 6-phosphate ratio tended to increase in untreated ischaemic myocardium relative to preischaemia (P = 0.09), consistent with the expected activation of phosphofructokinase during moderately severe ischaemia. During ischaemia, 8-SPT appreciably increased fructose 1,6-bisphosphate/fructose 6-phosphate, relative to both preischaemic 8-SPT-treated myocardium and untreated ischaemic myocardium (P < 0.05), indicating that ischaemia and adenosine receptor blockade synergistically activated phosphofructokinase. Although fructose 1,6-bisphosphate content increased in 8-SPT-treated ischaemic hearts, glucose-6-phosphate and fructose-6-phosphate content did not simultaneously decline (Fig. GR6 D). This result indicates that production of hexose monophosphates from exogenous glucose and endogenous glycogen stores must have increased to keep pace with phosphofructokinase activation. As noted above, glucose uptake was increased during adenosine receptor blockade (Fig. GR3 A).

3.5. Myocardial glycogen
Glycogen comprises a significant endogenous source of glucose equivalents in mammalian myocardium. To delineate the effects of ischaemia and adenosine receptor blockade on cardiac glycogen, myocardial glycogen content was measured in preischaemic and ischaemic myocardium. Adenosine receptor blockade did not alter glycogen content in preischaemic myocardium, which equalled 267 ± 27 µmol hexose equivalents·g dry mass–1 in untreated hearts and 307 ± 16 µmol hexose·g dry mass–1 in 8-SPT-treated hearts (P = n.s.). Myocardial glycogen content fell markedly in both treatment groups following 15 min mild hypoperfusion and 10 min ischaemia, to 161 ± 17 µmol hexose·g dry mass–1 in untreated controls and 193 ± 11 µmol hexose·g dry mass–1 in 8-SPT-treated hearts. The decline in glycogen content in 8-SPT-treated myocardium during the protocol (114 µmol hexose·g dry mass–1) was similar to that of untreated hearts (106 µmol hexose·g dry mass–1). Thus, adenosine receptor blockade did not appreciably alter glycogen content, despite the increased lactate production. This finding is not surprising even if it is assumed that the entire lactate formation from endogenous sources was generated from glycogen. Using the data from Table 1, 8-SPT increased lactate formation from endogenous precursors by 0.13 µmol·min–1·g wet wt–1 in preischaemia, by 0.17 µmol·min–1·g wet wt–1 in mild hypoperfusion, and by 0.25 µmol·min–1·g wet wt–1 in ischaemia. When each of these values is multiplied by the duration of 8-SPT perfusion in each phase and by the measured mean wet mass/dry mass ratio of 8, total lactate production from endogenous sources is computed to be increased by 50 µmol·g dry mass–1 by 8-SPT, which corresponds to 25 µmol·g dry mass–1 of hexose equivalents. This small difference of less than 10% of the total myocardial glycogen pool would be at or below the detection limit of the procedures used in this study.

3.6. Allosteric regulators of phosphofructokinase
Phosphofructokinase activity is modulated by cytosolic free adenylates and citrate. Table 3 summarises the effects of ischaemia and 8-SPT on myocardial levels of these regulatory metabolites of phosphofructokinase. Intracellular concentration of the phosphofructokinase inhibitor ATP tended to decrease, but not significantly, following mild hypoperfusion and ischaemia, and was unaltered by adenosine receptor blockade. Free cytosolic concentrations of ADP and AMP, which activate phosphofructokinase, did not increase during mild hypoperfusion, but rose appreciably during ischaemia (P < 0.05), due to cytosolic ATP degradation. Intracellular concentrations of these metabolites were not altered by 8-SPT treatment at any level of coronary perfusion. Cellular level of the phosphofructokinase inhibitor citrate tended to increase, albeit insignificantly, in ischaemic myocardium. Citrate content was elevated in 8-SPT-treated ischaemic myocardium relative to untreated normoxic controls (P < 0.05), but did not differ significantly from untreated ischaemic hearts.


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  		Table 3 Effects of adenosine receptor blockade on myocardial cytosolic free adenylates and citrate

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The objective of this study was to delineate the effects of endogenous adenosine on hexose metabolism in preischaemic, moderately hypoperfused, and more severely hypoperfused working guinea-pig myocardium. The principal new findings of the study are: (1) Adenosine receptor blockade by 8-p-sulfophenyl theophylline (8-SPT) markedly increased lactate production from exogenous glucose, especially during preischaemia and moderate hypoperfusion, but did not stimulate glucose oxidation in any condition. (2) Adenosine receptor blockade increased lactate formation from exogenous glucose and from endogenous precursors, but did not appreciably lower myocardial glycogen content. (3) Crossover plots and ratios of glycolytic intermediates revealed that phosphofructokinase, a key rate-controlling step in glycolysis, was activated by adenosine receptor blockade at every level of coronary flow.

4.1. Effects of adenosine on glucose metabolism
We recently investigated the role of endogenous adenosine on cytosolic energetics in isolated, working guinea-pig hearts subjected to moderately severe ischaemia [2]. In these experiments, 8-SPT increased lactate release and lessened the initial decline in cytosolic energetics during the first 10 min ischaemia, but blunted the subsequent recovery of cytosolic energetics. Maximum enhancement of lactate release by adenosine receptor blockade occurred at 5–10 min ischaemia, but subsided as ischaemia was prolonged. The carbon sources for 8-SPT-induced lactate formation were not defined in our recent investigation. Accordingly, the present study was designed to examine the effects of adenosine receptor blockade on glucose metabolism during 10 min moderately severe ischaemia. This study demonstrated that adenosine receptor blockade with 8-SPT promoted lactate release at graded rates of coronary perfusion, and that this lactate was generated in large part by metabolism of exogenous glucose.

The effects of exogenous adenosine on myocardial glucose metabolism have been the focus of intense research interest over the past several years. Several recent studies have demonstrated that exogenous adenosine inhibits glycolysis during myocardial ischaemia [5–9]. Finegan et al.[5] reported that pretreatment of crystalloid-perfused rat hearts with 100 µM adenosine inhibited glycolysis during ischaemia. Glucose oxidation was depressed during myocardial ischaemia, but was not altered by adenosine under these conditions. Dale et al. [6] also reported that the adenosine A1 receptor agonist N6-(L-2-phenylisopropyl)-adenosine (PIA) inhibited glycolysis by 50% in nonischaemic rat hearts metabolising glucose as sole exogenous substrate. Buxton et al. reported that 25 µM adenosine inhibited glycolytic flux by 15% in rat hearts perfused at constant flow [7]. Fralix et al. [8] found that 20 µM adenosine attenuated glycolysis during ischaemia and decreased lactate accumulation in nonworking rat hearts. Furthermore, Vander Heide et al. [9] found that intracoronary adenosine infusions (approximately 10 µmol·g wet wt–1·min–1) significantly slowed rates of glycogen utilisation and lactate accumulation during the first 20 min of coronary occlusion in in-situ canine myocardium.

Our results extend this knowledge to the effects of endogenous adenosine. In the present study, 8-SPT, applied at a concentration sufficient to effectively block adenosine A1 but not A2 receptors [2], augmented glucose uptake and stimulated degradation of this exogenous glucose to lactate. On the other hand, oxidation of exogenous glucose was not altered by 8-SPT. Taken together, the above described results of previous studies and our current findings indicate that adenosine, whether originating endogenously or applied via the coronary vasculature, inhibits glycolysis in normoxic and ischaemic myocardium.

In apparent disagreement with the findings summarised above, several studies reported that exogenous adenosine increased, not decreased, glycolytic flux in normoxic, ischaemic, and hypoxic myocardium [10–12]. Wyatt et al.[10] found that 100 µM adenosine increased glycolysis in normoxic and hypoxic nonworking rat hearts. Similarly, 10 µM adenosine increased lactate production in isolated perfused guinea-pig hearts [11]. Furthermore, Janier et al.[12] reported that 1 µM adenosine stimulated glycolysis by about 50% and delayed the onset of contracture in ischaemic rabbit hearts, effects which were blocked by the nonselective adenosine receptor antagonist, 8-phenyltheophylline. Buxton and co-workers [7] found that 25 µM adenosine increased glycolytic flux by 35% in rat hearts perfused at constant pressure. The causes of the discrepancies between these studies and those reporting adenosine inhibition of glycolysis are not clear, although differences in species, nutritional state, or method of estimating glycolysis may have been partially responsible. The discordant findings may also be due in part to the presence of insulin in the perfusion media of our studies and those of Fralix et al. [8] and Finegan et al. [5], whereas Wyatt et al.[10] did not perfuse hearts with insulin.

As described in our earlier report, the moderately severe ischaemic stress examined in the present study caused venous effluent total purine nucleoside (adenosine plus inosine) concentration to peak within 10 min at only about 3 µM [2]. This concentration is well below the arterial concentrations produced by exogenous adenosine infusion in most of the studies reporting a positive effect of adenosine on glycolysis. These higher adenosine concentrations may have exerted nonspecific effects that differed from those of endogenous adenosine. In the present study, glucose metabolism and glycolytic intermediates as well were measured in the same hearts; thus, the results from this investigation provide more direct evidence in support of the view that adenosine, including endogenous adenosine, inhibits glycolysis in normoxic and ischaemic myocardium.

Glycolysis is accelerated in myocardium during moderate ischaemia [16] or during increased workload [30]. As shown in Fig. GR6 B, all measured glycolytic intermediates either increased or tended to increase in ischaemic hearts not treated with 8-SPT relative to the corresponding preischaemic levels. As reported by Rovetto et al. [31] and Williamson et al. [32], phosphofructokinase activation in anoxic and ischaemic myocardium resulted in increased tissue levels of fructose 1,6-bisphosphate and dihydroxy-acetone phosphate. In the present study, ischaemia also increased the levels of these glycolytic intermediates. On the other hand, adenosine receptor blockade, which stimulated glycolysis although to a lesser degree than ischaemia (Table 1), did not uniformly increase all glycolytic intermediates.

Adenosine receptor blockade with 8-SPT increased myocardial lactate content and lactate release in preischaemic and mildly hypoperfused as well as ischaemic myocardium, indicating that endogenous adenosine inhibits glycolysis independent of ischaemia. In preischaemic myocardium, 8-SPT increased fructose 1,6-bisphosphate and decreased the products of aldolase, dihydroxyacetone phosphate + glyceraldehyde 3-phosphate (Fig. GR6 A). This result suggested that aldolase was inhibited concomitant with activation of phosphofructokinase. However, if adenosine receptor blockade had inhibited aldolase, glycolytic flux would have declined and the observed sustained increase in lactate production could not have occurred. Instead, the decrease in dihydroxyacetone phosphate may have occurred independent of aldolase activity according to the following scenario. Adenosine receptor blockade increased the intramyocytic lactate/pyruvate ratio (Fig. GR6), an index of cytosolic redox state (i.e., [NADH]/[NAD+]). As prescribed by the {alpha}-glycerophosphate dehydrogenase equilibrium, cytosolic reduction would shift this equilibrium from dihydroxyacetone phosphate toward {alpha}-glycerophosphate. In favor of this interpretation, although 8-SPT decreased dihydroxyacetone phosphate content in preischaemic myocardium, the sum of dihydroxyacetone phosphate and {alpha}-glycerophosphate was unchanged (0.9 ± 0.2 µmol·g dry mass–1 in untreated hearts vs. 0.7 ± 0.1 µmol·g dry mass–1 in 8-SPT-treated hearts; P > 0.1) while the {alpha}-glycerophosphate/dihydroxyacetone phosphate ratio was increased in 8-SPT-treated (2.6 ± 0.7) vs. untreated (1.2 ± 0.2) hearts (P < 0.05). The increase in fructose 1,6-bisphosphate content during 8-SPT treatment does indicate that phosphofructokinase was activated to a greater extent than aldolase. Moreover, increased ratios of fructose 1,6-bisphosphate/fructose 6-phosphate and 3-phosphoglycerate/dihydroxyacetone phosphate indicate that phosphofructokinase and the glyceraldehyde 3-phosphate dehydrogenase/phosphoglycerate kinase couple were activated by adenosine receptor blockade during preischaemia. As expected, phosphofructokinase was also activated by ischaemia; adenosine receptor blockade activated this key rate-controlling enzyme even further in the ischaemic setting (Table 2).

4.2. Regulation of phosphofructokinase by adenosine
The allosteric regulation of phosphofructokinase activity is extremely complex, and is characterised by the following features [33]: (1) inhibition by high concentrations of ATP; (2) synergistic inhibition by citrate at inhibitory concentrations of ATP [34]; (3) relief of ATP-mediated inhibition by inorganic phosphate, AMP, cyclic AMP, ADP, fructose 6-phosphate, and fructose 1,6-bisphosphate. Negative effectors decrease the enzyme's affinity for fructose 6-phosphate, and the positive effectors increase its affinity. The present ischaemic stress induced significant impairment of cytosolic energetics: free cytosolic ADP and AMP concentrations were elevated relative to preischaemic and mildly hypoperfused hearts. Adenosine receptor blockade with 8-SPT did not significantly affect free adenylate concentrations under any condition. On the other hand, 8-SPT increased citrate content about 70% in mildly hypoperfused and ischaemic myocardium. Such an increase in citrate would be expected to inhibit, not activate, phosphofructokinase [34]; however, it was not possible in this study to determine whether citrate increased in the mitochondrial compartment, the cytosol, or both. At present, the mechanism for the increase in citrate content induced by adenosine receptor blockade is unknown. 8-SPT did not increase cytosolic free ADP and AMP concentrations in ischaemic myocardium. Thus, adenosine receptor blockade with 8-SPT did not activate phosphofructokinase by altering cytosolic concentrations of the activator metabolites ADP and AMP.

The recent study of Mahrenholz et al. [35] demonstrated that phosphofructokinase from sheep heart was phosphorylated by Ca2+/calmodulin-dependent protein kinase (CaM-kinase) and by cyclic-AMP-dependent protein kinase (PKA). Phosphorylation by either CaM-kinase or PKA resulted in an increase in sensitivity of phosphofructokinase to ATP inhibition and a small but consistent decrease in Ki for ATP. Activation of adenosine A1 receptors in myocardium is known to inhibit cyclic AMP production [36] and, thus, would be expected to decrease phosphorylation of phosphofructokinase by PKA [37]. Via this mechanism, adenosine would activate phosphofructokinase, an effect inconsistent with the present findings. Hazen et al. [38] reported the rapid and reversible association of phosphofructokinase with cellular membranes during myocardial ischaemia. They demonstrated that the majority of phosphofructokinase activity is translocated from the cytosol to a membrane-associated compartment prior to the onset of irreversible myocytic injury and that membrane-associated phosphofructokinase is catalytically inactive. It is conceivable that adenosine could modulate glycolytic flux in myocardium by promoting phosphofructokinase translocation to membranes. Further investigation is required to test this hypothesis.

4.3. Possible effects of adenosine A2 receptor blockade
We previously demonstrated that 20 µM 8-SPT essentially prevents bradycardia, mediated by adenosine A1 receptors, due to a 150 µg adenosine bolus injection, but does not attenuate coronary vasodilation, mediated by A2 receptors on the vascular smooth muscle. In the present study, however, 20 µM 8-SPT produced a modest (ca. 17%) vasoconstriction in preischaemic hearts. It could therefore be proposed that lactate formation in 8-SPT-treated hearts was due to ischaemia secondary to A2 receptor antagonism. The present results do not support this interpretation. Energetic impairment due to ischaemia results in increased cytosolic free ADP and AMP concentrations[39] and concomitant increases in interstitial fluid purine nucleoside concentrations assessed in epicardial transudate [40]. Thus, if increased coronary resistance due to 8-SPT treatment were producing ischaemia sufficient to increase myocardial lactate release, cytosolic free ADP and AMP as well as epicardial transudate adenosine and inosine would have been elevated. As expected, cytosolic free ADP and AMP concentrations increased significantly in both treatment groups when coronary perfusion pressure was lowered to 22.5 cmH2O (Table 3), but were not increased by 8-SPT at any perfusion pressure. Total purine nucleoside (adenosine + inosine) concentrations (nM) in the epicardial transudate were also unaltered by 8-SPT. In preischaemic hearts, adenosine + inosine concentration was 420 ± 120 in untreated hearts and 570 ± 240 in 8-SPT-treated hearts (P > 0.05). Respective values were 330 ± 60 and 290 ± 120 in mild hypoperfusion (P > 0.05) and 820 ± 290 and 900 ± 410 in ischaemia (P > 0.05). These results indicate that adenosine receptor blockade did not impair cytosolic energetics or elevate interstitial purines. Moreover, myocardial O2 consumption (µmol·min–1·g–1) was not limited by 8-SPT in preischaemia (controls: 2.41 ± 0.13; 8-SPT: 2.50 ± 0.18) and mild hypoperfusion (controls: 1.46 ± 0.12; 8-SPT: 1.60 ± 0.14). Results from hearts subjected to graded reductions in coronary perfusion are also inconsistent with 8-SPT induced ischaemia. In these hearts, although 8-SPT moderately lowered coronary flow at the higher perfusion pressures (Fig. GR4 A), the blocker did not impair cardiac function at any perfusion pressure (Fig. GR4 B). The finding that 8-SPT markedly increased myocardial lactate formation at flows comparable to those of the untreated hearts (Fig. GR5), and the other results described above, strongly argue against the hypothesis that vasoconstriction-induced ischaemia was responsible for enhancement of glycolysis in 8-SPT-treated myocardium.

In hearts subjected to the hypoperfusion/ischaemia protocol of Fig. GR1, myocardial lactate formation during ischaemia was increased 50% by adenosine receptor blockade; on the other hand, adenosine receptor blockade did not increase lactate formation during ischaemia imposed by a more gradual lowering of Pa (Fig. GR5). This apparent discrepancy may have resulted from differences in the rate at which ischaemia was imposed in the two protocols. Recently, Ito [41] demonstrated in porcine myocardium that lactate formation was decreased almost 50% when severe ischaemia was produced by gradual lowering of coronary flow versus a sudden reduction in flow. Also, the protocol in which we gradually lowered Pa was much longer (105 min) than the hypoperfusion/ischaemia protocol (55 min), and the pool of glycolytic substrate responsive to adenosine receptor blockade may have been depleted during the longer protocol.

4.4. Implications of glycolytic inhibition by endogenous adenosine
During myocardial ischaemia, oxidative metabolism is inhibited [16]. Thus glycolysis is essential to produce ATP to prevent ischaemic injury and facilitate functional recovery upon reperfusion. However, high glycolytic rates associated with low coronary flow could also cause accumulation of lactate and protons, and thus prove detrimental. Since intracellular pH decreases during ischaemia, rates of H+/Na+ exchange and Na+/Ca2+ exchange could increase, resulting in intracellular Ca2+ accumulation [42, 43]. Ca2+ accumulation, in turn, can cause mitochondrial damage and cell swelling which can progress to cardiocyte death [44]. Preischaemic glycogen degradation or glycolytic inhibition was found to improve post-ischaemic recovery of hypertrophied rat hearts [45]. Thus adenosine, by inhibiting glycolysis and attenuating intracellular acidification during ischaemia, could exert a cardioprotective effect. We previously found that adenosine receptor blockade with 8-SPT intensified intracellular acidosis during myocardial ischaemia [2]. These previous results and the present findings suggest that endogenous adenosine could lessen myocardial acidosis and Ca2+ accumulation during ischaemia.

4.5. Conclusions
In summary, this study demonstrates that adenosine receptor blockade by 8-SPT increases lactate production from exogenous glucose by activating phosphofructokinase, and by increasing glucose uptake. Further investigation is required to delineate the specific mechanisms by which 8-SPT activates phosphofructokinase.


   Acknowledgements
 
This study was supported by grants to R.T.M. (R29 HL50441) and H.F.D. (R01 HL35056) from the National Heart, Lung and Blood Institute, and by a grant to H.F.D. from the Texas Advanced Research Program (009768-008). The skillful assistance of José Murillo is gratefully acknowledged.


   Notes
 
1 Present address: Department of Radiology/NMR Research, Johns Hopkins University School of Medicine, 217 Traylor Building, 720 Rutland Avenue, Baltimore, MD 21205, USA. Back

* Corresponding author. Tel. + 1 817 735-2260; Fax + 1 817 735-5084 Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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