Exogenous Mg-ATP Induces a Large Inhibition of Pyruvate Kinase in Intact Rat Hepatocytes
2001; Elsevier BV; Volume: 276; Issue: 9 Linguagem: Inglês
10.1074/jbc.m004169200
ISSN1083-351X
AutoresCarole Ichaï, Mohamad Y. El-Mir, Véronique Nogueira, M. Piquet, Christiane Chauvin, Éric Fontaine, Xavier Leverve,
Tópico(s)Mitochondrial Function and Pathology
ResumoMg-ATP infusion in vivo has been reported to be beneficial both to organ function and survival rate in various models of shock. Moreover, a large variety of metabolic effects has been shown to occur in several tissues due to purinergic receptor activation. In the present work we studied the effects of exogenous Mg-ATP in rat liver cells perifused with dihydroxyacetone to investigate simultaneously gluconeogenetic and glycolytic pathways. We found a significant effect on oxidative phosphorylation as characterized by a decrease in oxygen consumption rate and in the cellular ATP-to-ADP ratio associated with an increase in lactate-to-pyruvate ratio. In addition, exogenous Mg-ATP induced rapid and reversible inhibition of both gluconeogenesis and glycolysis. The main effect on gluconeogenesis was located at the level of the fructose cycle, whereas the decrease in glycolysis was due to a strong inhibition of pyruvate kinase. Although pyruvate kinase inhibition induced by exogenous Mg-ATP was allosteric when assessed in vitro after enzyme extraction, we found a large decrease in the apparent maximal velocity when kinetics were assessed in vivo in intact perifused hepatocytes. This newly described short-term regulation of pyruvate kinase occurs only in the intact cell and may open new potentials for the pharmacological regulation of pyruvate kinase in vivo. Mg-ATP infusion in vivo has been reported to be beneficial both to organ function and survival rate in various models of shock. Moreover, a large variety of metabolic effects has been shown to occur in several tissues due to purinergic receptor activation. In the present work we studied the effects of exogenous Mg-ATP in rat liver cells perifused with dihydroxyacetone to investigate simultaneously gluconeogenetic and glycolytic pathways. We found a significant effect on oxidative phosphorylation as characterized by a decrease in oxygen consumption rate and in the cellular ATP-to-ADP ratio associated with an increase in lactate-to-pyruvate ratio. In addition, exogenous Mg-ATP induced rapid and reversible inhibition of both gluconeogenesis and glycolysis. The main effect on gluconeogenesis was located at the level of the fructose cycle, whereas the decrease in glycolysis was due to a strong inhibition of pyruvate kinase. Although pyruvate kinase inhibition induced by exogenous Mg-ATP was allosteric when assessed in vitro after enzyme extraction, we found a large decrease in the apparent maximal velocity when kinetics were assessed in vivo in intact perifused hepatocytes. This newly described short-term regulation of pyruvate kinase occurs only in the intact cell and may open new potentials for the pharmacological regulation of pyruvate kinase in vivo. dihydroxyacetone dihydroxyacetone phosphate phosphoenolpyruvate Numerous publications have emphasized the beneficial role of Mg-ATP infusion in various models of shock or severe trauma in animals. Dysfunction of heart (1Robinson D.A. Wang P. Chaudry I.H. J. Surg. Res. 1997; 69: 159-165Abstract Full Text PDF PubMed Scopus (11) Google Scholar), kidney (2Wang P. Zhou M. Rana M.W. Singh G. Ba Z.F. Ayala A. Chaudry I.H. Can. J. Physiol. Pharmacol. 1992; 70: 349-357Crossref PubMed Scopus (21) Google Scholar), muscle (3Chaudry I.H. Sayeed M.M. Baue A.E. Can. J. Physiol. Pharmacol. 1975; 53: 859-865Crossref PubMed Scopus (6) Google Scholar), endothelial (4Wang P. Ba Z.F. Chaudry I.H. Am. J. Physiol. 1995; 268: H1390-H1396PubMed Google Scholar), and immune cells (5Wang P. Ba Z.F. Chaudry I.H. Am. J. Physiol. 1992; 263: G895-G900PubMed Google Scholar, 6Ayala A. Chaudry I.H. Shock. 1996; 6 Suppl. 1: S27-S38Crossref PubMed Google Scholar) are all improved by this treatment, which significantly increases the survival rate. Because such a benefit was independent of hemodynamic status (4Wang P. Ba Z.F. Chaudry I.H. Am. J. Physiol. 1995; 268: H1390-H1396PubMed Google Scholar, 7Chaudry I.H. Ann. N. Y. Acad. Sci. 1990; 603 (; discussion 140–141): 130-140Crossref PubMed Scopus (42) Google Scholar), a metabolic mechanism was proposed (8Chaudry I.H. Am. J. Physiol. 1983; 245: R117-R134PubMed Google Scholar). Because the liver plays a central role in the metabolic response to such severe illnesses, it may represent a major target for Mg-ATP (9Hayashi H. Chaudry I.H. Clemens M.G. Hull M.J. Baue A.E. Am. J. Physiol. 1986; 250: R573-R579PubMed Google Scholar, 10Mahmoud M.S. Wang P. Hootman S.R. Reich S.S. Chaudry I.H. Am. J. Physiol. 1994; 266: R1804-R1809PubMed Google Scholar).It is well known that purinergic receptor activation is responsible for a large variety of metabolic effects (11–24). ATP or adenosine or several analogues of purinergic receptors have been noted to affect liver glucose metabolism: i.e. stimulation of glycogenolysis (12Bartrons R. van Schaftingen E. Hers H.G. Biochem. J. 1984; 218: 157-163Crossref PubMed Scopus (35) Google Scholar, 13Buxton D.B. Robertson S.M. Olson M.S. Biochem. J. 1986; 237: 773-780Crossref PubMed Scopus (62) Google Scholar), increase (19Koike M. Kashiwagura T. Takegushi N. Biochem. J. 1992; 283: 265-272Crossref PubMed Scopus (33) Google Scholar, 25Mahmoud M.S. Wang P. Chaudry I.H. Biochim. Biophys. Acta. 1997; 1336: 546-549Google Scholar, 26Oetjen E. Schweickhardt C. Unthan Fechner K. Probst I. Biochem. J. 1990; 271: 337-344Crossref PubMed Scopus (29) Google Scholar) or decrease (11Asensi M. Lopez-Rodas A. Sastre J. Vina J. Estrela J.M. Am. J. Physiol. 1991; 261: R1522-R1526PubMed Google Scholar) of gluconeogenesis, and decrease of glycolysis (25Mahmoud M.S. Wang P. Chaudry I.H. Biochim. Biophys. Acta. 1997; 1336: 546-549Google Scholar). These effects have been related to a cAMP-dependent mechanism (12Bartrons R. van Schaftingen E. Hers H.G. Biochem. J. 1984; 218: 157-163Crossref PubMed Scopus (35) Google Scholar), a cAMP-independent inositol 3-phosphate/calcium-mediated signaling (13Buxton D.B. Robertson S.M. Olson M.S. Biochem. J. 1986; 237: 773-780Crossref PubMed Scopus (62) Google Scholar, 14Charest R. Blackmore P.F. Exton J.H. J. Biol. Chem. 1985; 260: 15789-15794Abstract Full Text PDF PubMed Google Scholar, 16Häussinger D. Stehle T. Gerok W. Eur. J. Biochem. 1987; 167: 65-71Crossref PubMed Scopus (96) Google Scholar, 18Keppens S. Gen. Pharmacol. 1993; 24: 283-289Crossref PubMed Scopus (36) Google Scholar, 21Lee J.W. Filkins J.P. Circ. Shock. 1987; 22: 205-219PubMed Google Scholar, 23Okajima F. Tokumitsu Y. Londo Y. Ui M. J. Biol. Chem. 1987; 262: 13483-13490Abstract Full Text PDF PubMed Google Scholar), a phospholipase C activation (18Keppens S. Gen. Pharmacol. 1993; 24: 283-289Crossref PubMed Scopus (36) Google Scholar), or a transcriptional effect (25Mahmoud M.S. Wang P. Chaudry I.H. Biochim. Biophys. Acta. 1997; 1336: 546-549Google Scholar). It must be noted, however, that if some effects are shared among the adenine nucleotide family, others appear to be specific (12Bartrons R. van Schaftingen E. Hers H.G. Biochem. J. 1984; 218: 157-163Crossref PubMed Scopus (35) Google Scholar, 14Charest R. Blackmore P.F. Exton J.H. J. Biol. Chem. 1985; 260: 15789-15794Abstract Full Text PDF PubMed Google Scholar, 18Keppens S. Gen. Pharmacol. 1993; 24: 283-289Crossref PubMed Scopus (36) Google Scholar), suggesting that different signaling pathways may be involved.Because the beneficial effects were observed in vivo with Mg-ATP but not with adenosine (8Chaudry I.H. Am. J. Physiol. 1983; 245: R117-R134PubMed Google Scholar, 27Sumpio B.E. Hull M.J. Baue A.E. Chaudry I.H. Am. J. Physiol. 1987; 252: R388-R393PubMed Google Scholar), we investigated the metabolic effects of exogenous Mg-ATP in rat liver cells perifused with DHA.1 Besides a significant effect on oxidative phosphorylation and on gluconeogenesis, we found that Mg-ATP was responsible for a large inhibition of pyruvate kinase. Interestingly, although pyruvate kinase (EC 2.7.1.40) inhibition induced by exogenous Mg-ATP was allosteric when assessed in vitro after enzyme extraction, we found a large decrease in the apparent maximal velocity when kinetics were assessed in vivo in intact perifused hepatocytes.MATERIALS AND METHODSMale Wistar rats (200–250 g), fasted for 24 h, were anesthetized intraperitoneally with sodium thiopental (125 mg/kg). Hepatocytes were isolated by the method of Berry and Friend (28Berry M.N. Friend D.S. J. Cell Biol. 1969; 43: 506-519Crossref PubMed Scopus (3607) Google Scholar) as modified by Groen et al. (29Groen A.K. Sips H.J. Vervoorn R.C. Tager J.M. Eur. J. Biochem. 1982; 122: 87-93Crossref PubMed Scopus (129) Google Scholar).Liver cells (200 mg of dry cells in 15 ml) were perifused by the method of van der Meer and Tager (30van der Meer R. Tager J.M. FEBS Lett. 1976; 67: 36-40Crossref PubMed Scopus (37) Google Scholar) as modified by Groen et al.(29Groen A.K. Sips H.J. Vervoorn R.C. Tager J.M. Eur. J. Biochem. 1982; 122: 87-93Crossref PubMed Scopus (129) Google Scholar, 31Groen A.K. Sips H.J. Vervoorn R.C. van der Meer R. Tager J.M. J. Biol. Chem. 1983; 258: 14346-14353Abstract Full Text PDF PubMed Google Scholar). Hepatocytes were perifused at 37 °C at a flow rate of 5 ml·min−1 with Krebs bicarbonate buffer (pH 7.4) continuously saturated with O2/CO2 (19:1) and containing calcium (1.3 mm) (32Argaud D. Roth H. Wiernsperger N. Leverve X.M. Eur. J. Biochem. 1993; 213: 1341-1348Crossref PubMed Scopus (140) Google Scholar, 33Leclercq P. Filippi C. Sibille B. Hamant S. Keriel C. Leverve X.M. Biochem. J. 1997; 325: 519-525Crossref PubMed Scopus (12) Google Scholar, 34Leverve X.M. Fontaine E. Putod-Paramelle F. Rigoulet M. Eur. J. Biochem. 1994; 224: 967-974Crossref PubMed Scopus (18) Google Scholar, 35Piquet M.A. Fontaine E. Sibille B. Filippi C. Keriel C. Leverve X.M. Biochem. J. 1996; 317: 667-674Crossref PubMed Scopus (24) Google Scholar).The time course of the effect of exogenous nucleotide addition was studied in hepatocytes perifused in the presence of a constant DHA concentration (9.6 mm). After an initial period of 45 min, a first steady state was reached and liver cells were exposed to a mixture of 100 μm MgCl2 and 100 μm ATP (Mg-ATP) for 30 min. After this period, Mg-ATP infusion was stopped, leading to a rapid decrease in ATP in the perifusate (3 min), and cells were further perifused for another 40 min in the absence of Mg-ATP. Perifusate samples were taken at different time intervals as indicated.To study the metabolic effect of exogenous ATP, perifused liver cells were titrated with DHA and in the presence or absence of exogenous Mg-ATP (100 μm). After an initial steady state had been reached (45 min) in the absence of DHA, seven successive steady states were obtained in the presence of increasing DHA concentrations (0.15, 0.30, 0.60, 1.20, 2.40, 4.80, and 9.60 mm) as indicated. Each of the successive steady states was obtained after 20 min, then both perifusate and cell samples were taken for subsequent analysis. The steady state was always confirmed by stable values of glucose, lactate, and pyruvate in three successive perifusate samples taken at 1-min intervals. Because these three values were always very close they have been averaged. Proteins in the perifusate were denatured by heating the samples (80 °C for 10 min) before centrifugation (36Leverve X.M. Verhoeven A.J. Groen A.K. Meijer A.J. Tager J.M. Eur. J. Biochem. 1986; 155: 551-556Crossref PubMed Scopus (39) Google Scholar). Glucose, lactate, and pyruvate were measured in the perifusate and DHAP; glucose 6-phosphate; fructose 6-phosphate; 3-phosphoglycerate; and PEP were measured in the cellular fraction as described previously (31Groen A.K. Sips H.J. Vervoorn R.C. van der Meer R. Tager J.M. J. Biol. Chem. 1983; 258: 14346-14353Abstract Full Text PDF PubMed Google Scholar, 32Argaud D. Roth H. Wiernsperger N. Leverve X.M. Eur. J. Biochem. 1993; 213: 1341-1348Crossref PubMed Scopus (140) Google Scholar, 33Leclercq P. Filippi C. Sibille B. Hamant S. Keriel C. Leverve X.M. Biochem. J. 1997; 325: 519-525Crossref PubMed Scopus (12) Google Scholar, 34Leverve X.M. Fontaine E. Putod-Paramelle F. Rigoulet M. Eur. J. Biochem. 1994; 224: 967-974Crossref PubMed Scopus (18) Google Scholar, 35Piquet M.A. Fontaine E. Sibille B. Filippi C. Keriel C. Leverve X.M. Biochem. J. 1996; 317: 667-674Crossref PubMed Scopus (24) Google Scholar). The net fluxes (micromoles/min/g of dry cells) of gluconeogenesis (Jglucose), glycolysis (Jlactate + pyruvate), and DHA metabolism (JDHA), were calculated from the total cell content of the perifusion chamber, the perifusate flow rate, and the concentration of glucose, lactate, and pyruvate in the perifusate. All determinations were made by enzymatic procedures (37Bergmeyer, H. U. (ed) (1974) Methods in Enzymatic Analysis, Vol. IV, pp. 1127-1624, Academic Press, New YorkGoogle Scholar) with either spectrophotometric or fluorometric determination of NADH.The effects of addition of exogenous Mg-ATP on cytosolic and mitochondrial adenine nucleotide content were studied in similar steady-state conditions as described above for the time course effect of Mg-ATP. After a first steady state in the presence of DHA (9.6 mm), Mg-ATP was added (100 μm) and, at 5, 10, 15, 20, and 30 min, cells were taken from the chamber for intracellular and mitochondrial nucleotide determinations. Experiments were performed with or without Mg-ATP. Samples of cell suspension were quickly removed from the chamber, and cellular content was separated from the extracellular medium by centrifugation of the cell suspension through a layer of silicone oil as described previously (21Lee J.W. Filkins J.P. Circ. Shock. 1987; 22: 205-219PubMed Google Scholar). The mitochondrial fraction was obtained by liver cell fractionation with digitonin as described previously (38Zuurendonk P.F. Tager J.M. Biochim. Biophys. Acta. 1974; 333: 393-399Crossref PubMed Scopus (262) Google Scholar). Cytosolic adenine nucleotide concentrations were calculated by subtraction of the mitochondrial from the total intracellular value. ATP, ADP, and AMP were determined by high pressure liquid chromatography (32Argaud D. Roth H. Wiernsperger N. Leverve X.M. Eur. J. Biochem. 1993; 213: 1341-1348Crossref PubMed Scopus (140) Google Scholar).After centrifugation of the cell suspension, pyruvate kinase activity was assessed on cell pellets resuspended in 1.5 ml of a buffer containing: 20 mm potassium phosphate (pH 7.4); 0.25m sucrose; 1 mm EDTA; 1 mmdithiothreitol. After homogenization for 1 min with an Ultraturax, this homogenate was centrifuged at 30,000 × g for 15 min (Beckman J 21). Pyruvate kinase activity in the supernatant was determined in 2 ml of a buffer containing 50 mm Tris-HCl (pH 7.4), 100 mm KCl, 5 mm MgCl2, and 10 μl of the supernatant. To obtain partially purified enzyme (L form), 0.4 ml of the supernatant obtained after homogenization was washed with 0.3 ml of 100% (NH4)2SO4 (final concentration 40%) and centrifuged at 30,000 × g for 15 min; the pellets were suspended in a medium (2 ml) containing 20 mmpotassium phosphate (pH 7.4), 30% glycerol, 1 mm EDTA, 1 mm dithiothreitol, 50 mm NaF, and pyruvate kinase activity was measured in a buffer containing 50 mmTris-HCl (pH 7.4), 20 mm KCl, 5 mmMgCl2 (39Llorente P. Marco R. Sols A. Eur. J. Biochem. 1970; 13: 45-54Crossref PubMed Scopus (145) Google Scholar, 40Riou J.P. Claus T.H. Pilkis S.J. J. Biol. Chem. 1978; 253: 656-659Abstract Full Text PDF PubMed Google Scholar). Enzyme activity was expressed as the ratio of activity measured at 0.4 mm PEP to that at 4 mm PEP (v/Vmax), because this expression of the results has been shown to accurately reflect the phosphorylated state of the enzyme (41Claus T.H. El Maghrabi M.R. Pilkis S.J. J. Biol. Chem. 1979; 254: 7855-7864Abstract Full Text PDF PubMed Google Scholar). The maximal velocity (Vmax, μmol/min/mg of proteins) was determined at 4 mm PEP. The velocity obtained at 4 mm PEP was very close to that obtained from a kinetic analysis of the saturation curve (data not shown). Protein content was determined in the supernatant by the Biuret method after homogenization. Due to the limited volume of samples and to the low protein content of the samples after partial purification by ammonium sulfate denaturation, we have determined in separate experiments the ratio of the protein content before and after purification in similar conditions. This ratio was 4.9 ± 0.2 (n = 4), and this value was used as the correcting factor for the protein content of the purified samples.The oxygen consumption rate in hepatocytes was determined after incubation in closed vials with Krebs-Ringer bicarbonate and DHA (20 mm) with or without 100 μm Mg-ATP. After 15 min, cells samples were removed from the vials and placed in an oxygraph vessel equipped with a Clark electrode at 37 °C for oxygen determination. Respiratory rate was expressed as micromoles of O2/min/g of dry cells.Results are expressed as means ± S.E.; Mg-ATP effect was assessed either by a one-way analysis of variance (StatView, Abacus Concepts, Inc., Berkley, CA) or by Student's t test.DISCUSSIONThe clear inhibitory effect of exogenous Mg-ATP addition on DHA metabolism in intact hepatocytes reported in this work consists of a decrease of gluconeogenesis associated with a potent inhibition of lactate-plus-pyruvate production. In addition, there seems to be a new mechanism for pyruvate kinase regulation after exogenous Mg-ATP addition occurring only in vivo in intact cells.By investigating DHA metabolism with successive steady states and measuring glucose and lactate-plus-pyruvate fluxes simultaneously with cellular intermediate concentrations, perifusion of liver cells is a suitable tool to determine the step(s) affected by exogenous Mg-ATP. Glucose production accurately reflects the rate of gluconeogenesis, because glycogen synthesis in these conditions is negligible as compared with the flux of glucose production. Moreover, glycogen synthesis is inhibited by exogenous ATP (18Keppens S. Gen. Pharmacol. 1993; 24: 283-289Crossref PubMed Scopus (36) Google Scholar). The flux of lactate-plus-pyruvate truly reflects the pyruvate kinase flux (i.e. glycolysis) only in the absence of significant pyruvate oxidation or transamination. Pyruvate oxidation or transamination may lead to an underestimate of glycolytic flux, but this is probably limited because of the very low pyruvate concentration due to the continuous rinsing of the perifusate.Moreover, if decrease of lactate-plus-pyruvate production by Mg-ATP had been the consequence of an increased pyruvate oxidation or transamination, PEP should not accumulate, in contrast to our findings (Fig. 5).Considering the pathway between DHAP and glucose production, it appears that exogenous Mg-ATP is responsible for a minor effect at the level of glucose/glucose 6-phosphate cycle (Fig. 4 A), whereas the main effect is located at the fructose 6-phosphate/fructose 1,6-bisphophate step (Fig. 4, C and D). A cAMP-dependent phosphorylation has been reported to inhibit 6-phosphofructo-1-kinase (47Castano L.G. Nieto A. Feliu J.E. J. Biol. Chem. 1979; 254: 5576-5579Abstract Full Text PDF PubMed Google Scholar), but this enzyme is not very active in hepatocytes from fasted rats (48Pilkis S.J. Granner D.K. Annu. Rev. Physiol. 1992; 54: 885-909Crossref PubMed Scopus (698) Google Scholar). On the other hand, fructose-1,6-bisphosphatase can be activated by a c-AMP-dependent phosphorylation (49Claus T.H. Schlumpf J.R. El-Maghrabi L.R. McGrane M. Pilkis S.J. Biochem. Biophys. Res. Commun. 1981; 100: 716-723Crossref PubMed Scopus (24) Google Scholar). Nevertheless, such effects of cAMP-related phosphorylation on 6-phosphofructo-1-kinase or on fructose-1,6-bisphosphatase seems to have a minor functional impact (48Pilkis S.J. Granner D.K. Annu. Rev. Physiol. 1992; 54: 885-909Crossref PubMed Scopus (698) Google Scholar), and it is believed that fructose 2,6-bisphosphate is the main regulator via changes in the bifunctional enzyme: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (50Hers H.G. van Schaftingen E. Biochem. J. 1982; 206: 1-12Crossref PubMed Scopus (308) Google Scholar). Bartronset al. (12Bartrons R. van Schaftingen E. Hers H.G. Biochem. J. 1984; 218: 157-163Crossref PubMed Scopus (35) Google Scholar) have reported that adenosine as well as the adenosine analogue 2-chloroadenosine are responsible for a decrease in fructose 2,6-bisphosphate due to an activation of fructose-2,6-bisphosphatase by adenylate cyclase activation and cAMP rise. Both adenosine and 2-chloroadenosine increase cAMP and decrease fructose 2,6-bisphosphate, but adenosine is responsible for a decreased rate of gluconeogenesis, whereas 2-chloroadenosine increases it (12Bartrons R. van Schaftingen E. Hers H.G. Biochem. J. 1984; 218: 157-163Crossref PubMed Scopus (35) Google Scholar). Adenosine is responsible for an inhibition of gluconeogenesis (11Asensi M. Lopez-Rodas A. Sastre J. Vina J. Estrela J.M. Am. J. Physiol. 1991; 261: R1522-R1526PubMed Google Scholar, 12Bartrons R. van Schaftingen E. Hers H.G. Biochem. J. 1984; 218: 157-163Crossref PubMed Scopus (35) Google Scholar,20Lavoinne A. Claeyssens S. Chedeville A. Eur. J. Biochem. 1990; 187: 403-407Crossref PubMed Scopus (10) Google Scholar, 22Lund P. Cornell N.W. Krebs H.A. Biochem. J. 1975; 152: 593-599Crossref PubMed Scopus (102) Google Scholar) at the level of fructose cycling. Hence, our results might support the view that the effect of exogenous Mg-ATP on gluconeogenesis from DHA are similar to, if not mediated by, the effect of adenosine as was suggested by Asensi et al. (11Asensi M. Lopez-Rodas A. Sastre J. Vina J. Estrela J.M. Am. J. Physiol. 1991; 261: R1522-R1526PubMed Google Scholar).The most striking finding of this work is a significant decrease of lactate-plus-pyruvate production resulting from a potent inhibition of pyruvate kinase. Cytosolic adenine nucleotides are potent regulators of glycolysis, but given the decrease in cytosolic ATP-to-ADP ratio following exogenous Mg-ATP, an activation of glycolysis would actually have been predicted (32Argaud D. Roth H. Wiernsperger N. Leverve X.M. Eur. J. Biochem. 1993; 213: 1341-1348Crossref PubMed Scopus (140) Google Scholar, 34Leverve X.M. Fontaine E. Putod-Paramelle F. Rigoulet M. Eur. J. Biochem. 1994; 224: 967-974Crossref PubMed Scopus (18) Google Scholar, 35Piquet M.A. Fontaine E. Sibille B. Filippi C. Keriel C. Leverve X.M. Biochem. J. 1996; 317: 667-674Crossref PubMed Scopus (24) Google Scholar). The increased cytosolic NADH/NAD ratio as shown by the increased lactate-to-pyruvate ratio (Fig. 7) could theoretically account, at least partly, for such an inhibition of lactate-plus-pyruvate production. But in the presence of Mg-ATP, the relationship between PEP and lactate-plus-pyruvate production reached a plateau indicating a clear saturation of the substrate PEP (Fig.5 A). Hence, the Mg-ATP inhibitory effect of lactate-plus-pyruvate production cannot be explained by a decrease in PEP concentration. Thus it can be concluded that acute administration of exogenous Mg-ATP strongly decreases the apparentVmax of pyruvate kinase when determined in vivo in intact cells. It has already been reported that Mg-ATP was responsible for a decrease in pyruvate kinase activity in vivo, but this was observed 4 h after exogenous Mg-ATP administration and was related to a transcriptional effect (25Mahmoud M.S. Wang P. Chaudry I.H. Biochim. Biophys. Acta. 1997; 1336: 546-549Google Scholar). In our experiments the effect was observed after a few minutes, indicating that another mechanism must be involved.The second striking finding of the present work is related to the fact that the change in apparent maximal velocity of pyruvate kinase was found only when determined in vivo but not in vitro after enzyme extraction and partial purification. This indicates that the amount of enzyme was not modified by such short-term effect, which is not surprising. Actually, we found an allosteric inhibition of this enzyme when assessed in vitro. An acute inhibitory effect of adenosine on pyruvate kinase, due to a cAMP-related phosphorylation, was already described by Bartronset al. (12Bartrons R. van Schaftingen E. Hers H.G. Biochem. J. 1984; 218: 157-163Crossref PubMed Scopus (35) Google Scholar). However, in the present work the allosteric inhibition was not apparent when enzyme activity was determined in intact cells. This finding indicates that another mechanism controls this step in vivo. Because this effect disappears after enzyme extraction, an intracellular metabolite could be involved. Indeed, there are several cellular metabolites, such as fructose 1,6-bisphosphate, alanine, ATP, and ADP known to regulate pyruvate kinase, but these effectors act via an allosteric mechanism (48Pilkis S.J. Granner D.K. Annu. Rev. Physiol. 1992; 54: 885-909Crossref PubMed Scopus (698) Google Scholar) and do not affect the Vmax in contrast to the present results. In the presence of Mg-ATP, the hypothetical inhibitor should be noncompetitive with PEP and does not bind abnormally tightly to the enzyme, because it is lost after extraction. Such a hypothesis opens the possibility of a new regulatory mechanism of pyruvate kinase, which can be detected only in vivo in intact cells.The question of the nature of the cellular effects of exogenous Mg-ATP is obviously complex. Although some effects of Mg-ATP seem to be similar to those of adenosine or adenosine agonists, others are different. Indeed, besides cAMP-related phosphorylation, inositol 1,4,5-trisphosphate, calcium, and phospholipase C signaling, other receptors and signaling pathways are also probably involved, resulting in a very complicated and subtle regulation leading to multiple metabolic responses as described in this paper (including changes in oxidative phosphorylation, redox state, phosphate potential, glycolysis, gluconeogenesis, etc.).Given the clear beneficial effect of exogenous Mg-ATP administration demonstrated by Chaudry (7Chaudry I.H. Ann. N. Y. Acad. Sci. 1990; 603 (; discussion 140–141): 130-140Crossref PubMed Scopus (42) Google Scholar, 8Chaudry I.H. Am. J. Physiol. 1983; 245: R117-R134PubMed Google Scholar) in several animal models of shock on survival and organ or cellular functions, a possible link with the present finding can be hypothesized. It has been recently demonstrated that isolated rat hepatocytes can signal to other hepatocytes by the release of ATP, suggesting a novel paracrine signaling pathway exists (51Schlosser S.F. Burgstahler A.D. Nathanson M.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9948-9953Crossref PubMed Scopus (192) Google Scholar). This finding favors a physiological role of exogenous ATP, which may trigger a protective effect. Numerous publications have emphasized the beneficial role of Mg-ATP infusion in various models of shock or severe trauma in animals. Dysfunction of heart (1Robinson D.A. Wang P. Chaudry I.H. J. Surg. Res. 1997; 69: 159-165Abstract Full Text PDF PubMed Scopus (11) Google Scholar), kidney (2Wang P. Zhou M. Rana M.W. Singh G. Ba Z.F. Ayala A. Chaudry I.H. Can. J. Physiol. Pharmacol. 1992; 70: 349-357Crossref PubMed Scopus (21) Google Scholar), muscle (3Chaudry I.H. Sayeed M.M. Baue A.E. Can. J. Physiol. Pharmacol. 1975; 53: 859-865Crossref PubMed Scopus (6) Google Scholar), endothelial (4Wang P. Ba Z.F. Chaudry I.H. Am. J. Physiol. 1995; 268: H1390-H1396PubMed Google Scholar), and immune cells (5Wang P. Ba Z.F. Chaudry I.H. Am. J. Physiol. 1992; 263: G895-G900PubMed Google Scholar, 6Ayala A. Chaudry I.H. Shock. 1996; 6 Suppl. 1: S27-S38Crossref PubMed Google Scholar) are all improved by this treatment, which significantly increases the survival rate. Because such a benefit was independent of hemodynamic status (4Wang P. Ba Z.F. Chaudry I.H. Am. J. Physiol. 1995; 268: H1390-H1396PubMed Google Scholar, 7Chaudry I.H. Ann. N. Y. Acad. Sci. 1990; 603 (; discussion 140–141): 130-140Crossref PubMed Scopus (42) Google Scholar), a metabolic mechanism was proposed (8Chaudry I.H. Am. J. Physiol. 1983; 245: R117-R134PubMed Google Scholar). Because the liver plays a central role in the metabolic response to such severe illnesses, it may represent a major target for Mg-ATP (9Hayashi H. Chaudry I.H. Clemens M.G. Hull M.J. Baue A.E. Am. J. Physiol. 1986; 250: R573-R579PubMed Google Scholar, 10Mahmoud M.S. Wang P. Hootman S.R. Reich S.S. Chaudry I.H. Am. J. Physiol. 1994; 266: R1804-R1809PubMed Google Scholar). It is well known that purinergic receptor activation is responsible for a large variety of metabolic effects (11–24). ATP or adenosine or several analogues of purinergic receptors have been noted to affect liver glucose metabolism: i.e. stimulation of glycogenolysis (12Bartrons R. van Schaftingen E. Hers H.G. Biochem. J. 1984; 218: 157-163Crossref PubMed Scopus (35) Google Scholar, 13Buxton D.B. Robertson S.M. Olson M.S. Biochem. J. 1986; 237: 773-780Crossref PubMed Scopus (62) Google Scholar), increase (19Koike M. Kashiwagura T. Takegushi N. Biochem. J. 1992; 283: 265-272Crossref PubMed Scopus (33) Google Scholar, 25Mahmoud M.S. Wang P. Chaudry I.H. Biochim. Biophys. Acta. 1997; 1336: 546-549Google Scholar, 26Oetjen E. Schweickhardt C. Unthan Fechner K. 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