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Metabolic regulation of ATP breakdown and of adenosine production in rat brain extracts
The International Journal of Biochemistry & Cell Biology 36 (2004) 2214–2225
Metabolic regulation of ATP breakdown and of adenosine production in rat brain extracts Catia Barsotti∗ , Piero L. Ipata Department of Physiology and Biochemistry, University of Pisa, Via San Zeno 51, 56127 Pisa, Italy Received 18 February 2004; received in revised form 28 April 2004; accepted 28 April 2004
Abstract ATP concentration is dramatically affected in ischemic injury. From previous studies on ATP mediated purine and pyrimidine salvage in CNS, we observed that when “post-mitochondrial” extracts of rat brain were incubated with ATP at 3.6 mM, a normoxic concentration, formation of IMP always preceded that of adenosine, a well known neuroactive nucleoside and a homeostatic cellular modulator. This observation prompted us to undertake a study aimed at assessing the precise pathways and kinetics of ATP breakdown, a process considered to be the major source of adenosine in rat brain. The results obtained using post-mitochondrial extracts strongly suggest that the breakdown of intracellular ATP at normoxic concentration follows almost exclusively the pathway ATPADPAMP → IMP → inosinehypoxanthine, with little, if any, intracellular adenosine production. At low ischemic concentration, intracellular ATP breakdown follows the pathway ATPADPAMP → adenosine → inosinehypoxanthine with little IMP formation. At the same time, extracellular ATP, whose concentration is known to be enhanced during ischemia, is actively broken down to adenosine through the pathway ATP → ADP → AMP → adenosine, catalysed by the well characterized ecto-enzyme cascade system. Moreover, we show that during intracellular GTP catabolism, xanthosine, in addition to guanosine, is generated through the so called “ribose 1-phosphate recycling for nucleoside interconversion”. These results considerably extend our knowledge on the long debated question of the extra or intracellular origin of adenosine in CNS, suggesting that at least in normoxic conditions, intracellular adenosine is of extracellular origin. © 2004 Elsevier Ltd. All rights reserved. Keywords: ATP breakdown; Adenosine metabolism; GTP breakdown; Guanosine; Xanthosine; Rat brain
1. Introduction Adenosine is an important neuroactive nucleoside and a homeostatic cellular modulator (Cunha, Abbreviations: Rib 1-P, ribose 1-phosphate; cN-II, cytosolic IMP-GMP specific 5 -nucleotidase; PEI, polyethyleneimine; Ado, adenosine; Ino, inosine; Hyp, hypoxanthine; Guo, guanosine; Gua, guanine; Xn, xanthine; Xao, xanthosine ∗ Corresponding author. Tel.: +39 050 2213181; fax: +39 050 2213170. E-mail address: [email protected] (C. Barsotti).
2001; Latini & Pedata, 2001). It is mainly produced by ATP breakdown, as it occurs in anoxic/ischemic conditions and to a lesser extent during strenuous physical exercise (Baldwin, Snow, Carey, & Febbraio, 1999; Broberg & Sahlin, 1989; Dagher, 2000; Phillis, O’Regan, Estevez, Song, & VanderHeide, 1996). The kinetics of ATP breakdown have been extensively investigated in whole organs and cell cultures, mainly by following the time course of the production of inosine and hypoxanthine (Dagher, 2000; Freedholm, Dunwiddie, Bergman, & Lindström, 1984; Kobayashi,
1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.04.015
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Fig. 1. Pathways of intracellular ATP catabolism in rat brain. The enzymes participating in these pathways are: (1) adenylate kinase; (2) adenylate deaminase; (3) cytosolic IMP-GMP specific 5 nucleotidase (cN-II); (4) purine nucleoside phosphorylase; (5) cytosolic AMP specific 5 nucleotidase; (6) adenosine deaminase. Enzymes 2 and 3 are activated by millimolar physiological ATP concentration (Chapman & Atkinson, 1973; Gysbers & Rathbone, 1996; Pesi et al., 1994, 1996; Schulz & Lowenstein, 1978; Setlow & Lowenstein, 1967; Van den Berghe, Van Pottelsberghe, & Hers, 1977). Enzyme 5 is activated by ADP (Skladanowski & Newby, 1990).
Yamada, & Okada, 1998; Ljunggren, Ratcheson, & Siesjö, 1974; Phillis et al., 1996; Pulsinelli & Duffy, 1983; Siesjö & Ljunggren, 1973), which are considered catabolites of adenosine. However, we emphasize that ATP breakdown may follow two distinct routes (Fig. 1). In the first route, ATP ADP AMP interconversion, catalysed by adenylate kinase, is followed by AMP deamination and IMP dephosphorylation to give inosine. In the second route, AMP is dephosphorylated to adenosine and then adenosine is deaminated to inosine. The two catabolic routes are referred to as the “IMP pathway” and the “adenosine pathway”, respectively, throughout this paper. In both of them, phosphorolysis of inosine to hypoxan-
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thine and ribose 1-phosphate (Rib 1-P) is considered the final step of ATP breakdown. However, adenosine is formed by only one of the two pathways. It is therefore important to establish the relative contribution of the two intracellular pathways of ATP breakdown. ATP is released in the extracellular space during ischemia (Sawynok & Sweeney, 1989; White & Hoehn, 1991; White & MacDonald, 1990). A set of membrane bound ecto-nucleotidases collectively functioning as an enzyme cascade, is responsible for extracellular ATP breakdown where adenosine is the major catabolite (Latini & Pedata, 2001; White & Hoehn, 1991; White & MacDonald, 1990; Zimmermann, 1996). Determining the relative contribution of the intracellular and extracellular pathways for ATP degradation is important in whole rat brain, because both intra and extracellular ATP are thought to be the source of adenosine (see Latini & Pedata, 2001 for review). In this paper, we have investigated both routes of ATP metabolism in whole rat brain, by comparing the kinetics of ATP breakdown and the time course of all the possible metabolites formed in post-mitochondrial extracts and membrane preparations of whole rat brain. The data show that the metabolic pathway of intracellular ATP breakdown is concentration dependent. Thus, at 3.6 mM (a normoxic concentration) ATP breakdown follows the IMP pathway with little, if any, adenosine production. Any intracellular adenosine must therefore be of extracellular origin. Only at ischemic concentration (less then 2 mM) does ATP breakdown follow the adenosine pathway. The IMP pathway does not appear to contribute to extracellular ATP degradation.
2. Materials and methods 2.1. Materials [8-14 C]-ATP (51 mCi/mmol) was obtained from ICN. [8-14 C]-GTP (60 mCi/mmol) was from Moravek Biochemicals. [8-14 C]-Adenosine (58 mCi/mmol), [8-14 C]-AMP (53 mCi/mmol), U73122, dithiothreitol, sucrose, nucleobases, nucleosides, and nucleotides were from Sigma Chemical Co. Hi Safe II Scintillation liquid was purchased from Wallac. Polyethyleneimine (PEI)-cellulose precoated
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thin-layer plastic sheets (0.1 mm thick) were purchased from Merck and prewashed once with 10% NaCl and three times with deionized water before use. All other chemicals were of reagent grade. Three-month-old male Sprague–Dawley rats (250 g) were killed according to the Guiding Principles in the Care and Use of Animals (DHWEW publication, NIH 86-23). The brain was removed and kept frozen at −80 ◦ C until needed. Storage times did not exceeded 3 months. 2.2. Preparation of rat brain extracts Whole rat brain was washed with 10 mM Tris–HCl buffer, pH 7.4, with 0.32 M sucrose and 44 M U73122, an inhibitor of phospholipase C used to prevent the displacement of ecto-5 -nucleotidase from membrane (Mackiewicz, Geiger, & Pack, 2000; Smallrige, Kiang, Gist, Fein, & Galloway, 1992). Homogenization (six up-and-down passes) was performed using the same medium in a glass Potter-Elvehjem homogenizer fitted with a Teflon pestle. The homogenate was centrifuged at 4 ◦ C at 39,000 × g for 1 h. The supernatant fluid obtained was dialyzed overnight at 4 ◦ C in dialysis bags against 10 mM Tris–HCl buffer, pH 7.4, supplemented with 1 mM dithiothreitol and is referred to as post-mitochondrial extract. This preparation proved to be extremely versatile in studies on ATP-mediated purine and pyrimidine salvage (Barsotti, Tozzi, & Ipata, 2002) and on purine nucleoside cycle (Barsotti, Pesi, Felice, & Ipata, 2003). Alternatively, the homogenate was fractionated to isolate membranes according to Delaney, Blackburn, and Geiger (1997) with minor modifications. The homogenate was centrifuged at 1000 × g for 10 min at 4 ◦ C, after which the supernatant was centrifuged at 11,000 × g for 20 min at 4 ◦ C. The supernatant was then collected and centrifuged at 105,000 × g for 1 h at 4 ◦ C. The pellet was collected and stirred for 90 min at 4 ◦ C in 10 mM Tris–HCl buffer, pH 7.4, containing Triton X-100 (2% (v/v)). The supernatant fluid, collected after centrifugation for 1 h at 105,000 × g at 4 ◦ C, is referred to as membrane preparation. Protein concentration of rat brain post-mitochondrial extract and membrane preparation were determined by Coomassie blue-binding assay, using bovine serum albumin as standard (Bradford, 1976).
2.3. Incubation with post-mitochondrial extracts The standard reaction mixtures contained 5 mM Tris–HCl buffer, pH 7.4, [8-14 C]-ATP (2500 dpm/ nmol), or [8-14 C]-GTP (15,000 dpm/nmol) at the concentrations indicated in the figure legends, 8.3 mM MgCl2 , 5 mM KH2 PO4 and brain post-mitochondrial extract, 1.15 mg of protein/ml. Modifications of the standard incubation mixture are indicated in the figure legends. The reaction was started by addition of the extract. At different time intervals the reaction was stopped by rapidly drying portions of 10 l of the incubation mixture on PEI-cellulose precoated thin-layer plastic sheets and a chromatogram was developed in n-butanol/glacial acetic acid/H2 O (4:2:1, v/v) (Mascia, Cappiello, Cherri, & Ipata, 2000) to separate adenosine, inosine and hypoxanthine, or with n-butanol/glacial acetic acid/aceton/NH3 / H2 O (35:15:25:2.3:22.7, v/v) to separate IMP, AMP, ADP, and ATP, or with n-propanol/NH3 /trichloroacetic acid (100%)/H2 O (75:0.7:5:20, v/v) (Cerletti, Ipata, & Tancredi, 1959; Giorgelli et al., 1997) to separate guanine, guanosine, xanthine, xanthosine, GMP, and GDP + GTP. In all separations, appropriate standards were used and detected as ultraviolet absorbing areas which were excised and counted for radioactivity with 8 ml of scintillation liquid. 2.4. Incubation with membrane preparations The standard reaction mixtures contained 5 mM Tris–HCl buffer, pH 7.4, [8-14 C]-ATP (10,000 dpm/ nmol), or [8-14 C]-GTP (10,000 dpm/nmol) at the concentrations indicated in the figure legends, 1.5 mM MgCl2 and brain membrane preparations, 0.5 mg of protein/ml. Modifications of the standard incubation mixture are indicated in the figure legends. The reactions were started by addition of membrane preparation. At different time intervals, the reactions were stopped and chromatographic analyses were performed as described above. 3. Results and discussion 3.1. ATP breakdown and adenosine metabolism in post-mitochondrial extracts of rat brain The results of our studies are illustrated in a series of figures. Fig. 2 shows the time courses of all
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Fig. 3. Time courses of adenosine disappearance and adenosine metabolites formation in the presence of 3.6 mM ATP in rat brain post-mitochondrial extracts. The incubation mixtures contained 1 M (A) or 100 M [8-14 C]-adenosine (B), 3.6 mM ATP, 8.3 mM MgCl2 , 5 mM KH2 PO4 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain extract, 1.15 mg of protein/ml. (䉬) ATP; ( ) ADP; (䉱) AMP; () IMP; (䊐) Ino; (䊊) Hyp. Insets: (䉱) AMP; (䊐) Ino; (䊊) Hyp. Fig. 2. Time courses of ATP breakdown and of ATP catabolites formation in rat brain post-mitochondrial extracts. The incubation mixtures contained 3.6 mM (A), or 2 mM (B), or 0.5 mM [8-14 C]-ATP (C), 8.3 mM MgCl2 , 5 mM KH2 PO4 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain extract, 1.15 mg of protein/ml. The reaction was initiated by addition of rat brain extract. (䉬) ATP; ( ) ADP; (䉱) AMP; (䊏) Ado; () IMP; (䊐) Ino; (䊊) Hyp.
two processes appear to differ in different brain cell types, depending on the specificity of the enzyme patterns (Parkinson, Sinclair, Othman, Haughey, & Geiger, 2002). Nevertheless, some aspects of ATP and adenosine metabolism, as demostrated in Figs. 2 and 3, to our knowledge, have never been previously rationalized and deserve a brief discussion.
possible intermediates formed during ATP breakdown in the post-mitochondrial extracts of whole rat brain. Fig. 3 shows the time courses of the adenosine anabolic and catabolic routes. The kinetics of these
3.1.1. At “normoxic” levels, ATP breakdown exclusively follows the IMP pathway The nature of the ATP catabolic route (i.e. if it follows the IMP or the adenosine pathway) is con-
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centration dependent. At 3.6 and 2 mM ATP initial concentration (Fig. 2A and B) IMP, but not AMP, accumulates in post-mitochondrial extracts with little nucleoside and base formation. Moreover, the ATP level was maintained at a fairly constant value of ca. 1.6 mol/mg of protein and the ATP/ADP ratio at a value of ca. 5 between 30 and 90 min of incubation. Thereafter, it gradually fell to ca. 2 at the eighth hour. Even after 8 h incubation, IMP, rather than inosine plus hypoxanthine, was the major ATP catabolite. This can be explained by the de-activation of cN-II prompted by the disappearance of ATP, a condition which also increases the Km value for the substrate IMP from micromolar to millimolar value (Pesi, Baiocchi, Tozzi, & Camici, 1996; Pesi et al., 1994). Interestingly, low levels of adenosine could be detected in post-mitochondrial extracts only after prolonged incubation in the experimental conditions of Fig. 2A and B. Thus, at 3.6 and 2 mM initial ATP, adenosine was produced after a delay period of about 120 and 90 min, respectively. The production of adenosine was clearly preceded by inosine and hypoxanthine formation (see insets to Fig. 2A and B), thus excluding a precursor–product relationship between adenosine and inosine plus hypoxanthine. In fact, when 1 M deoxycoformicine plus 50 M 5 -amino-5 -deoxyadenosine, which inhibits adenosine deaminase and adenosine kinase, respectively (Agarwal, Spector, & Parks, 1977; Miller et al., 1979), were added to an incubation mixture with 3.6 mM ATP, the results were almost identical to those of Fig. 2A (data not shown). This observation confirms that adenosine deaminase is not involved in inosine and hypoxanthine production during normoxic ATP breakdown and thus, in the recycling of adenosine into AMP and IMP. When considered together, our “in vitro” results suggest that under normoxic physiological conditions and possibly under mild anoxic conditions, ATP catabolism follows the IMP pathway. Even if “in vivo” some adenosine is uptaken from the external medium via the nucleoside transporters (Thorn & Jarvis, 1996), it would be readily recycled into AMP via adenosine kinase, whose Km for adenosine in whole rat brain is 2 M, about one order of magnitude lower than those calculated for adenosine deaminase (Phillips & Newsholme, 1979; Zimmermann, 1996). This idea is in accordance with the results presented in Fig. 3A which shows that adenosine at 1 M, a reasonable physiological nor-
moxic concentration (Delaney & Geiger, 1996; Latini & Pedata, 2001; Phillis, Perkins, Smith-Barbour, & O’Regan, 1995; Yamada, Kobayashi, & Okada, 1998), is anabolised to ATP and ADP or to IMP via the pathways adenosine → AMP ADP ATP and adenosine → AMP → IMP, respectively. Only at higher initial concentrations is adenosine mainly catabolised into inosine and hypoxanthine (Fig. 3B). 3.1.2. At “ischemic” levels, ATP breakdown follows the adenosine pathway At an initial concentration of 0.5 mM ATP, AMP immediately and transiently accumulates, followed by adenosine, inosine and hypoxanthine, well before the limited IMP formation (Fig. 2C). This suggests a precursor product relationship between AMP and adenosine plus inosine and hypoxanthine. Under these conditions, ATP catabolism mainly follows the adenosine pathway. Moreover, inosine and hypoxanthine, rather than IMP, were the major final products of 0.5 mM ATP catabolism. It cannot, however, be excluded “a priori” that both adenosine and IMP pathways are working, as the concentration of IMP rises gradually until the fifth hour of incubation. The ratio ATP/ADP reaches about 1 between 30 and 90 min incubation. It might be speculated then, that the brain selects the adenosine pathway under ischemic ATP concentration, despite the fact that the IMP pathway can replenish brain ATP, but not the adenosine pathway. Adenosine, which is produced exclusively in the adenosine pathway, exerts not only a direct neuroactive action (Latini & Pedata, 2001), but also an indirect neurotrophic action. Its deamination product, inosine, may be used as an energy source (Jurkowitz, Litsky, Browning, & Hohl, 1998) and as an activator of pyrimidine salvage synthesis (Mascia, Cotrufo, Cappiello, & Ipata, 1999). Our results show that at 3.6 and 2 mM initial concentration, ATP is catabolised through the IMP pathway, while at 0.5 mM ATP follows the adenosine catabolic route. This finding explains the apparently paradoxic observation that more adenosine (about four-fold) was produced at 0.5 mM than at 3.6 mM initial ATP concentration. Hydrolysis of S-adenosylmethionine by S-adenosylmethionine hydrolase, another possible intracellular source of adenosine, does not significantly contribute to adenosine production in the brain (Latini, Corsi, Pedata, & Pepeu, 1995; Pak, Haas, Decking, & Schrader, 1994).
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bation (Fig. 4A and B). Only 20% is degraded into bases and nucleosides after 8 h. In contrast, at 0.5 mM, ATP is 100% catabolised into hypoxanthine, inosine and adenosine (Fig. 4C). As the purine ring is maintained in its phosphorylated form it can be hypothesized that the ATP pool might be readily replenished in the cell cytoplasm during post-ischemic recovery via the purine nucleotide cycle, in which IMP is an obligatory intermediate (Barsotti et al., 2003; Schulz & Lowenstein, 1978), and the adenylate kinase reaction. However, a metabolic point of “no-return” should exist between 2 and 0.5 mM ATP concentration, where inosine and hypoxanthine accumulate, rather than IMP. It is difficult to envisage a complete intracellular salvage of accumulated inosine and hypoxanthine, since (i) inosine kinase does not exist in mammals (Barsotti et al., 2003; Stone & Simmonds, 1991) and (ii) the limited brain PRPP pool (Traut, 1994) would be insufficient to convert all accumulated hypoxanthine into IMP by hypoxanthine-guanine phosphoribosyltransferase. It is most likely that intracellular inosine and hypoxanthine are released “in vivo” to be salvaged in other tissues and organs, or excreted. 3.2. ATP directs AMP metabolism in rat brain
Fig. 4. Time courses of total nucleotides disappearance and of total purine bases plus nucleosides formation during ATP breakdown catalysed by rat brain post-mitochondrial extracts. The incubation mixtures contained 3.6 mM (A), or 2 mM (B), or 0.5 mM [8-14 C]-ATP (C), 8.3 mM MgCl2 , 5 mM KH2 PO4 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain extract, 1.15 mg of protein/ml. The reaction was initiated by addition of rat brain extract. (䊉) [ATP] + [ADP] + [AMP] + [IMP]; (䊊) [Ado] + [Ino] + [Hyp].
The data contained in Fig. 2 are reported as the time course of total purine nucleotides ([ATP] + [ADP] + [AMP] + [IMP]) and of total purine bases and nucleotides ([Ado] + [Ino] + [Hyp]) formation in Fig. 4. This figure shows that at both 3.6 and 2 mM ATP initial concentration, the purine pool is maintained mainly in its phosphorylated form, up to about 4 h of incu-
AMP is an obligatory intermediate of ATP breakdown and is either deaminated to IMP or dephosphorylated to adenosine (Fig. 1). The results presented in Fig. 5 show that ATP may influence AMP catabolism. Thus, in the presence of 3.6 and 2 mM ATP (Fig. 5A and B), AMP added at 0.1 mM, a physiological concentration (Traut, 1994), was either converted to IMP via adenylate deaminase or anabolised to ATP via adenylate kinase. We were unable to detect even trace amounts of adenosine (insets to Fig. 5A and B). By contrast, in the presence of 0.5 mM ATP (Fig. 5C) only a negligible amount of added AMP was deaminated to IMP, but adenosine formation started (inset to Fig. 5C). It is most likely that in these experimental conditions inosine plus hypoxanthine originated from adenosine. AMP was almost 100% converted into inosine and hypoxanthine via adenosine deaminase in the absence of ATP (Fig. 5D). This agrees with the results of Torrecilla et al. (2001). Our data therefore support the idea that the intracellular ATP level might indeed direct AMP catabolism either towards IMP or adenosine.
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Fig. 5. Effect of ATP on AMP metabolism in rat brain post-mitochondrial extracts. The incubation mixtures contained 100 M [8-14 C]-AMP, 3.6 mM (A), or 2 mM (B), or 0.5 mM (C), or no ATP (D), 8.3 mM MgCl2 , 5 mM KH2 PO4 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain extract, 1.15 mg of protein/ml. (䉬) ATP; ( ) ADP; (䉱) AMP; (䊏) Ado; () IMP; (䊐) Ino; (䊊) Hyp. In panel D, no ATP and ADP could be detected.
3.3. ATP breakdown by membrane preparation of rat brain A great deal of evidence suggests that ATP is released into the extracellular space either by regulated exocytosis together with a neurotransmitter, or through the plasma membrane by P-glycoprotein (for review see Zimmermann, 1996, and literature cited herein). In normal conditions, extracellular ATP concentration can be estimated to be between 2 and 100 M. However, since ATP is stored inside synaptic vesicles in high millimolar range, it might well reach transient a synaptic concentration of 1 mM or higher. ATP breakdown, as catalysed by our membrane preparations, follows a unique pathway. Interestingly, at 20 M ATP initial concentration (Fig. 6A), the pathway was almost super imposable to that catalysed by ectonucleotidases
of purified striatal cholinergic synaptosomes (James & Richardson, 1993). As discussed by Zimmermann (1996), the sigmoidal shape of the curve of adenosine production (see also Fig. 6B and C) is to be ascribed to the competitive feed-forward inhibition exerted by ADP and ATP on the ecto-5 -nucleotidase (Mallol & Bozal, 1988; Orford & Saggerson, 1996). This inhibition also causes AMP accumulation, before adenosine is produced. Accordingly, the delay between the breakdown of ATP and the formation of adenosine increases by increasing the initial ATP concentration and this may explain our paradoxical observation that more adenosine is generated from 20 M, than from 500 M ATP (Fig. 6A and C). Because we were unable to detect IMP formation, any inosine and hypoxanthine formed can be attributed to contaminating cytosolic adenosine deaminase and purine nucle-
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Fig. 7. Time courses of GTP breakdown and of GTP catabolites formation in rat brain post-mitochondrial extracts. (A) The incubation mixture contained 0.6 mM [8-14 C]-GTP, 8.3 mM MgCl2 , 5 mM KH2 PO4 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain extract, 1.15 mg of protein/ml. In B 3.6 mM ATP was added. (䉬) GTP + GDP; (䉱) GMP; (䊏) Guo; (䊉) Gua; (䊐) Xao; (䊊) Xn. Fig. 6. Time courses of ATP breakdown and of ATP catabolites formation in rat brain membrane preparations. The incubation mixtures contained 20 M (A), or 100 M (B), or 500 M [8-14 C]-ATP (C), 1.5 mM MgCl2 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain membrane preparation, 0.5 mg of protein/ml. (䉬) ATP; ( ) ADP; (䉱) AMP; (䊏) Ado; (䊐) Ino; (䊊) Hyp.
oside phosphorylase, or to membrane bound enzyme proteins. Adenosine deaminase has been shown to act as an ecto-enzyme in rat brain (Franco et al., 1997, 1998).
3.4. Pathways of GTP breakdown In addition to ATP, GTP has been shown to undergo severe reduction during ischemia in whole rat brain, with a concomitant increase in the intracellular level of guanosine (Onodera et al., 1986). The time course of all possible intermediates formed by the post-mitochondrial extract (Fig. 7A) showed that GTP added at 0.6 mM, a physiological concentration (Traut, 1994), is cabolised by the route presented in Fig. 8. In this route, xanthosine, in addition to guanosine, is
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Fig. 8. GTP catabolism in rat brain. Xanthosine, which may be considered the final product of the catabolic pathway, is formed through the so called “Rib1-P recycling for nucleoside interconversion” (Giorgelli et al., 1997). The enzymes participating in this pathways are: 1, cN-II; 2, purine nucleoside phosphorylase; 3, guanase.
generated. We suggest that the so called “Rib 1-P recycling for nucleoside interconversion” described by us in 1997 (Giorgelli et al., 1997) might be operative in rat brain (see also Torrecilla et al., 2001), especially in ischemic conditions when the ATP level is drastically reduced to 1–3% of control values (Phillis et al., 1996). In fact, 3.6 mM ATP (Fig. 7B) dramatically reduces the rate of GTP breakdown. No effects were observed from the 0.6 mM GTP on the rate of 3.6 mM ATP degradation (data not shown). Neither xanthine, nor xanthosine were formed, when GTP was incubated with rat brain membrane preparation (Fig. 9). The sigmoidal shape of the time couse of guanosine formation (Fig. 9) suggests that, in analogy to ATP, GTP exerts feed-forward inhibition on the dephosphorylation of the corresponding nucleoside-monophosphate. It is likely then, that ATP and GTP share the same set of ecto-nucleotidases.
4. Conclusions The ratios of inosine plus hypoxanthine levels to adenosine are generally considered to indicate the rate of metabolic degradation from adenosine to inosine and hypoxanthine. However, our “in vitro” results show that, at low millimolar physiological concentration, ATP is catabolised to inosine and hypoxanthine
Fig. 9. Time courses of GTP breakdown and of GTP catabolites formation in rat brain membrane preparations. The incubation mixtures contained 20 M (A), or 100 M (B), or 500 M [8-14 C]-GTP (C), 1.5 mM MgCl2 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain membrane preparation, 0.5 mg of protein/ml. (䉬) GTP + GDP; (䉱) GMP; (䊏) Guo; (䊉) Gua. No Xn and Xao formation were observed.
without detectable intermediate adenosine formation by post-mitochondrial extracts. We propose that, as long as intracellular ATP is maintained at normoxic concentration, its catabolism
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follows the IMP pathway and only a minimal amount of adenosine, if any, is generated in rat brain. Any inosine plus hypoxanthine would therefore originate from IMP, rather than adenosine. This view is in accordance
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with the strong activation exerted by millimolar ATP on both adenylate deaminase and cN-II. It is also supported by the low Km of adenosine kinase for adenosine, as compared with that of adenosine deaminase. In normoxic conditions, extracellular ATP is mainly catabolised to adenosine by the ecto-nucleotidase cascade system. The nucleoside is then taken up by the nucleoside transporter to be either anabolised to AMP via adenosine kinase, or degraded to inosine and hypoxanthine (Fig. 10A). During ischemic conditions, a different scenario can be envisaged. The drastic ATP drop relieves the activation of adenylate deaminase and of cN-II, causing ATP catabolism to be channelled towards the adenosine pathway. Enough adenosine becomes available to be released in the extracellular space (Fig. 10B). Moreover, the continued extracellular hydrolysis of nucleotides is important not only for the formation of adenosine, but also of other nucleosides, such as inosine and guanosine and for exerting trophic action in CNS. We are aware that not only ATP concentrations, but also other variables are affected during ischemia/hypoxia, such as intracellular pH and calcium. These might affect the enzymes of purine metabolism. For instance, the activity of the AMP specific cytosolic 5 -nucleotidase, the enzyme responsible of the intracellular adenosine production, has an acidic pH optimum (Zimmermann, 1992). It is therefore conceivable that its activity enhances during ATP breakdown under ischemic conditions. By contrast, the non muscular ATP activated adenylate deaminase, the enzyme responsible of intracellular IMP production in normoxic conditions, has a neutral pH optimum (Barsacchi, Ranieri-Raggi, & Bergamini, 1979). Further experiments in this direction would be helpful in the future.
Acknowledgements
Fig. 10. Pathways of ATP breakdown in rat brain during normoxic conditions (A) and ischemic conditions (B). The enzymes participating in these pathways are: (1) adenylate kinase; (2) adenylate deaminase; (3) cN-II; (4) purine nucleoside phosphorylase; (5) adenosine deaminase; (6) adenosine kinase; (7) ecto-ATPase; (8) ecto-ADPase; (9) ecto-5 -nucleotidase; (10) cytosolic 5 -nucleotidase specific for AMP.
This work is dedicated to Professor Paolo Cerletti, who 50 years ago introduced one of us (P.L.I.) to the fascination of nucleotide metabolism. Our work was supported by the Italian MURST National Interest Project “Molecular mechanisms of cellular and metabolic regulation of polynucleotides, nucleotides and analogs”.
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Freedholm, B. B., Dunwiddie, T. V., Bergman, B., & Lindström, K. (1984). Levels of adenosine and adenosine nucleotides in slices of rat hippocampus. Brain Research, 295, 127–136. Giorgelli, F., Bottai, C., Mascia, L., Scolozzi, C., Camici, M., & Ipata, P. L. (1997). Recycling of ␣-d-ribose 1-phosphate for nucleoside interconversion. Biochimica Biophysica Acta, 1335, 16–22. Gysbers, J. W., & Rathbone, M. P. (1996). GTP and guanosine synergistically enhance NGF-induced neurite outgrowth from PC12 cells. International Journal of Developmental Science, 14, 19–34. James, S., & Richardson, P. J. (1993). Production of adenosine from extracellular ATP at the striatal cholinergic synapse. Journal of Neurochemistry, 60, 219–227. Jurkowitz, M. S., Litsky, M. J., Browning, M. J., & Hohl, C. M. (1998). Adenosine, inosine and guanosine protect glial cells during glucose deprivation and mitochondrial inhibition: Correlation between protection and ATP preservation. Journal of Neurochemestry, 71, 535–548. Kobayashi, T., Yamada, T., & Okada, Y. (1998). The levels of adenosine and its metabolites in the guinea pig and rat brain during complete ischemia-in vivo study. Brain Research, 787, 211–219. Latini, C., & Pedata, F. (2001). Adenosine in the central nervous system: Release mechanisms and extracellular concentrations. Journal of Neurochemistry, 79, 463–484. Latini, S., Corsi, C., Pedata, F., & Pepeu, G. (1995). The source of brain adenosine outflow during ischemia and electrical stimulation. Neurochemistry International, 27, 239–244. Ljunggren, B., Ratcheson, R. A., & Siesjö, B. K. (1974). Cerebral metabolic state following complete compression ischemia. Brain Research, 73, 291–307. Mackiewicz, M., Geiger, J. D., & Pack, A. I. (2000). Simultaneous assessment of ecto- and cytosolic-5 -nucleotidase activities in brain micropunches. Journal of Neuroscience Methods, 104, 9–18. Mallol, J., & Boza, J. (1988). 5 -Nucleotidase from bovine brain cortex: Effect of solubilization on enzyme kinetics and modulation. Neurochemistry International, 12, 61–67. Mascia, L., Cappiello, M., Cherri, S., & Ipata, P. L. (2000). In vitro recycling of alpha-d-ribose 1-phosphate for the salvage of purine bases. Biochimica Biophysica Acta, 1474, 70–76. Mascia, L., Cotrufo, T., Cappiello, M., & Ipata, P. L. (1999). Ribose 1-phosphate and inosine activate uracil salvage in rat brain. Biochimica Biophysica Acta, 1472, 93–98. Miller, R. L., Adamczyk, D. L., Miller, W. H., Koszalka, G. W., Rideout, J. L., & Beacham III, L. M. et al., (1979). Adenosine kinase from rabbit liver. II. Substrate and inhibitor specificity. Journal of Biological Chemistry, 254, 2346–2346. Orford, M. R., & Saggerson, E. D. (1996). A low-Km 5 nucleotidase from rat brain cytosolic fraction: Purification, kinetic properties and description of regulation by a novel factor that increases sensitivity to inhibition by ATP and ADP. Journal of Neurochemistry, 67, 795–804. Pak, M. A., Haas, H. L., Decking, U. K. M., & Schrader, J. (1994). Inhibition of adenosine kinase increases endogenous adenosine and depresses neuronal activity in hippocampal slices. Neuropharmacology, 33, 1049–1053.
C. Barsotti, P.L. Ipata / The International Journal of Biochemistry & Cell Biology 36 (2004) 2214–2225 Parkinson, F. E., Sinclair, C. J. D., Othman, T., Haughey, N. J., & Geiger, J. D. (2002). Differences between rat primari cortical neurons and astrocytes in purine release evoked by ischemic conditions. Neuropharmacology, 43, 836–846. Pesi, R., Baiocchi, C., Tozzi, M. G., & Camici, M. (1996). Synergetic action of ADP and 2,3-bisphosphoglycerate on the modulation of cytosolic 5 -nucleotidase. Biochimica Biophysica Acta, 1294, 191–194. Pesi, R., Turriani, M., Allegrini, S., Scolozzi, C., Camici, M., & Ipata, P. L. (1994). The bifunctional cytosolic 5 -nucleotidase: Regulation of the phosphotransferase and nucleotidase activities. Archives of Biochemistry and Biophysics, 312, 75–80. Phillips, E., & Newsholme, E. A. (1979). Maximum activities, properties and distribution of 5 -nucleotidase, adenosine kinase and adenosine deaminase in rat and human brain. Journal of Neurochemistry, 33, 553–558. Phillis, J. W., O’Regan, M. H., Estevez, A. Y., Song, D., & VanderHeide, S. J. (1996). Cerebral energy metabolism during severe ischemia of varying duration and following reperfusion. Journal of Neurochemistry, 67, 1525–1531. Phillis, J. W., Perkins, L. M., Smith-Barbour, M., & O’Regan, M. H. (1995). Oxypurinol-enhanced postischemic recovery of the rat brain involves preservation of adenine nucleotides. Journal of Neurochemistry, 64, 2177–2184. Pulsinelli, W. A., & Duffy, T. E. (1983). Regional energy balance in rat brain after transient forebrain ischemia. Journal of Neurochemistry, 40, 1500–1503. Sawynok, J., & Sweeney, M. I. (1989). The role of the purines in nociception. Neuroscience, 32, 557–569. Schulz, V., & Lowenstein, J. M. (1978). The purine nucleotide cycle. Studies of ammonia production and interconversions of adenine and hypoxanthine nucleotides and nucleosides by rat brain in situ. Journal of Biological Chemistry, 253, 1938– 1943. Setlow, B., & Lowenstein, J. M. (1967). Adenylate deaminase. II. Purification and some regulatory properties of the enzyme from calf brain. Journal of Biological Chemistry, 242, 607–615. Siesjö, B. K., & Ljunggren, B. (1973). Cerebral energy reserves after prolonged hypoxia and ischemia. Archives of Neurology, 29, 400–403.
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Skladanowski, A. C., & Newby, A. C. (1990). Partial purification and properties of an AMP-specific soluble 5 -nucleotidase from pigeon heart. Biochemical Journal, 268, 117–122. Smallrige, R. C., Kiang, J. G., Gist, I. D., Fein, H. G., & Galloway, R. J. (1992). U73122, an aminosteroid phospholipase C antagonist, noncompetitively inhibits thyrotropin-releasing effects in G113 rat pituitary cells. Endocrinology, 131, 1883– 1888. Stone, T. W., & Simmonds, H. A. (1991). Metabolism of endogenous purines. In Purines: Basic and clinical aspects (pp. 8–22). Dordrecht: Kluwer Academic. Thorn, J. A., & Jarvis, S. M. (1996). Adenosine transporters. General Pharmacology, 27, 613–620. Torrecilla, A., Marques, A. F. P., Buscalioni, R. D., Oliviera, J. M. A., Teixeira, N. A., & Atencia, E. A. et al., (2001). Metabolic fate of AMP, IMP, GMP and XMP in the cytosol of rat brain: An experimental and theoretical analysis. Journal of Neurochemistry, 76, 1291–1307. Traut, T. W. (1994). Physiological concentrations of purines and pyrimidines. Molecular Cell Biochemistry, 140, 1–22. Van den Berghe, G., Van Pottelsberghe, C., & Hers, H. G. (1977). A kinetic study of the soluble 5 -nucleotidase of rat liver. Biochemical Journal, 162, 611–616. White, T. D., & Hoehn, K. (1991). Release of adenosine and ATP from nervous tissue. In T. Stone (Ed.), Adenosine in the nervous system (pp. 173–195). London: Academic Press. White, T. D., & MacDonald W. F. (1990). Neural release of ATP and adenosine. In G. R. Dubyak & J. S. Fedan (Eds.), Biological actions of extracellular ATP (Vol. 603, pp. 287–299). New York: New York Academy of Sciences. Yamada, T., Kobayashi, T., & Okada, Y. (1998). The effect of simulated ischemia on the levels of adenosine and its metabolites in slices of cerebellum, superior colliculus and hippocampus in the guinea pig-in vitro study. Brain Research, 787, 220–225. Zimmermann, H. (1992). 5 -Nucleotidase: Molecular structure and functional aspects. Biochemical Journal, 285, 345–365. Zimmermann, H. (1996). Biochemistry, localization and functional roles of ecto-nucleotidases in the nervous system. Progress in Neurobiology, 49, 589–618.
Metabolic regulation of ATP breakdown and of adenosine production in rat brain extracts Catia Barsotti∗ , Piero L. Ipata Department of Physiology and Biochemistry, University of Pisa, Via San Zeno 51, 56127 Pisa, Italy Received 18 February 2004; received in revised form 28 April 2004; accepted 28 April 2004
Abstract ATP concentration is dramatically affected in ischemic injury. From previous studies on ATP mediated purine and pyrimidine salvage in CNS, we observed that when “post-mitochondrial” extracts of rat brain were incubated with ATP at 3.6 mM, a normoxic concentration, formation of IMP always preceded that of adenosine, a well known neuroactive nucleoside and a homeostatic cellular modulator. This observation prompted us to undertake a study aimed at assessing the precise pathways and kinetics of ATP breakdown, a process considered to be the major source of adenosine in rat brain. The results obtained using post-mitochondrial extracts strongly suggest that the breakdown of intracellular ATP at normoxic concentration follows almost exclusively the pathway ATPADPAMP → IMP → inosinehypoxanthine, with little, if any, intracellular adenosine production. At low ischemic concentration, intracellular ATP breakdown follows the pathway ATPADPAMP → adenosine → inosinehypoxanthine with little IMP formation. At the same time, extracellular ATP, whose concentration is known to be enhanced during ischemia, is actively broken down to adenosine through the pathway ATP → ADP → AMP → adenosine, catalysed by the well characterized ecto-enzyme cascade system. Moreover, we show that during intracellular GTP catabolism, xanthosine, in addition to guanosine, is generated through the so called “ribose 1-phosphate recycling for nucleoside interconversion”. These results considerably extend our knowledge on the long debated question of the extra or intracellular origin of adenosine in CNS, suggesting that at least in normoxic conditions, intracellular adenosine is of extracellular origin. © 2004 Elsevier Ltd. All rights reserved. Keywords: ATP breakdown; Adenosine metabolism; GTP breakdown; Guanosine; Xanthosine; Rat brain
1. Introduction Adenosine is an important neuroactive nucleoside and a homeostatic cellular modulator (Cunha, Abbreviations: Rib 1-P, ribose 1-phosphate; cN-II, cytosolic IMP-GMP specific 5 -nucleotidase; PEI, polyethyleneimine; Ado, adenosine; Ino, inosine; Hyp, hypoxanthine; Guo, guanosine; Gua, guanine; Xn, xanthine; Xao, xanthosine ∗ Corresponding author. Tel.: +39 050 2213181; fax: +39 050 2213170. E-mail address: [email protected] (C. Barsotti).
2001; Latini & Pedata, 2001). It is mainly produced by ATP breakdown, as it occurs in anoxic/ischemic conditions and to a lesser extent during strenuous physical exercise (Baldwin, Snow, Carey, & Febbraio, 1999; Broberg & Sahlin, 1989; Dagher, 2000; Phillis, O’Regan, Estevez, Song, & VanderHeide, 1996). The kinetics of ATP breakdown have been extensively investigated in whole organs and cell cultures, mainly by following the time course of the production of inosine and hypoxanthine (Dagher, 2000; Freedholm, Dunwiddie, Bergman, & Lindström, 1984; Kobayashi,
1357-2725/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2004.04.015
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Fig. 1. Pathways of intracellular ATP catabolism in rat brain. The enzymes participating in these pathways are: (1) adenylate kinase; (2) adenylate deaminase; (3) cytosolic IMP-GMP specific 5 nucleotidase (cN-II); (4) purine nucleoside phosphorylase; (5) cytosolic AMP specific 5 nucleotidase; (6) adenosine deaminase. Enzymes 2 and 3 are activated by millimolar physiological ATP concentration (Chapman & Atkinson, 1973; Gysbers & Rathbone, 1996; Pesi et al., 1994, 1996; Schulz & Lowenstein, 1978; Setlow & Lowenstein, 1967; Van den Berghe, Van Pottelsberghe, & Hers, 1977). Enzyme 5 is activated by ADP (Skladanowski & Newby, 1990).
Yamada, & Okada, 1998; Ljunggren, Ratcheson, & Siesjö, 1974; Phillis et al., 1996; Pulsinelli & Duffy, 1983; Siesjö & Ljunggren, 1973), which are considered catabolites of adenosine. However, we emphasize that ATP breakdown may follow two distinct routes (Fig. 1). In the first route, ATP ADP AMP interconversion, catalysed by adenylate kinase, is followed by AMP deamination and IMP dephosphorylation to give inosine. In the second route, AMP is dephosphorylated to adenosine and then adenosine is deaminated to inosine. The two catabolic routes are referred to as the “IMP pathway” and the “adenosine pathway”, respectively, throughout this paper. In both of them, phosphorolysis of inosine to hypoxan-
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thine and ribose 1-phosphate (Rib 1-P) is considered the final step of ATP breakdown. However, adenosine is formed by only one of the two pathways. It is therefore important to establish the relative contribution of the two intracellular pathways of ATP breakdown. ATP is released in the extracellular space during ischemia (Sawynok & Sweeney, 1989; White & Hoehn, 1991; White & MacDonald, 1990). A set of membrane bound ecto-nucleotidases collectively functioning as an enzyme cascade, is responsible for extracellular ATP breakdown where adenosine is the major catabolite (Latini & Pedata, 2001; White & Hoehn, 1991; White & MacDonald, 1990; Zimmermann, 1996). Determining the relative contribution of the intracellular and extracellular pathways for ATP degradation is important in whole rat brain, because both intra and extracellular ATP are thought to be the source of adenosine (see Latini & Pedata, 2001 for review). In this paper, we have investigated both routes of ATP metabolism in whole rat brain, by comparing the kinetics of ATP breakdown and the time course of all the possible metabolites formed in post-mitochondrial extracts and membrane preparations of whole rat brain. The data show that the metabolic pathway of intracellular ATP breakdown is concentration dependent. Thus, at 3.6 mM (a normoxic concentration) ATP breakdown follows the IMP pathway with little, if any, adenosine production. Any intracellular adenosine must therefore be of extracellular origin. Only at ischemic concentration (less then 2 mM) does ATP breakdown follow the adenosine pathway. The IMP pathway does not appear to contribute to extracellular ATP degradation.
2. Materials and methods 2.1. Materials [8-14 C]-ATP (51 mCi/mmol) was obtained from ICN. [8-14 C]-GTP (60 mCi/mmol) was from Moravek Biochemicals. [8-14 C]-Adenosine (58 mCi/mmol), [8-14 C]-AMP (53 mCi/mmol), U73122, dithiothreitol, sucrose, nucleobases, nucleosides, and nucleotides were from Sigma Chemical Co. Hi Safe II Scintillation liquid was purchased from Wallac. Polyethyleneimine (PEI)-cellulose precoated
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thin-layer plastic sheets (0.1 mm thick) were purchased from Merck and prewashed once with 10% NaCl and three times with deionized water before use. All other chemicals were of reagent grade. Three-month-old male Sprague–Dawley rats (250 g) were killed according to the Guiding Principles in the Care and Use of Animals (DHWEW publication, NIH 86-23). The brain was removed and kept frozen at −80 ◦ C until needed. Storage times did not exceeded 3 months. 2.2. Preparation of rat brain extracts Whole rat brain was washed with 10 mM Tris–HCl buffer, pH 7.4, with 0.32 M sucrose and 44 M U73122, an inhibitor of phospholipase C used to prevent the displacement of ecto-5 -nucleotidase from membrane (Mackiewicz, Geiger, & Pack, 2000; Smallrige, Kiang, Gist, Fein, & Galloway, 1992). Homogenization (six up-and-down passes) was performed using the same medium in a glass Potter-Elvehjem homogenizer fitted with a Teflon pestle. The homogenate was centrifuged at 4 ◦ C at 39,000 × g for 1 h. The supernatant fluid obtained was dialyzed overnight at 4 ◦ C in dialysis bags against 10 mM Tris–HCl buffer, pH 7.4, supplemented with 1 mM dithiothreitol and is referred to as post-mitochondrial extract. This preparation proved to be extremely versatile in studies on ATP-mediated purine and pyrimidine salvage (Barsotti, Tozzi, & Ipata, 2002) and on purine nucleoside cycle (Barsotti, Pesi, Felice, & Ipata, 2003). Alternatively, the homogenate was fractionated to isolate membranes according to Delaney, Blackburn, and Geiger (1997) with minor modifications. The homogenate was centrifuged at 1000 × g for 10 min at 4 ◦ C, after which the supernatant was centrifuged at 11,000 × g for 20 min at 4 ◦ C. The supernatant was then collected and centrifuged at 105,000 × g for 1 h at 4 ◦ C. The pellet was collected and stirred for 90 min at 4 ◦ C in 10 mM Tris–HCl buffer, pH 7.4, containing Triton X-100 (2% (v/v)). The supernatant fluid, collected after centrifugation for 1 h at 105,000 × g at 4 ◦ C, is referred to as membrane preparation. Protein concentration of rat brain post-mitochondrial extract and membrane preparation were determined by Coomassie blue-binding assay, using bovine serum albumin as standard (Bradford, 1976).
2.3. Incubation with post-mitochondrial extracts The standard reaction mixtures contained 5 mM Tris–HCl buffer, pH 7.4, [8-14 C]-ATP (2500 dpm/ nmol), or [8-14 C]-GTP (15,000 dpm/nmol) at the concentrations indicated in the figure legends, 8.3 mM MgCl2 , 5 mM KH2 PO4 and brain post-mitochondrial extract, 1.15 mg of protein/ml. Modifications of the standard incubation mixture are indicated in the figure legends. The reaction was started by addition of the extract. At different time intervals the reaction was stopped by rapidly drying portions of 10 l of the incubation mixture on PEI-cellulose precoated thin-layer plastic sheets and a chromatogram was developed in n-butanol/glacial acetic acid/H2 O (4:2:1, v/v) (Mascia, Cappiello, Cherri, & Ipata, 2000) to separate adenosine, inosine and hypoxanthine, or with n-butanol/glacial acetic acid/aceton/NH3 / H2 O (35:15:25:2.3:22.7, v/v) to separate IMP, AMP, ADP, and ATP, or with n-propanol/NH3 /trichloroacetic acid (100%)/H2 O (75:0.7:5:20, v/v) (Cerletti, Ipata, & Tancredi, 1959; Giorgelli et al., 1997) to separate guanine, guanosine, xanthine, xanthosine, GMP, and GDP + GTP. In all separations, appropriate standards were used and detected as ultraviolet absorbing areas which were excised and counted for radioactivity with 8 ml of scintillation liquid. 2.4. Incubation with membrane preparations The standard reaction mixtures contained 5 mM Tris–HCl buffer, pH 7.4, [8-14 C]-ATP (10,000 dpm/ nmol), or [8-14 C]-GTP (10,000 dpm/nmol) at the concentrations indicated in the figure legends, 1.5 mM MgCl2 and brain membrane preparations, 0.5 mg of protein/ml. Modifications of the standard incubation mixture are indicated in the figure legends. The reactions were started by addition of membrane preparation. At different time intervals, the reactions were stopped and chromatographic analyses were performed as described above. 3. Results and discussion 3.1. ATP breakdown and adenosine metabolism in post-mitochondrial extracts of rat brain The results of our studies are illustrated in a series of figures. Fig. 2 shows the time courses of all
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Fig. 3. Time courses of adenosine disappearance and adenosine metabolites formation in the presence of 3.6 mM ATP in rat brain post-mitochondrial extracts. The incubation mixtures contained 1 M (A) or 100 M [8-14 C]-adenosine (B), 3.6 mM ATP, 8.3 mM MgCl2 , 5 mM KH2 PO4 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain extract, 1.15 mg of protein/ml. (䉬) ATP; ( ) ADP; (䉱) AMP; () IMP; (䊐) Ino; (䊊) Hyp. Insets: (䉱) AMP; (䊐) Ino; (䊊) Hyp. Fig. 2. Time courses of ATP breakdown and of ATP catabolites formation in rat brain post-mitochondrial extracts. The incubation mixtures contained 3.6 mM (A), or 2 mM (B), or 0.5 mM [8-14 C]-ATP (C), 8.3 mM MgCl2 , 5 mM KH2 PO4 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain extract, 1.15 mg of protein/ml. The reaction was initiated by addition of rat brain extract. (䉬) ATP; ( ) ADP; (䉱) AMP; (䊏) Ado; () IMP; (䊐) Ino; (䊊) Hyp.
two processes appear to differ in different brain cell types, depending on the specificity of the enzyme patterns (Parkinson, Sinclair, Othman, Haughey, & Geiger, 2002). Nevertheless, some aspects of ATP and adenosine metabolism, as demostrated in Figs. 2 and 3, to our knowledge, have never been previously rationalized and deserve a brief discussion.
possible intermediates formed during ATP breakdown in the post-mitochondrial extracts of whole rat brain. Fig. 3 shows the time courses of the adenosine anabolic and catabolic routes. The kinetics of these
3.1.1. At “normoxic” levels, ATP breakdown exclusively follows the IMP pathway The nature of the ATP catabolic route (i.e. if it follows the IMP or the adenosine pathway) is con-
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centration dependent. At 3.6 and 2 mM ATP initial concentration (Fig. 2A and B) IMP, but not AMP, accumulates in post-mitochondrial extracts with little nucleoside and base formation. Moreover, the ATP level was maintained at a fairly constant value of ca. 1.6 mol/mg of protein and the ATP/ADP ratio at a value of ca. 5 between 30 and 90 min of incubation. Thereafter, it gradually fell to ca. 2 at the eighth hour. Even after 8 h incubation, IMP, rather than inosine plus hypoxanthine, was the major ATP catabolite. This can be explained by the de-activation of cN-II prompted by the disappearance of ATP, a condition which also increases the Km value for the substrate IMP from micromolar to millimolar value (Pesi, Baiocchi, Tozzi, & Camici, 1996; Pesi et al., 1994). Interestingly, low levels of adenosine could be detected in post-mitochondrial extracts only after prolonged incubation in the experimental conditions of Fig. 2A and B. Thus, at 3.6 and 2 mM initial ATP, adenosine was produced after a delay period of about 120 and 90 min, respectively. The production of adenosine was clearly preceded by inosine and hypoxanthine formation (see insets to Fig. 2A and B), thus excluding a precursor–product relationship between adenosine and inosine plus hypoxanthine. In fact, when 1 M deoxycoformicine plus 50 M 5 -amino-5 -deoxyadenosine, which inhibits adenosine deaminase and adenosine kinase, respectively (Agarwal, Spector, & Parks, 1977; Miller et al., 1979), were added to an incubation mixture with 3.6 mM ATP, the results were almost identical to those of Fig. 2A (data not shown). This observation confirms that adenosine deaminase is not involved in inosine and hypoxanthine production during normoxic ATP breakdown and thus, in the recycling of adenosine into AMP and IMP. When considered together, our “in vitro” results suggest that under normoxic physiological conditions and possibly under mild anoxic conditions, ATP catabolism follows the IMP pathway. Even if “in vivo” some adenosine is uptaken from the external medium via the nucleoside transporters (Thorn & Jarvis, 1996), it would be readily recycled into AMP via adenosine kinase, whose Km for adenosine in whole rat brain is 2 M, about one order of magnitude lower than those calculated for adenosine deaminase (Phillips & Newsholme, 1979; Zimmermann, 1996). This idea is in accordance with the results presented in Fig. 3A which shows that adenosine at 1 M, a reasonable physiological nor-
moxic concentration (Delaney & Geiger, 1996; Latini & Pedata, 2001; Phillis, Perkins, Smith-Barbour, & O’Regan, 1995; Yamada, Kobayashi, & Okada, 1998), is anabolised to ATP and ADP or to IMP via the pathways adenosine → AMP ADP ATP and adenosine → AMP → IMP, respectively. Only at higher initial concentrations is adenosine mainly catabolised into inosine and hypoxanthine (Fig. 3B). 3.1.2. At “ischemic” levels, ATP breakdown follows the adenosine pathway At an initial concentration of 0.5 mM ATP, AMP immediately and transiently accumulates, followed by adenosine, inosine and hypoxanthine, well before the limited IMP formation (Fig. 2C). This suggests a precursor product relationship between AMP and adenosine plus inosine and hypoxanthine. Under these conditions, ATP catabolism mainly follows the adenosine pathway. Moreover, inosine and hypoxanthine, rather than IMP, were the major final products of 0.5 mM ATP catabolism. It cannot, however, be excluded “a priori” that both adenosine and IMP pathways are working, as the concentration of IMP rises gradually until the fifth hour of incubation. The ratio ATP/ADP reaches about 1 between 30 and 90 min incubation. It might be speculated then, that the brain selects the adenosine pathway under ischemic ATP concentration, despite the fact that the IMP pathway can replenish brain ATP, but not the adenosine pathway. Adenosine, which is produced exclusively in the adenosine pathway, exerts not only a direct neuroactive action (Latini & Pedata, 2001), but also an indirect neurotrophic action. Its deamination product, inosine, may be used as an energy source (Jurkowitz, Litsky, Browning, & Hohl, 1998) and as an activator of pyrimidine salvage synthesis (Mascia, Cotrufo, Cappiello, & Ipata, 1999). Our results show that at 3.6 and 2 mM initial concentration, ATP is catabolised through the IMP pathway, while at 0.5 mM ATP follows the adenosine catabolic route. This finding explains the apparently paradoxic observation that more adenosine (about four-fold) was produced at 0.5 mM than at 3.6 mM initial ATP concentration. Hydrolysis of S-adenosylmethionine by S-adenosylmethionine hydrolase, another possible intracellular source of adenosine, does not significantly contribute to adenosine production in the brain (Latini, Corsi, Pedata, & Pepeu, 1995; Pak, Haas, Decking, & Schrader, 1994).
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bation (Fig. 4A and B). Only 20% is degraded into bases and nucleosides after 8 h. In contrast, at 0.5 mM, ATP is 100% catabolised into hypoxanthine, inosine and adenosine (Fig. 4C). As the purine ring is maintained in its phosphorylated form it can be hypothesized that the ATP pool might be readily replenished in the cell cytoplasm during post-ischemic recovery via the purine nucleotide cycle, in which IMP is an obligatory intermediate (Barsotti et al., 2003; Schulz & Lowenstein, 1978), and the adenylate kinase reaction. However, a metabolic point of “no-return” should exist between 2 and 0.5 mM ATP concentration, where inosine and hypoxanthine accumulate, rather than IMP. It is difficult to envisage a complete intracellular salvage of accumulated inosine and hypoxanthine, since (i) inosine kinase does not exist in mammals (Barsotti et al., 2003; Stone & Simmonds, 1991) and (ii) the limited brain PRPP pool (Traut, 1994) would be insufficient to convert all accumulated hypoxanthine into IMP by hypoxanthine-guanine phosphoribosyltransferase. It is most likely that intracellular inosine and hypoxanthine are released “in vivo” to be salvaged in other tissues and organs, or excreted. 3.2. ATP directs AMP metabolism in rat brain
Fig. 4. Time courses of total nucleotides disappearance and of total purine bases plus nucleosides formation during ATP breakdown catalysed by rat brain post-mitochondrial extracts. The incubation mixtures contained 3.6 mM (A), or 2 mM (B), or 0.5 mM [8-14 C]-ATP (C), 8.3 mM MgCl2 , 5 mM KH2 PO4 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain extract, 1.15 mg of protein/ml. The reaction was initiated by addition of rat brain extract. (䊉) [ATP] + [ADP] + [AMP] + [IMP]; (䊊) [Ado] + [Ino] + [Hyp].
The data contained in Fig. 2 are reported as the time course of total purine nucleotides ([ATP] + [ADP] + [AMP] + [IMP]) and of total purine bases and nucleotides ([Ado] + [Ino] + [Hyp]) formation in Fig. 4. This figure shows that at both 3.6 and 2 mM ATP initial concentration, the purine pool is maintained mainly in its phosphorylated form, up to about 4 h of incu-
AMP is an obligatory intermediate of ATP breakdown and is either deaminated to IMP or dephosphorylated to adenosine (Fig. 1). The results presented in Fig. 5 show that ATP may influence AMP catabolism. Thus, in the presence of 3.6 and 2 mM ATP (Fig. 5A and B), AMP added at 0.1 mM, a physiological concentration (Traut, 1994), was either converted to IMP via adenylate deaminase or anabolised to ATP via adenylate kinase. We were unable to detect even trace amounts of adenosine (insets to Fig. 5A and B). By contrast, in the presence of 0.5 mM ATP (Fig. 5C) only a negligible amount of added AMP was deaminated to IMP, but adenosine formation started (inset to Fig. 5C). It is most likely that in these experimental conditions inosine plus hypoxanthine originated from adenosine. AMP was almost 100% converted into inosine and hypoxanthine via adenosine deaminase in the absence of ATP (Fig. 5D). This agrees with the results of Torrecilla et al. (2001). Our data therefore support the idea that the intracellular ATP level might indeed direct AMP catabolism either towards IMP or adenosine.
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Fig. 5. Effect of ATP on AMP metabolism in rat brain post-mitochondrial extracts. The incubation mixtures contained 100 M [8-14 C]-AMP, 3.6 mM (A), or 2 mM (B), or 0.5 mM (C), or no ATP (D), 8.3 mM MgCl2 , 5 mM KH2 PO4 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain extract, 1.15 mg of protein/ml. (䉬) ATP; ( ) ADP; (䉱) AMP; (䊏) Ado; () IMP; (䊐) Ino; (䊊) Hyp. In panel D, no ATP and ADP could be detected.
3.3. ATP breakdown by membrane preparation of rat brain A great deal of evidence suggests that ATP is released into the extracellular space either by regulated exocytosis together with a neurotransmitter, or through the plasma membrane by P-glycoprotein (for review see Zimmermann, 1996, and literature cited herein). In normal conditions, extracellular ATP concentration can be estimated to be between 2 and 100 M. However, since ATP is stored inside synaptic vesicles in high millimolar range, it might well reach transient a synaptic concentration of 1 mM or higher. ATP breakdown, as catalysed by our membrane preparations, follows a unique pathway. Interestingly, at 20 M ATP initial concentration (Fig. 6A), the pathway was almost super imposable to that catalysed by ectonucleotidases
of purified striatal cholinergic synaptosomes (James & Richardson, 1993). As discussed by Zimmermann (1996), the sigmoidal shape of the curve of adenosine production (see also Fig. 6B and C) is to be ascribed to the competitive feed-forward inhibition exerted by ADP and ATP on the ecto-5 -nucleotidase (Mallol & Bozal, 1988; Orford & Saggerson, 1996). This inhibition also causes AMP accumulation, before adenosine is produced. Accordingly, the delay between the breakdown of ATP and the formation of adenosine increases by increasing the initial ATP concentration and this may explain our paradoxical observation that more adenosine is generated from 20 M, than from 500 M ATP (Fig. 6A and C). Because we were unable to detect IMP formation, any inosine and hypoxanthine formed can be attributed to contaminating cytosolic adenosine deaminase and purine nucle-
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Fig. 7. Time courses of GTP breakdown and of GTP catabolites formation in rat brain post-mitochondrial extracts. (A) The incubation mixture contained 0.6 mM [8-14 C]-GTP, 8.3 mM MgCl2 , 5 mM KH2 PO4 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain extract, 1.15 mg of protein/ml. In B 3.6 mM ATP was added. (䉬) GTP + GDP; (䉱) GMP; (䊏) Guo; (䊉) Gua; (䊐) Xao; (䊊) Xn. Fig. 6. Time courses of ATP breakdown and of ATP catabolites formation in rat brain membrane preparations. The incubation mixtures contained 20 M (A), or 100 M (B), or 500 M [8-14 C]-ATP (C), 1.5 mM MgCl2 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain membrane preparation, 0.5 mg of protein/ml. (䉬) ATP; ( ) ADP; (䉱) AMP; (䊏) Ado; (䊐) Ino; (䊊) Hyp.
oside phosphorylase, or to membrane bound enzyme proteins. Adenosine deaminase has been shown to act as an ecto-enzyme in rat brain (Franco et al., 1997, 1998).
3.4. Pathways of GTP breakdown In addition to ATP, GTP has been shown to undergo severe reduction during ischemia in whole rat brain, with a concomitant increase in the intracellular level of guanosine (Onodera et al., 1986). The time course of all possible intermediates formed by the post-mitochondrial extract (Fig. 7A) showed that GTP added at 0.6 mM, a physiological concentration (Traut, 1994), is cabolised by the route presented in Fig. 8. In this route, xanthosine, in addition to guanosine, is
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Fig. 8. GTP catabolism in rat brain. Xanthosine, which may be considered the final product of the catabolic pathway, is formed through the so called “Rib1-P recycling for nucleoside interconversion” (Giorgelli et al., 1997). The enzymes participating in this pathways are: 1, cN-II; 2, purine nucleoside phosphorylase; 3, guanase.
generated. We suggest that the so called “Rib 1-P recycling for nucleoside interconversion” described by us in 1997 (Giorgelli et al., 1997) might be operative in rat brain (see also Torrecilla et al., 2001), especially in ischemic conditions when the ATP level is drastically reduced to 1–3% of control values (Phillis et al., 1996). In fact, 3.6 mM ATP (Fig. 7B) dramatically reduces the rate of GTP breakdown. No effects were observed from the 0.6 mM GTP on the rate of 3.6 mM ATP degradation (data not shown). Neither xanthine, nor xanthosine were formed, when GTP was incubated with rat brain membrane preparation (Fig. 9). The sigmoidal shape of the time couse of guanosine formation (Fig. 9) suggests that, in analogy to ATP, GTP exerts feed-forward inhibition on the dephosphorylation of the corresponding nucleoside-monophosphate. It is likely then, that ATP and GTP share the same set of ecto-nucleotidases.
4. Conclusions The ratios of inosine plus hypoxanthine levels to adenosine are generally considered to indicate the rate of metabolic degradation from adenosine to inosine and hypoxanthine. However, our “in vitro” results show that, at low millimolar physiological concentration, ATP is catabolised to inosine and hypoxanthine
Fig. 9. Time courses of GTP breakdown and of GTP catabolites formation in rat brain membrane preparations. The incubation mixtures contained 20 M (A), or 100 M (B), or 500 M [8-14 C]-GTP (C), 1.5 mM MgCl2 , 5 mM Tris–HCl buffer, pH 7.4, and rat brain membrane preparation, 0.5 mg of protein/ml. (䉬) GTP + GDP; (䉱) GMP; (䊏) Guo; (䊉) Gua. No Xn and Xao formation were observed.
without detectable intermediate adenosine formation by post-mitochondrial extracts. We propose that, as long as intracellular ATP is maintained at normoxic concentration, its catabolism
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follows the IMP pathway and only a minimal amount of adenosine, if any, is generated in rat brain. Any inosine plus hypoxanthine would therefore originate from IMP, rather than adenosine. This view is in accordance
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with the strong activation exerted by millimolar ATP on both adenylate deaminase and cN-II. It is also supported by the low Km of adenosine kinase for adenosine, as compared with that of adenosine deaminase. In normoxic conditions, extracellular ATP is mainly catabolised to adenosine by the ecto-nucleotidase cascade system. The nucleoside is then taken up by the nucleoside transporter to be either anabolised to AMP via adenosine kinase, or degraded to inosine and hypoxanthine (Fig. 10A). During ischemic conditions, a different scenario can be envisaged. The drastic ATP drop relieves the activation of adenylate deaminase and of cN-II, causing ATP catabolism to be channelled towards the adenosine pathway. Enough adenosine becomes available to be released in the extracellular space (Fig. 10B). Moreover, the continued extracellular hydrolysis of nucleotides is important not only for the formation of adenosine, but also of other nucleosides, such as inosine and guanosine and for exerting trophic action in CNS. We are aware that not only ATP concentrations, but also other variables are affected during ischemia/hypoxia, such as intracellular pH and calcium. These might affect the enzymes of purine metabolism. For instance, the activity of the AMP specific cytosolic 5 -nucleotidase, the enzyme responsible of the intracellular adenosine production, has an acidic pH optimum (Zimmermann, 1992). It is therefore conceivable that its activity enhances during ATP breakdown under ischemic conditions. By contrast, the non muscular ATP activated adenylate deaminase, the enzyme responsible of intracellular IMP production in normoxic conditions, has a neutral pH optimum (Barsacchi, Ranieri-Raggi, & Bergamini, 1979). Further experiments in this direction would be helpful in the future.
Acknowledgements
Fig. 10. Pathways of ATP breakdown in rat brain during normoxic conditions (A) and ischemic conditions (B). The enzymes participating in these pathways are: (1) adenylate kinase; (2) adenylate deaminase; (3) cN-II; (4) purine nucleoside phosphorylase; (5) adenosine deaminase; (6) adenosine kinase; (7) ecto-ATPase; (8) ecto-ADPase; (9) ecto-5 -nucleotidase; (10) cytosolic 5 -nucleotidase specific for AMP.
This work is dedicated to Professor Paolo Cerletti, who 50 years ago introduced one of us (P.L.I.) to the fascination of nucleotide metabolism. Our work was supported by the Italian MURST National Interest Project “Molecular mechanisms of cellular and metabolic regulation of polynucleotides, nucleotides and analogs”.
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