JOURNAL OF CELLULAR PHYSIOLOGY 143563-568 (1990)
Studies on the Energy Metabolism of Opossum (Didelphis virginiana) Erythrocytes: V. Utilization of Hypoxanthine for the Synthesis of Adenine and Guanine Nucleotides In Vitro N.C. BETHLENFALVAY,* J.C. WHITE, E. CHADWICK, AND J.E. LIMA Department of Primary Care (N.C.B.) and C/inica/ Investigation C).C.W., E.C., I . E . L . ) , Fitzsirnons Army Medical Center, Aurora, Colorado 80045
High pressure liquid radiochromatographywas used to test the ability of opossum erythrocytes to incorporate tracer amounts of [ G 3 H I hypoxanthine (Hy) into [’HI labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate I”H1 ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient H v are more favorable in the venous circulation.
Comparative erythrocyte physiology has yet to address the task of a n inquiry into the salvage and de novo synthesis of purine compounds in a systematic manner. It was nearly three decades ago that rabbit red cells were shown to be able to incorporate [14C] hypoxanthine (Hy) into both ATP and GTP (Lowy e t al., 1961; Hershko et al., 1967). More recently Moyer and Henderson (1983)showed that mouse erythrocytes, in the presence of supraphysiologic Pi, were also able to incorporate radiolabelled Hy into ATP and GTP. Nucleotide biosynthesis from preformed purines in mammalian cells has been the subject of excellent comprehensive reviews (Murray et al., 1970; Murray, 1971). IMP is a pivotal compound for the metabolic balance between adenine and guanine nucleotides. Human erythrocytes, which lack S-AMP synthetase (Lowy and Dorfman, 1970), are unable to convert IMP to AMP, a n enzymatic capacity inherent to rabbit and mouse red cells. We have presented evidence of net ATP synthesis in opossum erythrocytes with inosine as a source of energy for metabolism. In that study, ATP was measured enzymatically (Bethlenfalvay et al., 1984).Later radiochromatographic investigations revealed that red cells of this species are deficient in ADA activity, and contain large amounts of dATP (Bethlenfalvay et al., 1989).The objective of this study was to investigate the metabolic trail of tracer amounts of Hy provided to opossum erythrocytes. The results of the experiments show extensive incorporation of [‘HI Hy into ATP and GTP not only in the presence of supraphysiologic but also a t physiologic Pi concentrations if intracellular hemoglobin was deoxygenated. Our observations suggest that in the opossum erythrocyte ATP and GTP 8 1990 WILEY-LISS, INC.
may, to some extent, be produced from ambient Hy in the venous circulation.
MATERIALS AND METHODS Animals Adult D . virginiana species were obtained from commercial vendors and were shipped from Florida and Texas via air. The animals were allowed to adjust to Denver altitude (5,400 feet above sea level) for 3 months before use. Chemicals [G-’H] hypoxanthine (6.7 Ci/mmole) was obtained from Amersham (Arlington Heights, IL). ATP, GTP, dATP, hypoxanthine, and mycophenolic acid were purchased from Sigma Chemical Company (St. Louis, Received July 27, 1989; accepted February 28, 1990. *To whom reprint requestskorrespondence should be addressed. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army and The Department of Defense. Abbreviations used: ADA, adenosine deaminase; ADP, adenosine 5’-diphosphate; AMP, adenosine 5’-monophosphate; ATP, adenosine 5’-triphosphate; dADP, deoxyadenosine 5’-diphosphate; dATP, deoxyadenosine 5’-triphosphate; GTP, guanosine 5’-triphosphate; HGPRT, hypoxanthine-guanine phosphoribosyl transferase; Hy, hypoxanthine; IMP, inosine 5’-monophosphate; Pi, Sinorganic phosphate; PRPP, 5-phosphoribosyl-1-pyrophosphate; AMP, succinyladenosine-5‘-monophosphate; 2-3 DPG, 2-3 diphosphoglycerate.
BETHLENFALVAYETAL TABLE 1. Incorporation of 50 KCi (7.46 nmoles) of [G-3Hl hypoxanthine in the presence of 30 mM Pi into ATP and GTP in opossum erythrocvtes with and without inhibitors in 4 h
Baseline (n = 8 ) Control (n=8) Hadacidin4 (n = 7) Mycophenolic acid5 (n=4)
ATP 3,082 2 216"
nmoles Specific activity" nmoles Specific activity nmoles Specific activity
2,177 -t 291 720 f 119 2,043 ? 251 155 -t 27 2,227 L 354 801 ? 145
'nmoleslg hemoglobin. 'Values are mean 2 SEM. "CPMinanomole (2,500 r l flow-cell counting efficiency 45 x ~ o - ~ M . '5 x 10 'M
TABLE 2. Incorporation of 25 kC1 (3.35 nmoles) of L'H] hypoxanthine in the presence of 1 rnM Pi into ATP and GTP in opossum erythrocytes under oxygen or nitrogen as gas phase in 4 h
Baseline Oxygen Nitrogen
Condition nmoles' nmoles Radioactivity? Specific activity' nrnoles Radioactivity SDecific activitv
ATP 1,[email protected]
1,290 179,995 139 1,906 515,565 27 1
GTP 581 538
69,630 129 600 412,628 688
nmoledg hemoglohin . 'Values are the average obtained in red cells from two animals. 'CPM!g hemoglobin. 4CPM/nanomole (2,500 ~1 flow-cell counting eFficiency = 4.5%.).
c m 911
1,038 ? 46 619 2 151 1,219 f 28 574 2 130 1,012 + 48 0
2,242 IT 347 2,180 2 310 0 2,320 IT 320 0 2,200 ? 300 0
bles. After a 4 h incubation at 37'C (air gas phase), the suspensions were extracted with KOH (Stocchi et al., 1985) and the neutralized material was kept at -70°C until analyzed by radiochromatography. Physiologic medium Pi. Aliquots (1ml) of cell suspensions (1mM Pi) were provided with 3.7 nmoles (25 FCi) of IG-3Hl Hy. Cells were incubated parallel under a n atmosphere of nitrogen or oxygen. Individual suspensions were extracted a t 0.5, 1, 2, and 4 h a s described above. Parallel 1 ml suspensions not containing radioactivity were also kept at 37"C, and at the end of the incubation periods, hematologic parameters were determined.
HPLC/radiochromatography The chromatographic system (Waters) consisted of a n 840 chromatography station, three model 510 pumps, a digital professional model 380 solvent proPreparation of red cell suspensions grammer, a model 710-B sample processor, and a model Opossums were anesthetized with ketamine and 490 programmable multi-wavelength detector. Radioblood (15 ml) was obtained by cardiocenthesis (Beth- activity was measured by using a Flo-one beta model lenfalvay et al., 1983). Red cells sedimented from whole 1-C flow detector equipped with a 2500 p.1 flow-cell (Radiomatic Instruments, Addison, IL, USA). The deblood were washed x 3 in saline, removing the top 2-3 mm of cells each time, and resuspended as detailed tector unit was connected to the HPLC system in series enabling the column effluent to pass from the UV debelow: Supraphysiologic medium Pi. Cells (20-30%) tector directly to the radioactivity detector. Miniscint were suspended in a n electrolyte solution containing (Radiomatic Instruments) liquid scintillator fluid was (mmoles/l): 150 NaC1, 30 Na,HP04, 5 KCL, 1.5 MgCl,, used for all analyses in a ratio recommended by the manufacturer. Flow cell counting efficiencies were cal15 D-glucose, and 100 TRIS-HC1 (pH 7.4 460 mosM). Physiologic medium Pi. Packed red cells (0.35 ml) culated as described by Webster and Whaun (1981). were added to 0.65 ml of a n electrolyte solution con- Separation of nucleotide di- and triphosphates was taining (mmolesll): 140 NaCl, 5 KC1, 1 Na2HP04, 1.5 done in a Beckman Ultrasil AX (4.6 x 250 mm) anion MgCl,, 8 D-glucose, and 15 TRIS-HC1 (pH 7.3, 297 exchange column with 0.2M phosphate buffer, pH 6.4 as mobile phase, in a n isocratic mode at 19°C a s demosM). Aliquots of these suspensions were used for the de- scribed by Arezzo (1987). Peak identities were contermination of baseline ATP, dATP, and GTP concen- firmed by retention time of and coelution with stantrations and of hematologic parameters within 45 min- dards, by enzymatic peak shift techniques, and by resistance of deoxynucleotides to oxidative destruction utes of cardiocenthesis. by periodate (Garrett and Santi, 1979). Total area integration of radioactivity (CPM) was possible by using Incorporation of [G-3Hlhypoxanthine into ATP the Flo-one system microcomputer. and GTP Supraphysiologicmedium Pi. To assess the incorOther determinations poration of tracer amounts of Hy into ATP and GTP 1 ml aliquots of cell suspensions were preincubated for Hematologic parameters of whole blood and of cell 30 minutes a t 37°C and were then provided with 7.46 suspensions were determined with a Coulter model ST nmoles (50 FCi) of [G-'H] Hy in the absence or presence apparatus. pH measurements were done a t 37°C with a of inhibitors of S-AMP synthetase or IMP dehydroge- Corning #135 pHlion meter. Osmolarity of incubation nase in concentrations shown in figure legends and ta- buffers was determined by using a Wescor 5100 C va-
MO). Hadacidin was a gift from Merck, Sharp, and Dohme (Rahway, NJ). All other chemicals were of highest quality commercially available.
SYNTHESIS OF ATP AND G-TPFROM HYPOXANTHINE
por pressure osmometer. Static ‘H radioactivity in cell extracts was measured with a Packard Tri-Carb model 4640 instrument. I 1
RESULTS Hematology Leukocytes in cell suspensions were consistently below 0.7 x lO’/~l, and platelets were not seen. We found it necessary to use a hypertonic buffer (460 mosM) to prevent lysis of metabolizing cells in suspensions (Bethlenfalvay et al., 1988, 1989). Surprisingly opossum erythrocytes do not shrink in this hyperosmolar medium; they in fact expand in volume. Thus, red cell count, hemoglobin, and mean cellular hemoglobin remain stable in such a buffer during a 4 h incubation while mean cell volume and hematocrit increase to 120 and 125% of control value. A corresponding decline of mean corpuscular hemoglobin concentration to 79% of control value occurs in 4 h at 37°C. I n the physiologic (297 mosM) buffer 2-3% red cell lysis occurs in 4 h, but the cells increase only by 5-6% in volume. Incorporation of [3Hl hypoxanthine into ATP and GTP The incubation of opossum erythrocytes in a medium containing glucose, supraphysiologic (30 mM) Pi, and Hy (7.7 nmoles, 50 pCi/ml) resulted in extensive labelling of ATP and GTP. As is shown in Figure 1A and Table 1, the extent of [’HI incorporation into ATP and GTP is similar. Hadacidin, a n inhibitor of S-AMP synthetase (Shigeura and Gordon, 1962), decreased synthesis of L3H]ATP to 21% of control value (Fig. lB, Table 1).Mycophenolic acid, a potent inhibitor of IMP dehydrogenase (Sweeney et al., 1972), abolished the synthesis of [‘HI guanine nucleotides (Fig. lC, Table 1). Figure 2 (lower) shows t h a t in the presence of physiologic (1 mM) Pi, erythrocytes under nitrogen atmosphere incorporate [3H] Hy into [3H] ATP in a linear fashion with time. In contrast, under oxygen as gas phase, synthesis of labelled ATP is blunted. The rate of net [‘HI ATP formation, however, exceeds the rate (8%/ h) of ATP catabolism. Stability of ATP, dATP, and of GTP Opossum red cells using D-glucose as provided energy are unable to maintain their baseline ATP content during incubations at physiologic or at supraphysiologic medium Pi concentrations under a n atmosphere of air or oxygen. In contrast, when nitrogen serves a s gas phase, ATP is stable on metabolizing cells (Fig. 2 upper, Tables 1 and 2). The above data show t h a t in opossum erythrocytes incubated in a n electrolyte solution under “physiologic” conditions, T1/2 of ATP is about 6 h despite their brisk (3 pmoles/ml/h) utilization of D-glucose (Bethlenfalvay et al., 1984).In contrast, a s Tables 1 and 2 indicate, dATP and GTP are relatively stable in erythrocytes during incubations at both physiologic and supraphysiologic medium Pi concentrations. Relative concentrations of ATP, dATP, and of GTP in erythrocytes of individual opossums Figure 3 shows a wide range of concentrations of ATP and dATP in red cells of individual animals, re-
150 100 50
Fig. 1. A representative radiochromatogram (-UV, - - - - -:3H trace) of opossum RBC incubated for 4 h with 7.46 nmoles (50 pCi) of [G-’H] hypoxanthine per ml suspension (Pi 30 mM). Adenosine, deoxyadenosine, and guanosine di- and triphosphates were separated isocratically in 50 pl of extract, corresponding to 1.2 mg of hemoglobin. A No inhibitors present. B Hadacidin 500 pM. C: Mycophenolic acid 50 p M . Flow rates: 1.5 ml/min (HPLC); 6.5 mlimin (Flo-1).Flow cell (2,500 p1) counting efficiency is 4.5-4.84 under the conditions used. AUFS = 0.25.
sulting in high SEM (Table 1).These levels, however, were fairly constant on repeated sampling of blood for other studies (data not shown). In contrast to ATP and to dATP the concentration of GTP in opossum erythrocytes is narrow in range (Table 1). The levels detected are similar to those determined in the rabbit, dog, and cat and are about six times higher than those observed in human erythrocytes (Brown et al., 1972).
DISCUSSION The data presented show that hypoxanthine, a product of purine nucleotide catabolism, can serve as a pre-
A T P
TIME (HOURS) S N L
Fig. 2. Changes in 1'Hl ATP and ATP in red cells provided with 3.7 nmoles (25 p,CI) 13HJ hypoxanthine per rnl suspension in the presence of physiologic (I rnM) phosphate in 4 h. Details of incubation and chromatography are under Materials and Methods.
cursor for adenine and guanine nucleotides in the opossum erythrocyte in vitro and may contribute to their renewal in vivo. The extensive labelling of ATP and GTP in red cells provided with [3H] Hy occurs through successive steps catalyzed by HGPRT, S-AMP synthetase, and lyase respectively (Fig. l A , Table 1). Indeed, the use of specific enzyme inhibitors of S-AMP synthetase and IMP dehydrogenase resulted in a decrease of ['HI-labelled ATP and abolished the formation of ['HI GTP (Fig. 1B,C, Table l ) . The synthesis of purine nucleotides in red cells is regulated by their endogenous supply of PRPP (Hersh-
ko et al., 1969; Planet and Fox, 1977). The formation of PRPP in mammalian cells has been the subject of a recent excellent review (Becker et al., 1979).Elsewhere it was shown that for the formation of PRPP, dATP can fully replace ATP as a pyrophosphoryl donor with equal activity (Fox and Kelley, 1971; Roth et al., 1974). Pi is a n absolute requirement for PRPP synthetase a.ctivity. The inhibition of the synthetase in red cells by free intracellular 2-3 DPG (Bunn et al., 1971) is readily overcome by use of supraphysiologic (5-60 mM) Pi concentrations (Hershko et al., 1969). On the other hand, the mechanism of how red cells
SYNTHESIS OF ATP AND GTP FROM HYPOXANTHINE
the gas atmosphere (Hershko et al., 1967). In contrast, Whelan and Bagnara (1979) reported ATP to be stable in human red cells during 90 minutes of incubation irrespective of the two gas atmospheres used. Marked depletion of red cell ATP associated with a a dATP accumulation is reported in human severe coma. bined immunodeficiency characterized by profound ADA deficiency (Morgan et al., 1987; Simmonds et al., 0 1982). Similar observations have been made in patients following therapeutic inhibition of cellular ADA with deoxycoformycin a s a treatment modality of lym, , , phoreticular malignancy (Siaw et al., 1980; Koller and Mitchell, 1983; Ho et al., 1988). In vitro treatment of 1 2 3 4 human red cells with deoxycoformycin and then provided with deoxyadenosine and D-glucose similarly re)I moles/g hgb sulted in a marked decline of ATP and concomitant dATP accumulation (Koller et al., 1984; Buc et al., Fig. 3. Scatter plot of ATP and deoxy ATP concentrations in eryth1986). The mechanism of induced ATP breakdown in rocytes. The symbols represent individual animals. No correlation human hemopoietic cells accumulating dATP is the was observed. subject of three excellent recent reviews (Bagnara and Hershfield, 1982; Valentine et al., 1985; Bontemps et al., 1986). make enough PRPP for nucleotide synthesis in vivo (1 The resolution of the issue about the instability of mM Pi) has remained problematic. A decrease of oxy- ATP in the opossum erythrocyte metabolizing under genation of hemoglobin in human red cells results in a air or oxygen in vitro must await the precise definition decrease of unbound inhibitors of PRPP synthetase, of the kinetics of enzymes involved in concert in PRPP i.e., 2-3 DPG and MgATP (Bunn et al., 1971). Sciaky and purine metabolism a s well as their catabolism. Our and coworkers (1974) proposed that the venous circu- findings suggest that adenosine may be a n absolute lation provided an environment favorable for the syn- requirement for the maintenance of ATP in the oposthesis of PRPP and the formation of purine nucleotides. sum erythrocyte in vivo, and that the processes of synRecently, Salerno and Giacomello (1986) confirmed thesis and catabolism of adenine ribonucleotides may that human red cells under a n atmosphere of nitrogen be different in the arterial and venous circulation of and in the presence of physiologic (1 mM) Pi showed this animal. The extent to which dATP participates in brisk IMP synthesis from provided Hy, presumably due the grand scheme of energy metabolism in the red cell to the binding of 2-3 DPG to deoxygenated hemoglo- of this marsupial species is unknown a t present. Furbin. ther studies are needed to provide these insights. The results presented in this report support the vaACKNOWLEDGMENTS lidity of the assumption that purine nucleotide formation may occur (albeit in a limited “pulse fashion”) in We are grateful to Ms. K. Wyatt for the preparation the venous phase of circulation. Under a n atmosphere of figures and to Ms. C. Montoya for typing the manuof nitrogen and at physiologic (1 mM) Pi, a sustained script. This research was supported by grant 801650 increase of labelled ATP is seen in red cells provided from the Department of Clinical Investigation, Fitzsiwith I3H] Hy, whereas less accumulation of the radio- mons Army Medical Center. label is seen in cells for whom oxygen served as gas phase suggesting inhibition of PRPP synthetase in LITERATURE CITED fully oxygenated erythrocytes. (Fig. 2, Table 2). At Arezzo, F. (1987) Determination of ribonucleoside triphophates and Denver altitude, 2-3 DPG concentrations are similar deoxyribonucleotide triphophates in Novikoff hepatoma cells by in human and opossum erythrocytes (Bethlenfalvay e t high-performance liquid chromatography. Anal. Biochem., 160:6764. al., 1983). Mixed venous opossum blood has a PO, of 38 A S . , and Hershfield, M.S. (1982) Mechanism of deoxyademm Hg, and is 42% saturated with O2 (Hoversland e t Bagnara, nosine-induced catabolism of adenine ribonucleotides in adenosine al., 1975). The extent to which 2-3 DPG binds to oposdeaminase-inhibited human T lymphoblastoid cells. Proc. Natl. sum hemoglobin at venous oxygen saturation as well Acad. Sci. USA, 792673-2677. as the extent to which 2-3 DPG might inhibit opossum Becker, M.A., Raivio, K.O., and Seegemiller, J.E. (1979)Synthesis of phosphoribosyl pyrophosphate in mammalian cells. Adv. Enzymol., PRPP synthetase remains to be determined. 49:281-305. Under a n atmosphere of air or oxygen, and in con- Bethlenfalvay, N.C., Waterman, M.R., Lima, J.E., and Waldrup, T. trast to dATP and GTP, ATP is unstable in opossum (1983)Comparative aspects of methemoglobin formation and reduction in opossum (Didelphis virginiana) and human erythrocytes. red cells metabolizing glucose in vitro as the sole Comp. Biochem. Physiol., 75Ar635-639. source of energy irrespective of medium Pi concentraN.C., Lima, J.E., and Waldrup, T. (1984) Studies on tion (Tables 1 and 2, Fig. 2 upper). Interestingly and Bethlenfalvay, the energy metabolism of opossum (Didelphis virginiana) erythroperhaps significantly, the stability of adenine nuclecytes I. Utilization of carbohydrates and purine nucleosides. J. Cell. Physiol., 120:69-74. otides in opossum red cells under the two different gas phases utilized in this study parallels that exhibited by Bethlenfalvay, N.C., Lima, J.E., Chadwick, E., and Stewart, I. 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5 1 a
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