Regulation of cardiac AMP-specific 5'-nucleotidase during ischemia mediates ATP resynthesis on reflow

Marianna I. Bak and Joanne S. Ingwall

Nuclear Magnetic Resonance Laboratory for Physiological Chemistry, Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The ability to resynthesize ATP during recovery from ischemia is limited to the size of endogenous pool of adenine nucleotides. Cytosolic AMP-specific 5'-nucleotidase (5'-NT) plays a key role in ATP degradation and hence the capacity for ATP resynthesis. We have suggested (J. Clin. Invest. 93: 40-49, 1994) that intracellular acidosis [intracellular pH (pHi)] is a potent inhibitor of 5'-NT under in vivo conditions. To test this hypothesis further, we used the hyperthyroid rat heart because we could alter pHi during ischemia and determine the consequences of lower pHi on AMP accumulation (by chemical assay) and ATP resynthesis (by 31P nuclear magnetic resonance spectroscopy) during reperfusion. Global no-flow ischemia caused pHi to decrease from 7.1 under well-oxygenated control perfusion to 6.7. We found that decreasing pHi further from pH 6.7 to 6.4 leads to increased accumulation (30%) of AMP during ischemia and to a 2.5-fold increase in ATP resynthesis during reperfusion. Analysis of all known substrates, products, activators, and inhibitors of the 5'-NT suggests that 5'-NT is activated primarily by Mg2+ and ADP and is inhibited by H+. Thus these observations provide evidence for a salutary effect of intracellular acidosis on preserving the AMP pool due to inhibition of 5'-NT and suggest a novel role of H+ in protecting ischemic tissue.

ATP resynthesis; acidosis; reperfusion; rat myocardium

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE ABSOLUTE REQUIREMENT OF mechanical performance, synthesis of biological molecules, and transport of ions in cardiac myocytes for ATP is well established. In oxygenated tissues, the rate of ATP utilization equals the rate of ATP production. This balance is disrupted during acute ischemia or hypoxia, and, as a consequence, the ATP concentration decreases. The primary pathway for ATP degradation in heart is ATP right-arrow ADP right-arrow AMP right-arrow adenosine right-arrow inosine right-arrow hypoxanthine right-arrow xanthine (Fig. 1). The phosphorylated purines remain in the myocytes, but the nonphosphorylated purines diffuse down their concentration gradients to the extracellular space. Because de novo synthesis of the purine ring in mammalian myocardium is very slow (1, 34), resynthesis of ATP during the first few minutes of reperfusion occurs only by rephosphorylation of the remaining pool of cytosolic adenine nucleotides, mainly AMP. Therefore, the preservation of the adenine nucleotide pool, especially the AMP pool, during ischemic episodes is essential for maintaining cell function and viability. The size of the AMP pool in ischemic heart cells is set primarily by the activity of the cytosolic AMP-specific 5'-nucleotidase (5'-NT, EC 3.1.3.5). Thus, by dephosphorylation of AMP to adenosine, this enzyme plays a major role in ATP degradation in mammalian myocytes (Fig. 1).

To date, four types of 5'-NTs have been identified in the heart. Two of these are cytosolic; in a recent review, Zimmerman (35) named them c-N-I and c-N-II. c-N-I uses primarily AMP, and c-N-II uses primarily IMP (Fig. 1). Since the initial discovery of 5'-NT activity in 1934 (26), ecto-nucleotidases have been the subject of extensive studies, but these ecto-enzymes are not responsible for the degradation of cytosolic AMP during hypoxia or ischemia (12, 20, 22, 35). It has been recognized only recently that AMP-specific cytosolic 5'-NT plays the major role in the production of adenosine from cytosolic AMP in mammalian heart (6, 27, 29, 31, 33), including human myocardium (5, 28). With that recognition, several in vitro studies have established the substrate, activators, and inhibitors of cytosolic 5'-NT (5, 6, 12, 22, 27, 28, 31). Recent in vitro work, aimed at elucidating the regulation of 5'-NT, has shown that the activity of this enzyme is decreased at acidic pH (6, 27). If this mechanism also operates in the intact heart, then regulation of 5'-NT by intracellular pH (pHi) would be a major determinant of cardiac function and cell survival. Inhibition of 5'-NT activity would limit the conversion of AMP to the purines (Fig. 1). In this way, the cell would retain the capacity to use AMP to resynthesize ATP, if and when reperfused. Therefore, defining the regulators of 5'-NT activity in cardiac tissue in vivo is central to understanding the pathophysiology of some of the important biochemical consequences of ischemia (production of adenosine and accumulation of AMP) and the recovery of reperfused tissue (ATP resynthesis).

In a prior report that used the isolated perfused rat heart (3), we presented evidence for activation of 5'-NT early during energy-deprived states, followed by inhibition of 5'-NT during prolonged ischemia. Because AMP accumulated during ischemia (where pHi falls to 6.1) but not during hypoxia (where pHi remains near 7.0), we suggested that the underlying mechanism for this observation was inhibition of cytosolic 5'-NT by accumulation of H+. In this way, intracellular acidosis, a feature of ischemia common to all tissues, leads to the preservation of cytosolic AMP during ischemia and enhanced capacity for resynthesis of ATP from AMP on reperfusion. This mechanism also explains the ability of reperfused myocardium to resynthesize ATP to ~40% of its preischemic value and the lack of ATP resynthesis on reoxygenation after hypoxia.


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Fig. 1.   Principal pathways of purine metabolism in the mammalian heart. De novo refers to the synthetic pathway for IMP synthesis from nonpurine precursors. Thick lines represent dominant pathways; thin and dashed lines represent quantitatively minor pathways. All phosphorylated compounds remain in the myocyte, whereas inosine (INO), hypoxanthine (HYPO), and adenosine (ADO) diffuse from the myocyte to the extracellular space and vessels. The multi-step reactions using HYPO, ribose-1-phosphate (ribose-1-P) and 5-phosphoribosyl-1-pyrophosphate (PRPP) are indicated by dashed lines. The broken arrows show the inhibitory effects of H+ and Pi. See text for further description. 5'-NT (c-I), cytosolic AMP-specific 5'-nucleotidase; 5'-NT (c-II), cytosolic IMP-specific 5'-nucleotidase.

The aim of the present study was to provide a further test of the hypothesis that inhibition of 5'-NT activity by H+ during ischemia determines the size of the AMP pool and hence the capacity for ATP resynthesis in the intact heart. To accomplish this, we have varied and defined the quantitative changes in pHi between the values of 7.0 and 6.0 during global no-flow ischemia; measured the concentrations of substrate, products, activators, and inhibitors of 5'-NT; and determined the amount of ATP resynthesized on reperfusion.

We chose to study this process in hearts from hyperthyroid rats [T3 hearts, that is, induced by 3,5,3'-triiodo-L-thyronine (T3)] for two reasons. First, T3 hearts exposed to no-flow ischemia for the same time duration accumulated less intracellular H+ compared with hearts from euthyroid rats. Second, the pHi in ischemia in T3 hearts can be changed by both pharmacological and chemical maneuvers. In hearts supplied with amiloride, an inhibitor of Na+/H+ exchange, before ischemia, the pHi became more acidic. Similarly, by replacing HCO-3 in the perfusate with HEPES, we were able to lower the pHi in these T3 ischemic hearts. In this way, we were able to determine the in vivo regulation of 5'-NT in the heart.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals

Forty-five male Sprague-Dawley rats (mean weight 386 ± 5 g; obtained from Charles River Laboratory, Wilmington, MA) were housed in a light- and temperature-controlled room. Food and water were given ad libitum. Hyperthyroidism was induced by intraperitoneal injection of T3 (200 µg/kg body wt, dissolved in 0.01 N NaOH, once daily for 8 days). Rats were weighed daily and again immediately before death. The experimental protocol for the present study was approved by the Standing Committee on Animals of Harvard Medical Area and followed the recommendations of current National Institutes of Health and American Physiological Society guidelines in Guide for the Care and Use of Laboratory Animals.

Rat Heart Preparation

Anesthesia was achieved by intraperitoneal injection of pentobarbital sodium (20 mg/kg). Beating hearts were rapidly removed and placed into cold Krebs-Henseleit buffer. The heart was attached to a perfusion apparatus via the aorta and perfused in the isovolumic, Langendorff mode at a constant temperature of 37°C and constant perfusion pressure of 100 mmHg as was described previously (2, 3). The standard perfusate was phosphate-free Krebs-Henseleit buffer of the following composition (in mM): 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.75 CaCl2, 0.5 EDTA, 11 glucose, and 25 NaHCO3. This buffer was gassed with a 95% O2-5% CO2 gas mixture, giving a pH of 7.4. In one set of experiments, HCO-3 was replaced by 10 mM HEPES. In this case, perfusate was saturated with 100% O2 to give a final pH of 7.4.

A small polyethylene tube was placed through the apex to drain flow from the thebesian veins. A water-filled balloon was inserted into the left ventricle through the mitral valve and connected to a pressure transducer (Statham P23Db, Gould Instruments, Oxnard, CA) for continuous measurement of heart function. Initial end-diastolic pressure was set at 10-12 mmHg. Heart performance was estimated as the rate pressure product (RPP), which is the product of heart rate and left ventricular developed pressure (LVDP). Coronary flow was determined by periodically collecting and measuring the coronary sinus effluent.

Characteristics of Hyperthyroid Rats and Hearts

Daily T3 injections did not cause a significant decrease in body weight of the animals (386 ± 5 vs. 375 ± 8 g after 8 days of T3 injections, n = 45). There was no significant difference in cardiac function of T3 rats measured by the product of heart rate and LVDP during well-oxygenated perfusion (RPP; 30,400 ± 2,000 mmHg) compared with hearts from euthyroid rats studied previously (28,400 ± 2,200 mmHg) (3). Coronary flow in T3 hearts was also not different from that in euthyroid hearts after the degree of hypertrophy was taken into account (~15.6 ml · min-1 · g wet weight-1).

Experimental Protocols

The hearts were immersed in their effluent inside a 20-mm (ID) glass nuclear magnetic resonance (NMR) tube (Wilmad Glass, Buena, NJ). After being placed in the magnet, each heart underwent a stabilization period of ~20 min during which the magnet was shimmed. Baseline data defining cardiac performance, pHi, and high-energy phosphate content were then collected during the next 12 min. Hearts were then divided into groups for use in one of three protocols.

In the first protocol, untreated hyperthyroid hearts (untreated T3; n = 13) were subjected to 28 min of global, no-flow normothermic (37°C) ischemia. Ischemia was induced by occlusion of the cannula connected to the aorta. For eight of these hearts, the ischemic period was followed by a reperfusion period of 30 min.

In the second protocol, 13 hearts were perfused with 1 mM amiloride (which was dissolved in Krebs-Henseleit perfusate) for 4 min before ischemia (T3+AMI). For five of these T3+AMI hearts, the experiment was terminated at the end of ischemia; in the remaining eight hearts, the ischemic period was followed by 30 min of reperfusion.

In the third protocol, 10 hearts were exposed to a period of 16 min of perfusion, with perfusate in which HCO-3 was replaced by HEPES before ischemia (T3+HEP). For four of these T3+HEP hearts, the protocol was terminated at the end of ischemia; in the remaining group of hearts, the ischemic period was followed by 30 min of reperfusion.

At the end of the experiment, each heart was freeze-clamped with aluminum tongs, precooled in liquid nitrogen, and stored at -80°C until purine and protein contents were assayed. In addition, for the purpose of measuring the content and distribution of purines and creatine in well-oxygenated tissue, we analyzed nine additional hearts from T3 rats that were freeze-clamped after 16 min of standard oxygenated perfusion.

NMR Measurements

31P-NMR spectra were collected at a 31P frequency of 145.75 MHz using a 9.4-T superconducting magnet with a 5.7-cm bore and a GE-400 Omega spectrometer (Fremont, CA). The perfused heart was inserted into a 1H/31P double-tuned probe. During a 20-min equilibration period before data acquisition, the magnetic field homogeneity was optimized by shimming on the 1H signal of the heart and the surrounding perfusate contained within the sensitive volume of the NMR probe (line width was 16-22 Hz). For 31P-NMR spectroscopy, the pulse angle used was 45° (23 µs pulse time) using a sweep width of ±3,000 Hz and 2,000 data points. At the beginning of each study, a fully relaxed spectrum was acquired using a 90° pulse and a 10-s repetition time. Subsequently, partially saturated 31P-NMR spectra (2.18-s interpulse delay) were obtained, averaging data from 104 free induction decays (total time of 4 min) throughout each protocol. A comparison of fully relaxed and partially saturated spectra showed that the nucleotide 5'-triphosphate (NTP) resonances were fully relaxed (±5%); for phosphocreatine (PCr) and Pi resonance areas, correction factors for saturation were required (1.2 and 1.15, respectively). Spectra were analyzed using exponential multiplication of 20 Hz and phasing with zero and first-order phase corrections. Peak areas were estimated by fitting with lorentzian line shapes using the NMR1 software curve-fitting program (NMR1, Syracuse, NY). As we previously stated, the values for ATP content in perfused hearts under diverse physiological conditions measured by 31P-NMR spectroscopy and by chemical assay are indistinguishable (2, 3).

Cytosolic concentrations of ATP measured by NMR spectroscopy were calculated from the areas of the [beta -P]NTP resonances by using results from an external ATP standard and knowing the NMR-sensitive volume and measured values of heart weight and protein content (2). The area of the [beta -P]NTP spectrum obtained under control perfusion was set to this experimentally determined value (see below) and used as the reference value for all resonances in all spectra. The ATP concentration determined by chemical assay in T3 hearts (n = 9) perfused for 16 min at a constant pressure and temperature was 31.6 ± 2.4 nmol/mg protein. With the use of values of 0.162 mg protein/mg wet weight determined experimentally for T3 hearts and a value of 0.48 µl intracellular water/mg wet weight (23), a corresponding cytosolic ATP concentration of 10.7 mM was calculated. This value was taken as the internal standard to calculate intracellular concentrations of PCr, Pi, and ATP.

Cystosolic ADP concentrations were calculated using a creatine kinase equilibrium expression (17), with values determined from 31P-NMR and biochemical assays
[ADP] = <FR><NU>[ATP] × [creatine]</NU><DE>[PCr] × [H<SUP>+</SUP>] × <IT>K</IT><SUB>CK</SUB></DE></FR>
where the creatine kinase equilibrium constant (KCK) is equal to 1.66 × 109 M-1 and brackets indicate concentration. pHi was measured by comparing the chemical shift between Pi and PCr with values obtained from a standard curve. No adjustments were made for any changes in Mg2+ concentration.

Chemical Assay of Metabolites

Frozen ventricular tissue from each heart was pulverized and extracted with 0.6 N perchloric acid. A portion of this homogenate was taken for protein assay, using the method of Lowry et al. (18). After neutralization and centrifugation, one aliquot of the supernatant (200 µl) was analyzed by high-pressure liquid chromatography (HPLC) using a Partisil 10 SAX column (Whatman, Clifton, NJ) for measurement of nucleotides, as described previously (3). A second aliquot (150 µl), used for measurements of AMP, adenosine, inosine, hypoxanthine, and xanthine, was injected into a reverse-phase C18 µBondapak column (Whatman). In other experiments, we used a third aliquot of supernatant (70 µl) for measurement of total creatine using the method of Kammermeier (14). Results are expressed as nanomoles per milligram protein.

All chemicals were obtained from Sigma Chemical (St. Louis, MO).

Statistical Analysis

All data are presented as mean values ± SD. Statistical significance of the results was determined by using analysis of variance followed by a Student's unpaired t-test to determine differences between groups by using the StatView TM 512+ (Brainpower) computer program. A value of P < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of Thyroid Hormone on Cardiac Metabolite Contents During Oxygenated Perfusion

The adenine nucleotide content and the distribution of nucleosides and nucleobases measured in T3 hearts, which were freeze-clamped after 16 min of oxygenated perfusion (Table 1), were not different from results described previously (3) for euthyroid rat hearts analyzed at the end of the stabilization period. These results show that the concentrations of ATP and its metabolites are indistinguishable in T3 and control hearts during conditions of well-oxygenated perfusion.

                              
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Table 1.   Content of adenine nucleotides, nucleosides, and purine bases in hearts from T3 rats

In contrast, compared with euthyroid hearts studied previously (3), T3 hearts contained ~1.8 times more Pi (9.4 ± 1.2 vs. 4.5 ± 1.1 mM) and one-half of the PCr (8.6 ± 1.2 vs. 17.6 ± 2.2 mM) and 70% of the creatine (20.4 ± 0.9 vs. 28.7 ± 2.0 mM) concentrations. pHi was the same as for euthyroid hearts under oxygenated perfusion (7.12 ± 0.02 vs. 7.12 ± 0.02).

Effects of Myocardial Ischemia on pHi and ATP in T3 Hearts, Measured by 31P-NMR Spectroscopy

Figure 2 shows the time course for the development of intracellular acidosis during no-flow ischemia in all treatment groups. For untreated T3 hearts during the first 4 min of ischemia, pHi declined from 7.14 ± 0.02 during control perfusion to 6.82 ± 0.04. Thereafter, however, pHi declined less rapidly. By 8 min of ischemia, pHi fell to 6.66 ± 0.06 and there was no further decline of pHi in untreated T3 hearts during the next 20 min of ischemia. When reperfusion occurred, pHi returned to the preischemic level.


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Fig. 2.   Changes in intracellular pH (pHi) in hearts from hyperthyroid rats [T3 hearts, that is, induced by 3,5,3'-triiodo-L-thyronine (T3)] exposed to 28 min of ischemia followed by a 28-min period of reperfusion. bullet , Untreated T3 hearts; square , T3 ischemic hearts perfused with amiloride (T3+AMI hearts); triangle , T3 ischemic hearts perfused with HEPES (T3+HEP hearts). Data represent mean values ± SD.

Figure 3 illustrates the time dependence of ATP depletion during ischemia and recovery of ATP on reperfusion following ischemic injury in all groups studied. For untreated T3 hearts, the cessation of flow caused a steep decrease in ATP content, to ~2% of preischemic values by 8 min. When reperfusion occurred, recovery of ATP content was observed and the resynthesis of ATP was very rapid and occurred early, within the first 4 min of reperfusion. Untreated T3 hearts recovered <20% of their preischemic ATP amount (P < 0.001).


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Fig. 3.   Changes in ATP concentrations [in mM; 31P nuclear magnetic resonance (31P-NMR) spectroscopy] in hearts from T3 rats exposed to 28 min of ischemia followed by a 28-min period of reperfusion. bullet , Untreated T3 hearts; square , T3+AMI hearts; triangle , T3+HEP hearts. Data represent mean values ± SD.

Consequences of Varying H+ Accumulation on Metabolite Distribution During Ischemia in T3 Hearts

31P-NMR measurements. Results presented above show that pHi falls to 6.7 during total ischemia in the T3 hearts. On the basis of the in vitro observations of other investigators (6, 27) and our whole heart data (3), it seemed likely that lowering the pHi in T3 hearts during ischemia below 6.7 would further inhibit the activity of 5'-NT and thereby lead to greater accumulation of AMP in ischemic tissue. In turn, this could result in greater ATP resynthesis on reperfusion. To test this hypothesis, we attempted to produce significantly greater acidosis during ischemia in T3 hearts using two methods: inhibition of Na+/H+ exchange by the administration of amiloride and removal of HCO-3 from the perfusate before the onset of ischemia. Results shown in Fig. 2 demonstrate that both maneuvers, designed to increase intracellular acidosis during ischemia in hyperthyroid hearts, were successful. The accumulation of protons in myocardium during 8 min of ischemia was significantly faster (0.074 and 0.079 pH units/min) in T3+AMI and T3+HEP hearts, respectively, compared with hearts from untreated T3 rats (0.060 pH units/min) (P < 0.05). The degree of intracellular acidosis in both the amiloride-treated and HCO-3-free perfused hearts was similar: after 12 min of ischemia, pHi fell to 6.45 ± 0.15 and 6.41 ± 0.13 for T3+AMI and T3+HEP, respectively, and remained significantly below that observed in untreated T3 hearts (6.70 ± 0.06, P < 0.05) throughout the remaining time of ischemia.

The time course of ATP changes during the experimental protocol (Fig. 3) shows that, in T3+AMI and T3+HEP hearts, the degradation of ATP during ischemia was slower compared with untreated T3 hearts. Eight minutes after the onset of ischemia, these hearts had 32 ± 17 and 36 ± 9% of their preischemic content of ATP, respectively, whereas untreated T3 hearts had only 2% of their preischemic ATP levels (P < 0.001). After 16 min of ischemia, ATP measured by NMR spectroscopy was <1% of preischemic values for all three groups of hearts. The ATP concentration increased rapidly at the onset of reperfusion in all groups. Importantly, a greater recovery of ATP was observed in T3+HEP (48 ± 9% of the preischemic value) and T3+AMI (41 ± 10% of the preischemic value) hearts than in untreated T3 hearts (19 ± 6%) (P < 0.001).

Figure 4 depicts the time course of PCr changes in the three groups of T3 hearts. Note that there is a small but significant increase of PCr during well-oxygenated perfusion (preischemia) in T3+HEP hearts. In all hearts, PCr fell rapidly during ischemia and was no longer detectable after 8 min. The PCr concentration increased rapidly, although to different extents, at the onset of reperfusion in all three groups to values close to their preischemic values, demonstrating that oxidative phosphorylation is intact in these reperfused hearts.


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Fig. 4.   Changes in phosphocreatine (PCr) concentrations (in mM, 31P-NMR spectroscopy) in hearts from T3 rats exposed to 28 min of ischemia followed by a 28-min period of reperfusion. bullet , Untreated T3 hearts; square , T3+AMI hearts; triangle , T3+HEP hearts. Data represent mean values ± SD.

As illustrated in Fig. 5, with the onset of ischemia, the fastest production of Pi was observed in untreated T3 hearts. By 8 min of ischemia, the concentrations of Pi were 43.4 ± 2.8, 34.3 ± 6.0, and 35.3 ± 5.1 mM in untreated T3, T3+AMI, and T3+HEP hearts, respectively (P < 0.05, untreated T3 vs. T3+AMI and vs. T3+HEP). After 12 min of ischemia, there were no significant differences in the Pi concentrations among the three groups. When restoration of flow following ischemia occurred, Pi returned toward preischemic values in all groups. Pi recovery was incomplete only in the untreated T3 hearts.


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Fig. 5.   Changes in Pi concentrations (in mM, 31P-NMR spectroscopy) in hearts from T3 rats exposed to 28 min of ischemia followed by a 28-min period of reperfusion. bullet , Untreated T3 hearts; square , T3+AMI hearts; triangle , T3+HEP hearts. Data represent mean values ± SD.

Purine metabolites. The tissue contents of ATP and its degradation products measured by chemical assay for all T3 groups studied are shown in Table 1.

In untreated T3 hearts, 28 min of ischemia markedly reduced the content of ATP, from 31.1 to 0.4 nmol/mg protein. The dominant nucleotide remaining at the end of ischemia was AMP (12.7 ± 1.6 nmol/mg protein, 77% of the remaining adenine nucleotide pool). Degradation of AMP led to the accumulation of purine nucleosides and nucleobases. More than one-half of the total purine pool (55%) was converted to nucleosides and nucleobases. When reperfusion occurred, the ATP content measured by chemical assay increased from 0.4 to 4.8 nmol/mg protein in reperfused T3 hearts. Thus 4.4 nmol/mg protein of AMP was used for the resynthesis of ATP and the remaining 5.9 nmol/mg protein of AMP was dephosphorylated and lost from the cell. This is also shown by the decrease in the total adenine nucleotide pool during reperfusion (from 16.5 at the end of ischemia to 11.9 nmol/mg protein at the end of reperfusion). These results show that, during reperfusion of previously ischemic T3 myocardium, we observed not only a loss of nonphosphorylated purines that had accumulated during ischemia but also further degradation of nucleotides. As a result, the total purine pool at the end of the reperfusion was reduced by 66% (from 37.7 to 12.9 nmol/mg protein).

Similar to data from untreated T3 hearts and as was measured by 31P-NMR spectroscopy (Fig. 3), HPLC data show that ischemia caused a depletion of ATP in T3+AMI and T3+HEP hearts. At the end of 28 min of ischemia, the remaining ATP in these hearts was only 2.6 and 5.1% of the preischemic ATP concentration, respectively. In contrast to untreated T3 hearts, a markedly increased accumulation of AMP was observed in ischemic T3 myocardium when pHi was decreased. Hearts from the T3+AMI and T3+HEP groups accumulated 30 and 40% more AMP (Table 1), respectively (P < 0.01), than untreated T3 hearts. The relationship between AMP accumulation and pHi during global ischemia in T3 hearts is shown in Fig. 6. The results suggest that AMP accumulation increases as the pHi falls during ischemia. An excellent correlation was found between AMP levels measured in myocardium at the end of the 28 min of ischemia and the lowest pHi measured during the ischemic period (r2= 0.939, n = 14, P < 0.001). Consistent with higher AMP levels, the total pool of purines after 28 min of ischemia was smaller and the total nucleotide pool was higher in both the T3+AMI and T3+HEP groups (P < 0.05, Table 1) compared with untreated T3 hearts.


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Fig. 6.   Relationship between pHi and AMP (in nmol/mg protein, measured by high-performance liquid chromatography) in all T3 hearts, measured at end of control period and at end of 28 min of ischemia. Data represent 4 conditions. open circle , T3 hearts at end of 16 min of oxygenated perfusion; bullet , untreated T3 hearts at the end of 28 min of ischemia; square , T3+AMI hearts at the end of 28 min of ischemia; triangle , T3+HEP hearts at the end of 28 min of ischemia. Value of r2 = 0.939, P < 0.001.

Our results also show that a greater fraction of the accumulated AMP (primarily cytosolic) in ischemic tissue from treated T3 hearts was utilized for resynthesis of ATP in reperfused myocardium (Table 1). When reperfusion occurred, as was also shown by the NMR measurements (Fig. 3), the recovery of ATP in T3+AMI and T3+HEP groups was greater than for the untreated T3 group. We observed more than a 2.5-fold increase of ATP content in T3+AMI and T3+HEP groups (11.2 and 11.9 nmol/mg protein, respectively) when compared with untreated T3 hearts (4.4 nmol/mg protein, P < 0.001). This increase in postischemic ATP resynthesis was not only a result of increased AMP accumulation at the end of ischemia but also due to better AMP utilization for ATP resynthesis. More than 74 and 93% of the AMP that had accumulated at the end of ischemia in T3+AMI and T3+HEP hearts, respectively, were used for ATP resynthesis compared with only 49% used in untreated T3 hearts (Table 1). As a result, compared with untreated T3 hearts, the total adenine nucleotide pool at the end of reperfusion was higher in T3+AMI and T3+HEP hearts (P < 0.05).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The primary enzyme responsible for the dephosphorylation of AMP to adenosine in the mammalian myocyte is the AMP-specific cytosolic 5'-NT enzyme. The literature indicates that, although different isoenzymes exist in various tissues, the enzymatic properties of AMP-specific 5'-NT persist in all species. The enzyme has been well characterized in vitro. In the late 1980s, two groups studying the enzyme, isolated from pigeon, rat, and rabbit heart homogenates, showed that the Michaelis-Menten constant (Km) for AMP is ~4.8 mM and the enzyme requires Mg2+ for its activity, has a pH optimum near 7, and is inhibited by Pi and H+ (20, 22, 27, 31, 33). More recent studies of a highly purified enzyme isolated from dog (6), rat (28), and human (5, 28) hearts have shown similar kinetic properties, with a Km of 1.5 mM under maximally stimulated conditions, and have shown that the enzyme is activated by AMP, ADP, and Mg2+ and is active between pH 6.5 and 7.8. If the same regulation occurs in intact heart, then pHi has an important role in the determination of the size of the AMP pool in the ischemic myocardium.

The present study was designed to address the postulate that pH is a major regulator of 5'-NT activity during ischemia. We identified and used a heart model in which pH could be manipulated during ischemia and its consequences on AMP accumulation during ischemia and ATP resynthesis during reperfusion could be measured. We presented evidence from in vivo studies showing that intracellular acidification of ischemic myocardium inhibits cytosolic 5'-NT, thereby increasing AMP accumulation during ischemia and ATP resynthesis during reperfusion.

The notion that acidosis may play a salutary effect during ischemia may seem contradictory to the widely held premise that acidosis leads to cell death. It must be emphasized that the role of pH studied here is regulation of a specific pathway in acutely injured cardiomyocytes, whereas the classic relation between pHi and irreversible ischemic injury applies to a much longer time scale of ischemic insult. Consistent with pH regulation within injured, but not dying, cells are our observations that in the reperfused myocardium studied here there is a recovery of PCr and return of pHi to normal values. On reperfusion for all hearts, PCr content recovered to values close to their preischemic levels, showing that the ability of mitochondria to rephosphorylate ADP was intact, even though the duration of the energy-poor state varied. Rapid recovery of normal pHi shows that the mechanisms for the maintenance of pHi are functional. Furthermore, there was no loss of cytosolic enzymes during the experimental protocol (data not shown).

By combining results from the in vivo 31P-NMR spectroscopy experiments with chemical analysis of hearts freeze-clamped at the end of the experiment, each of the substrates, products, activators, and inhibitors of the cytosolic AMP-specific 5'-NT reaction could be determined for the intact heart. There are few enzymes that can be studied in such a complete way. The discussion that follows presents an analysis of the regulation of the 5'-NT by each of these metabolites in the ischemic T3 myocardium in vivo.

Substrate and Products of the 5'-NT Reaction

5'-NT catalyzes the hydrolysis of AMP to form adenosine and Pi. The amount of substrate that accumulates during ischemia can be determined in three ways: 1) from the increase in AMP measured in tissue extracts from ischemic compared with well-oxygenated myocardium, 2) from the adenylate kinase reaction if the free ADP concentration is known, and 3) from the 31P-NMR spectrum, under some conditions (3). Because adenosine is rapidly deaminated in vivo to form inosine, hypoxanthine, and xanthine (Fig. 1), the sum of purine nucleosides and nucleobases must be used to estimate the amount of purine product formed by this reaction. Accumulation of Pi can be determined in real time using 31P-NMR spectroscopy, but it cannot be readily used to calculate the rate of product formation by the 5'-NT reaction because of the presence of multiple sources (e.g., hydrolysis of PCr) and sinks (such as sarcolemmal efflux, movement into the mitochondria, and accumulation of phosphorylated glycolytic intermediates).

Both substrate (AMP) and products (Pi and adenosine and its degradation products) of the 5'-NT reaction accumulated during ischemia in all hearts. The accumulation of the reaction products shows that 5'-NT was activated during ischemia. The fastest rate of Pi accumulation and the highest accumulation of adenosine and its degradation products were observed in untreated T3 hearts, suggesting that 5'-NT activity was either activated earlier and/or inhibited less than in the T3+AMI and T3+HEP hearts. Accumulation of both products and substrate during ischemia shows that the activity of 5'-NT was not limited by substrate availability. Instead, the enzyme was unable to use substrate during ischemia. Central to the hypothesis being tested, our results show a substantially greater accumulation of AMP during ischemia, by 30% and 40%, when the pHi was lowered from 6.7 to 6.45 in the T3+AMI and T3+HEP hearts, respectively. Thus, after being activated early during ischemia, 5'-NT activity was then inhibited. Moreover, the extent of this inhibition is pH dependent. Consistent with greater 5'-NT activity in untreated T3 hearts, a greater fraction of the AMP that had accumulated during ischemia was converted to purine nucleosides and nucleobases (which were lost from myocytes during reperfusion) in the untreated compared with treated T3 hearts. About one-half of the AMP that had accumulated during ischemia in untreated T3 hearts was lost during reperfusion, whereas only 26 and 7% of the AMP was converted to adenosine and its degradation products in the T3+AMI and T3+HEP hearts, respectively. Together, these results suggest that 5'-NT was more active during both ischemia and reperfusion in the untreated T3 hearts than in the T3+AMI and T3+HEP hearts.

Activators of the 5'-NT Reaction

Two potent activators of 5'-NT are known, Mg2+ and ADP. Activity of the cytosolic enzyme isolated from hearts of rat, rabbit, pigeon, and dog shows an absolute requirement for Mg2+ (6, 12, 20, 22, 27, 31, 33, 35). Although the 31P-NMR spectrum can be used to estimate cytosolic Mg2+ concentrations, the method is based on the chemical shifts of the three ATP resonance areas. Because ATP hydrolysis was essentially complete within 8-12 min of ischemia in all hearts studied, this method cannot be used here. However, Murphy et al. (21), using another approach, have shown that intracellular Mg2+ rose nearly threefold during 15 min of ischemia in rat myocardium (to 2.1 mM). Results using 5'-NT purified from dog heart show that the enzyme was fully activated at ~3.5 mM Mg2+. It seems likely that under the experimental conditions described here Mg2+ concentrations increased and contributed to the activation of 5'-NT during ischemia. Because the rate of ATP hydrolysis was faster, one would predict that Mg2+ concentrations accumulated faster in untreated T3 hearts than in T3+AMI and T3+HEP hearts. Consistent with greater Mg2+-stimulated 5'-NT activity, the untreated T3 hearts accumulated Pi faster and they accumulated more adenosine and its degradation products than T3+AMI and T3+HEP hearts.

In vitro studies with 5'-NT purified from rabbit (33) and dog (6) myocardium have shown that the enzyme is also activated by free ADP, especially at low ATP concentrations. In addition, the affinity of the enzyme for AMP is increased in the presence of ADP (6). For the purified heart enzyme from all species studied, the maximum activation of the enzyme was achieved at ~80 µM ADP in the absence of ATP but little activation by ADP occurs at physiological ATP concentrations of 10 mM. To assess the effects of changing concentrations of ADP in our experimental conditions, we calculated free ADP concentration by using the creatine kinase equilibrium expression. The mean estimates of free ADP are 116, 116, and 94 µM in untreated T3, T3+AMI, and T3+HEP hearts, respectively. By 4 min of ischemia, ADP increased 11-fold in T3 hearts (1,280 µM) but only 3- to 4-fold in T3+HEP and T3+AMI hearts (447 and 280 µM, respectively). From these results, we would make three predictions: 1) high ADP concentrations in all hearts during preischemia did not activate the enzyme because ATP content is high; 2) the concentration of free ADP in all hearts is sufficiently high to activate 5'-NT during ischemia when the ATP concentration is low; 3) because all hearts had high ATP levels at the beginning of ischemia, 5'-NT activity would be activated most rapidly in hearts that showed the fastest rate of ATP hydrolysis (and hence the fastest rate of ADP accumulation), namely, the untreated T3 hearts. Our data are in accord with each of these predictions. First, we did not detect nucleoside production in any of the hearts during preischemia, showing that 5'-NT activity is low. Second, all hearts demonstrated increased accumulation of products during ischemia, showing that 5'-NT is activated during ischemia. Third, the untreated T3 hearts, which had the fastest rate of ATP depletion, also had the highest rate of Pi accumulation during early ischemia and the largest accumulation of purine nucleosides and nucleobases during ischemia, consistent with the fastest activation of the enzyme.

Taken together, our results suggest that 5'-NT was activated at the beginning of ischemia in all T3 hearts and that increased concentrations of the primary activators, especially ADP, explain the greater activity of 5'-NT in untreated T3 hearts than in the T3+AMI and T3+HEP hearts.

Inhibitors of the 5'-NT Reaction

The primary inhibitors of 5'-NT are Pi and H+. Several in vitro studies have shown that Pi inhibits both ecto- (12, 35) and cytosolic 5'-NTs (12, 20, 22, 31). Studies of the cytosolic 5'-NT purified from rat hearts showed that inhibition by Pi varies from <10 (31) to 50% (20). If Pi would play a significant role in 5'-NT inhibition under the in vivo conditions described here, the highest degree of inhibition of 5'-NT should have occurred in untreated T3 hearts, which showed the fastest rate of Pi accumulation during ischemia and also the slowest rate of recovery of Pi during reperfusion (Fig. 5). However, as described above, this is the group that showed the highest activity of 5'-NT both during ischemia and reperfusion. Thus we conclude that total Pi is unlikely to be a major inhibitor of 5'-NT in the ischemic heart. It seems more likely that H+ plays this role.

One way that protons can function to inhibit phosphate-dependent enzyme activity is to shift the phosphate equilibrium toward formation of H2PO-4. To determine if H2PO-4 could be responsible for 5'-NT inhibition, we calculated the production of H2PO-4 during ischemia using measured values of Pi and pHi and pKa = 6.9. Neither the rate of H2PO-4 production nor its extent of accumulation was very different among the three T3 groups (not shown), suggesting that accumulation of H2PO-4 does not explain inhibition of 5'-NT activity in ischemia.

Other mechanisms for acidosis-mediated enzyme inhibition include denaturation (24), proton-dependent shifts in the equilibrium between monomer and active aggregates (10, 24), and titration of an imidazole group in the active site (25, 30). Although we have no direct evidence regarding these possible mechanisms as they apply to 5'-NT, we can draw some likely conclusions and indicate possible mechanisms of action. First, we observed no evidence that acidosis denatures the enzyme during acute ischemia. On the contrary, the conversion of some of the AMP that had accumulated during ischemia to purine nucleosides during reperfusion argues that 5'-NT is either still active or reactivated early during reperfusion. Second, recently published data on the enzyme purified from dog myocardium suggest that 5'-NT is a tetramer (6). In some enzymes, pH regulation occurs by shifting the equilibrium among aggregated states, only some of which are active. For example, Carpenter and Hand (4) showed that in perfused rat hearts the decrease of pHi that accompanies ischemia led to a tetramer-to-dimer shift of phosphofructokinase, leading to phosphofructokinase inhibition. Finally, the protonation state of the active site of the enzyme can shift the equilibrium position of the reaction and even prevent substrate binding. The imidazole group of histidine residues is the usual target (30). Whether any of these mechanisms apply to 5'-NT remains to be determined.

Implications

Our data show that when pHi was lowered during acute zero-flow ischemia the heart accumulated more AMP during ischemia and there was increased resynthesis of ATP during reperfusion. Although the etiology of myocardial dysfunction after ischemia is multifactorial, a causal relation between substrate availability for ATP repletion in ischemic tissue and the capacity of the reperfused myocardium to resynthesize ATP is clear (1, 7, 8, 11, 32). The requirement of contractile function (myofibrillar ATPase) for ATP is absolute.

The results presented here allow us to assess the pH range that regulates 5'-NT activity in the intact rat heart. Darvish and Metting (6) suggest that 5'-NT in the dog heart is active only between pH 6.8 and 7.8 (maximal activity was at pH 7.5 and at pH 6.7 it has <20% of its maximal activity). Skladanowski and Newby (27) showed in pigeon heart an activity-pH curve that shifted to lower pH values (maximal activity was at pH 7.0 and at pH 6.7 it has ~67% of its maximal activity). Because we observed that decreasing pHi from 6.7 to 6.4 in T3 hearts led to increased accumulation of substrate and decreased accumulation of product, our results show that the activity-pH curve for rat heart 5'-NT resembles the profile of pigeon heart 5'-NT (27). One explanation for these apparently conflicting results is that there are species differences in the regulation of 5'-NT by pH. If this alternative explanation is correct, then 5'-NT activity in myocardium of large animal and human hearts would be inhibited by much smaller changes in pHi than observed here for the rat heart. As a consequence, even relatively small decreases in pHi would confer protection for these hearts. This has potentially important clinical implications, for example, for designing cardioplegic solutions and for developing the strategies to optimize preservation of the purine nucleotide pool during early reperfusion following myocardial infarction.

In this context, the greater tolerance of the neonatal vs. mature myocardium to ischemia-reperfusion injury is noteworthy (9, 17, 19). This phenomenon has been attributed to lower concentrations of 5'-NT (9, 13) and presumably lower capacity to hydrolyze AMP. Recent human studies showing higher accumulation of AMP in ischemic tissue and smaller loss of ATP after heart surgery in patients <18 mo old compared with patients to 8 years of age (17) were explained by lower tissue concentrations of 5'-NT. In addition, an inverse relationship between 5'-NT activity and ventricular functional recovery after ischemia was observed (9, 13). These clinical results are consistent with the premise put forth in this report that the preservation of the precursors of ATP (namely AMP) during ischemia is an important factor in postischemic recovery of ATP, which in turn contributes to improved function of the heart.

Role of 5'-NT Inhibition on Adenine Nucleotide Metabolism in T3 Hearts

In this report, we have confined our analysis of the whole heart regulation of 5'-NT to hyperthyroid hearts and have not compared results from T3 and euthyroid hearts. This is because thyroid treatment is known to alter the isoenzyme composition of many proteins important for myocyte function, for example, myosin, creatine kinase, and the Na+ pump. Although there is no evidence that either the amount or activity of 5'-NT is altered in the T3 hearts, its environment is certainly different. However, some comparisons may be useful. Our previous report (3) studying 5'-NT in euthyroid rat hearts made ischemic for 28 min showed that pHi fell from 7.1 to 6.2 and that nearly all of the AMP that had accumulated during ischemia (13.7 nmol/mg protein) was used for ATP resynthesis during reperfusion (12.5 nmol/mg protein). Because pHi is already so low, it is not surprising that neither supplying amiloride nor changing the perfusate to HEPES-containing buffer changed the fall in pHi. These maneuvers also had no effect on the extent of AMP accumulation during ischemia or ATP resynthesis during reperfusion (unpublished results). As shown in the present study, pHi in untreated T3 hearts made ischemic for 28 min fell to only 6.7 but pHi fell to 6.4 in hearts supplied with amiloride and perfused with HEPES. If pHi plays a major role in regulating AMP degradation in the ischemic rat heart (and all modulators are the same), we would expect that the extent of ATP resynthesis would be greatest in the euthyroid hearts, that the two treated T3 groups would be similar to the euthyroid hearts, and that the untreated T3 hearts would have the lowest ATP recovery. Together with our previously published work, the results presented here show that these predictions are satisfied. The amount of ATP synthesized during reperfusion was 12.5, 11.2, and 11.9 nmol/mg protein in euthyroid, T3+AMI, and T3+HEP groups, respectively, whereas it was only 4.8 nmol/mg protein for the untreated T3 hearts.

In summary, this study provides evidence for a salutary effect of intracellular acidosis on preserving the AMP pool due to inhibition of 5'-NT. The finding that intracellular acidosis in vivo alters the fate of AMP suggests that cytosolic 5'-NT plays a unique role in mediating ATP degradation during ischemia and setting the capacity for ATP resynthesis during reperfusion. We suggest that modulating the activity of cytosolic 5'-NT by altering pHi may constitute a novel target for pharmacological interventions. Finally, these results should lead to reassessment of the classical concept of a detrimental role of acidosis in the ischemic tissues.

    ACKNOWLEDGEMENTS

We thank Ilana Reis for excellent technical assistance.

    FOOTNOTES

This research was supported by National Heart, Lung, and Blood Institute Grants HL-43170 and HL-50594.

Address for reprint requests: M. I. Bak, NMR Laboratory for Physiological Chemistry, Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115.

Received 30 June 1997; accepted in final form 12 December 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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