Stimulus- and cell-type-specific release of purines in cultured rat forebrain astrocytes and neurons

Adenosine is formed during conditions that deplete ATP, such as ischemia. Adenosine deaminase converts adenosine into inosine, and both adenosine and inosine can be beneficial for postischemic recovery. This study investigated adenosine and inosine release from astrocytes and neurons during chemical hypoxia or oxygen-glucose deprivation. In both cell types, 2-deoxyglucose was the most effective stimulus for depleting cellular ATP and for evoking inosine release; in contrast, oxygen-glucose deprivation evoked the greatest adenosine release. alpha,beta-Methylene ADP, an inhibitor of ecto-5'nucleotidase, significantly reduced adenosine release from astrocytes but not neurons. Dipyridamole, an inhibitor of equilibrative nucleoside transporters, inhibited both adenosine and inosine release from neurons. Erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase, reduced neuronal inosine release evoked by oxygen-glucose deprivation but not by 2-deoxyglucose treatment. These data indicate that (1). astrocytes release adenine nucleotides that are hydrolyzed extracellularly to adenosine, whereas neurons release adenosine per se, (2). inosine is formed intracellularly and released via nucleoside transporters, and (3). inosine is formed by an adenosine deaminase-dependent pathway during oxygen-glucose deprivation but not during 2-deoxyglucose treatment. In summary, the metabolic pathways for adenosine formation and release were cell-type dependent whereas the pathways for inosine formation were stimulus dependent.     

Purine uptake and release in rat C6 glioma cells: nucleoside transport and purine metabolism under ATP-depleting conditions

Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.     

Targeting a novel Plasmodium falciparum purine recycling pathway with specific immucillins

Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of polyamine synthesis are recycled in a novel pathway in which 5'-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5'-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5'-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not been described previously. 5'-Methylthio-immucillin-H, a transition state analogue inhibitor that is selective for malarial relative to human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may also have application as anti-malarials.     

Metabolism of Exogenous Purine Bases and Nucleosides by Salmonella typhimurium

Purine-requiring mutants of Salmonella typhimurium LT2 containing additional mutations in either adenosine deaminase or purine nucleoside phosphorylase have been constructed. From studies of the ability of these mutants to utilize different purine compounds as the sole source of purines, the following conclusions may be drawn. (i) S. typhimurium does not contain physiologically significant amounts of adenine deaminase and adenosine kinase activities. (ii) The presence of inosine and guanosine kinase activities in vivo was established, although the former activity appears to be of minor significance for inosine metabolism. (iii) The utilization of exogenous purine deoxyribonucleosides is entirely dependent on a functional purine nucleoside phosphorylase. (iv) The pathway by which exogenous adenine is converted to guanine nucleotides in the presence of histidine requires a functional purine nucleoside phosphorylase. Evidence is presented that this pathway involves the conversion of adenine to adenosine, followed by deamination to inosine and subsequent phosphorolysis to hypoxanthine. Hypoxanthine is then converted to inosine monophosphate by inosine monophosphate pyrophosphorylase. The rate-limiting step in this pathway is the synthesis of adenosine from adenine due to lack of endogenous ribose-l-phosphate.     

Adenosine, Inosine, and Guanosine Protect Glial Cells During Glucose Deprivation and Mitochondrial Inhibition: Correlation Between Protection and ATP Preservation

Abstract: The purpose of this study was to determine the mechanism by which adenosine, inosine, and guanosine delay cell death in glial cells (ROC-1) that are subjected to g lucose d eprivation and m itochondrial respiratory chain inhibition with amobarbital (GDMI). ROC-1 cells are hybrid cells formed by fusion of a rat oligodendrocyte and a rat C6 glioma cell. Under GDMI, ATP was depleted rapidly from ROC-1 cells, followed on a much larger time scale by a loss of cell viability. Restoration of ATP synthesis during this interlude between ATP depletion and cell death prevented further loss of viability. Moreover, the addition of adenosine, inosine, or guanosine immediately before the amobarbital retarded the decline in ATP and preserved cell viability. The protective effects on ATP and viability were dependent on nucleoside concentration between 50 and 1,500 µ M . Furthermore, protection required nucleoside transport into the cell and the continued presence of nucleoside during GDMI. A significant positive correlation between ATP content at 16 min and cell viability at 350 min after the onset of GDMI was established ( r = 0.98). Modest increases in cellular lactate levels were observed during GDMI (1.2 nmol/mg/min lactate produced); however, incubation with 1,500 µ M inosine or guanosine increased lactate accumulation sixfold. The protective effects of inosine and guanosine on cell viability and ATP were >90% blocked after treatment with 50 µ M BCX-34, a nucleoside phosphorylase inhibitor. Accordingly, lactate levels also were lower in BCX-34-treated cells incubated with inosine or guanosine. We conclude that under GDMI, the ribose moiety of inosine and guanosine is converted to phosphorylated glycolytic intermediates via the pentose phosphate pathway, and its subsequent catabolism in glycolysis provides the ATP necessary for maintaining plasmalemmal integrity.     

Ribose 1-phosphate and inosine activate uracil salvage in rat brain

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.     

Is inosine the physiological energy source of pig erythrocytes?

Pig erythrocytes are unable to metabolize glucose and their physiological energy source is unknown. These cells have a high-capacity nucleoside transport system with similar properties to that responsible for nucleoside transport in other species. Nucleoside transport is sufficiently rapid to allow the possibility that inosine and/or adenosine may represent major energy substrates for pig erythrocytes in vivo. Normal and adenosine deaminase-deficient pig erythrocytes have similar ATP levels, suggesting that adenosine is not important in this respect. However, it was calculated that an extracellular inosine concentration of only 40 nM could support the cells' entire energy requirement, a value 40-fold lower than plasma levels of this nucleoside.     

Efficiency of purine utilization by Helicobacter pylori: roles for adenosine deaminase and a NupC homolog

The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase.     

Uptake and metabolism of purine nucleosides and nucleotides in isolated frog skeletal muscle.

When isolated frog skeletal muscles were incubated with ¹⁴C-labeled adenosine, the nucleoside was rapidly taken up by the cells and was either immediately incorporated into adenine nucleotides or deaminated to inosine. Incorporation was predominant at low (micromolar) concentrations whereas, deamination was the major route of metabolism at high (millimolar) concentrations. When muscles were incubated with ¹⁴C-labeled inosine the nucleoside, after entry into the cells, was metabolized to a lesser extent than adenosine. ATP and hypoxanthine were the major products of its metabolism. Intracellular concentrations were calculated using ³H-labeled sorbitol to measure the extracellular space.Because of its lower rate of intracellular metabolism inosine was used to investigate the characteristics of the nucleoside transport system. The uptake of inosine was saturable at high concentrations and was specifically inhibited by the presence of adenosine or uridine in the incubation media. Persantin, a well known specific inhibitor of nucleoside transport, also competitively inhibited inosine uptake, as did theophylline [1, Woo et al. Can J. Physiol. Pharmacol. $\text{52}$, 1063, 1974]. These data, along with the knowledge that in a well-oxygenated muscle, inosine entry follows a downhill chemical potential gradient, strongly support the view that the transport mechanism is facilitated diffusion.The muscle cell membrane does not appear to be permeable to ¹⁴C-labeled ATP under the conditions studied. Investigations of the permeability to the major extracellular degradation products of ATP suggest that AMP was the compound most likely to cross the cell membrane.     

Nucleoside transport and metabolism in erythrocytes from the Yucatan miniature pig. Evidence that inosine functions as an in vivo energy substrate

Erythrocytes from the Yucatan miniature pig, like those from the normal domestic pig, lack functional glucose transporters and were unable to utilize plasma glucose as an energy source. In contrast, inosine and adenosine entered the cells rapidly. The nucleoside transporter responsible for this uptake was identified as a band 4.5 polypeptide (5000 copies per cell; apparent Mr 45 000-66 000). Inosine concentrations in the physiological plasma range (1.6-2.5 microM) were found to maintain normal erythrocyte ATP levels and ATP/ADP ratios during prolonged in vitro incubation of cells at 37 degrees C, an effect that was blocked by the specific nucleoside transport inhibitor, nitrobenzylthioguanosine. In the absence of extracellular nucleoside, cells 'protected' themselves against some of the consequences of deprivation of energy substrate by glycolyzing the ribose moiety of inosine produced during ATP catabolism. Although erythrocytes from the miniature pig were capable of utilizing extracellular adenosine as an energy substrate, plasma samples from these animals contained less than 0.4 microM adenosine. It is concluded that inosine is a major physiological energy source of pig erythrocytes.     

Adenosine kinase mediates high affinity adenosine salvage in Trypanosoma brucei

African sleeping sickness is caused by Trypanosoma brucei. This extracellular parasite lacks de novo purine biosynthesis, and it is therefore dependent on exogenous purines such as adenosine that is taken up from the blood and other body fluids by high affinity transporters. The general belief is that adenosine needs to be cleaved to adenine inside the parasites in order to be used for purine nucleotide synthesis. We have found that T. brucei also can salvage this nucleoside by adenosine kinase (AK), which has a higher affinity to adenosine than the cleavage-dependent pathway. The recombinant T. brucei AK (TbAK) preferably used ATP or GTP to phosphorylate both natural and synthetic nucleosides in the following order of catalytic efficiencies: adenosine > cordycepin > deoxyadenosine > adenine arabinoside (Ara-A) > inosine > fludarabine (F-Ara-A). TbAK differed from the AK of the related intracellular parasite Leishmania donovani by having a high affinity to adenosine (K m = 0.04-0.08 microm depending on [phosphate]) and by being negatively regulated by adenosine (K i = 8-14 microm). These properties make the enzyme functionally related to the mammalian AKs, although a phylogenetic analysis grouped it together with the L. donovani enzyme. The combination of a high affinity AK and efficient adenosine transporters yields a strong salvage system in T. brucei, a potential Achilles' heel making the parasites more sensitive than mammalian cells to adenosine analogs such as Ara-A. Studies of wild-type and AK knockdown trypanosomes showed that Ara-A inhibited parasite proliferation and survival in an AK-dependent manner by affecting nucleotide levels and by inhibiting nucleic acid biosynthesis.     

Metabolism and salvage of adenine and hypoxanthine by myocytes isolated from mature rat heart

Adenine and hypoxanthine can be utilised by cardiac muscle cells as substrates for the synthesis of ATP. A possible therapeutic advantage of these compounds as high-energy precursors is their lack of vasoactive properties. Myocytes isolated from mature rat heart have been used to establish in kinetic detail the capacity of the heart to incorporate adenine, hypoxanthine and ribose into cellular nucleotides. Maximum rates of catalysis by enzymes on the salvage pathways have been established. Whilst the rate of incorporation of adenine into the ATP pool appears to depend upon intracellular concentrations of adenine and phosphoribosylpyrophosphate, for hypoxanthine the pattern is more complex. Hypoxanthine is salvaged at a slow rate compared with adenine, and is incorporated into GTP and IMP as well as into adenine nucleotides. The rate of incorporation of hypoxanthine into both IMP and ATP is accelerated in myocytes incubated with ribose. However, the rate-limiting reaction appears to be that catalysed by adenylosuccinate synthetase, for the rate of ATP synthesis is not accelerated when hypoxanthine concentration is increased from 10 to 50 microM, while the rate of IMP synthesis is more than doubled. Adenine and hypoxanthine phosphoribosyl transferases are present in equal catalytic amounts, but rat cardiac myocytes have very little adenylosuccinate synthetase activity. Exogenous ribose is incorporated into adenine nucleotides in amounts equimolar with adenine or hypoxanthine.     

Structures of human purine nucleoside phosphorylase complexed with inosine and ddI

Human purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effect on B-cell function. PNP is highly specific for 6-oxopurine nucleosides and exhibits negligible activity for 6-aminopurine nucleosides. The catalytic efficiency for inosine is 350,000-fold greater than for adenosine. Adenine nucleosides and nucleotides are deaminated by adenosine deaminase and AMP deaminase to their corresponding inosine derivatives which, in turn, may be further degraded. Here we report the crystal structures of human PNP in complex with inosine and 2('),3(')-dideoxyinosine, refined to 2.8A resolution using synchrotron radiation. The present structures provide explanation for ligand binding, refine the purine-binding site, and can be used for future inhibitor design.     

Adenine nucleotides and adenosine metabolism in pig kidney proximal tubule membranes

Exogenous adenosine triphosphate (ATP) added to brush-border membrane vesicles was rapidly degraded mainly to inosine according to the high ecto-nucleotidase activities in these vesicles. In the absence of phosphate, inosine was slowly transformed into hypoxanthine, and xanthine oxidase and dehydrogenase activities were not detected. The presence of ecto-adenosine deaminase and ecto-adenosine monophosphate (AMP) nucleotidase was shown. The ecto-adenosine deaminase was inhibited by deoxycoformycin and was also detected in rat renal brush-border membrane vesicles. Using orthovanadate, levamisole, and α, β-methylene adenosine diphosphate as possible inhibitors, alkaline phosphatase was shown to be the main agent responsible for ecto-AMP nucleotidase activity. In pig renal basolateral membrane vesicles and in whole cell extracts from pig renal cortex, ecto-AMP nucleotidase was the limiting factor in ATP degradation. Comparing the ATP catabolism in the whole cell cortical extract with the catabolism in the same sample precleared of membranes, it was shown that ectonucleotidase activity is mainly bound to the membranous components. It is also shown that the whole cell extract of pig renal cortex has hypoxanthine phosphoribosyl transferase activity, and it seems probable that the rapid and specific formation of luminal inosine and its transport into the cell in competition with adenosine may start the purine salvage pathway through the synthesis of IMP from hypoxanthine. © Wiley-Liss, Inc.     

Adenine and adenosine salvage pathways in erythrocytes and the role of S-adenosylhomocysteine hydrolase. A theoretical study using elementary flux modes

This article is devoted to the study of redundancy and yield of salvage pathways in human erythrocytes. These cells are not able to synthesize ATP de novo. However, the salvage (recycling) of certain nucleosides or bases to give nucleotide triphosphates is operative. As the salvage pathways use enzymes consuming ATP as well as enzymes producing ATP, it is not easy to see whether a net synthesis of ATP is possible. As for pathways using adenosine, a straightforward assumption is that these pathways start with adenosine kinase. However, a pathway bypassing this enzyme and using S-adenosylhomocysteine hydrolase instead was reported. So far, this route has not been analysed in detail. Using the concept of elementary flux modes, we investigate theoretically which salvage pathways exist in erythrocytes, which enzymes belong to each of these and what relative fluxes these enzymes carry. Here, we compute the net overall stoichiometry of ATP build-up from the recycled substrates and show that the network has considerable redundancy. For example, four different pathways of adenine salvage and 12 different pathways of adenosine salvage are obtained. They give different ATP/glucose yields, the highest being 3:10 for adenine salvage and 2:3 for adenosine salvage provided that adenosine is not used as an energy source. Implications for enzyme deficiencies are discussed.     

Metabolic regulation of ATP breakdown and of adenosine production in rat brain extracts

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 ATP<=>ADP<=>AMP --> IMP --> inosine<=>hypoxanthine, with little, if any, intracellular adenosine production. At low ischemic concentration, intracellular ATP breakdown follows the pathway ATP<=>ADP<=>AMP --> adenosine --> inosine<=>hypoxanthine 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.     

Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1

Like all parasitic protozoa, the human malaria parasite Plasmodium falciparum lacks the enzymes required for de novo synthesis of purines and it is therefore reliant upon the salvage of these compounds from the external environment. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a nucleoside transporter that has been localized to the plasma membrane of the intraerythrocytic form of the parasite. In this study we have characterized the transport of purine and pyrimidine nucleosides across the plasma membrane of 'isolated' trophozoite-stage P. falciparum parasites and compared the transport characteristics of the parasite with those of PfENT1 expressed in Xenopus oocytes. The transport of nucleosides into the parasite: (i) was, in the case of adenosine, inosine and thymidine, very fast, equilibrating within a few seconds; (ii) was of low affinity [K(m) (adenosine) = 1.45 +/- 0.25 mM; K(m) (thymidine) = 1.11 +/- 0.09 mM]; and (iii) showed 'cross-competition' for adenosine, inosine and thymidine, but not cytidine. The kinetic characteristics of nucleoside transport in intact parasites matched very closely those of PfENT1 expressed in Xenopus oocytes [K(m) (adenosine) = 1.86 +/- 0.28 mM; K(m) (thymidine) = 1.33 +/- 0.17 mM]. Furthermore, PfENT1 transported adenosine, inosine and thymidine, with a cross-competition profile the same as that seen for isolated parasites. The data are consistent with PfENT1 serving as a major route for the uptake of nucleosides across the parasite plasma membrane.     

Adenosine stimulates anabolic metabolism in developing castor bean (Ricinus communis L.) cotyledons

In previous experiments it was shown that Castor-bean (Ricinus communis) endosperm releases carbohydrates, amino acids and nucleoside derivatives, which are subsequently imported into the developing cotyledons (Kombrink and Beevers in Plant Physiol 73:370-376, 1983). To investigate the importance of the most prominent nucleoside adenosine for the metabolism of growing Ricinus seedlings, we supplied adenosine to cotyledons of 5-days-old seedlings after removal of the endosperm. This treatment led to a 16% increase in freshweight of intact seedlings within 16 h, compared to controls. Using detached cotyledons, we followed uptake of radiolabelled adenosine and identified 40% of label in solubles (mostly ATP and ADP), 46% incorporation in RNA and 2.5% in DNA, indicating a highly active salvage pathway. About 7% of freshly imported adenosine entered the phloem, which indicates a major function of adenosine for cotyledon metabolism. Import and conversion of adenosine improved the energy content of cotyledons as revealed by a substantially increased ATP/ADP ratio. This effect was accompanied by slight increases in respiratory activity, decreased levels of hexose phosphates and increased levels of fructose-1,6-bisphosphate and triose phosphates. These alterations indicate a stimulation of glycolytic flux by activation of phosphofructokinase, and accordingly we determined a higher activity of this enzyme. Furthermore the rate of [(14)C]-sucrose driven starch biosynthesis in developing castor-bean is significantly increased by feeding of adenosine. In conclusion, our data indicate that adenosine imported from mobilizing endosperm into developing castor-bean cotyledons fulfils an important function as it promotes anabolic reactions in this rapidly developing tissue.     

Extracellular inosine modulates ERK 1/2 and p38 phosphorylation in cultured Sertoli cells: possible participation in TNF-alpha modulation of ERK 1/2

Extracellular ATP and adenosine modulation of MAPKs is well described in different cells types, but few studies have addressed the effects of extracellular inosine on these kinases. Previous results showed that hydrogen peroxide and TNF-alpha increase extracellular inosine concentration in cultured Sertoli cells and this nucleoside protects Sertoli cells against hydrogen peroxide induced damage and participates in TNF-alpha induced nitric oxide production. In view of the fact that MAPKs are key mediators of the cellular response to a large variety of stimuli, we investigated the effect of extracellular inosine on the phosphorylation of ERK 1/2 and p38 MAPKs in cultured Sertoli cells. The involvement of this nucleoside in the activation of ERK 1/2 by TNF-alpha was also investigated. Inosine and the selective A1 adenosine receptor agonist R-PIA increases the phosphorylation of ERK 1/2 and p38, and this was blocked by the selective A1 adenosine receptors antagonists, CPT and DPCPX. These antagonists also inhibited TNF-alpha increase in the phosphorylation of ERK 1/2. TNF-alpha also rapidly augmented extracellular inosine concentration in cultured Sertoli cells. These results show that extracellular inosine modulates ERK 1/2 and p38 in cultured Sertoli cells, possible trough A1 adenosine receptor activation. This nucleoside also participates in TNF-alpha modulation of ERK 1/2.     

Adenosine metabolism in kidney slices under normoxic conditions

The effect of adenosine in rat kidney under normoxic conditions has been studied. It is demonstrated that adenosine modulates cell nucleotide levels. HPLC analysis of the purine compounds inside the cell indicates that adenosine improves the ATP/ADP ratio, whereas it diminishes the adenine content. This behaviour is not due to mediation by specific receptors, as agonists at P1 purinoceptors did not have any effect. Further evidence using inosine as well as dipyridamole and deoxycoformycin indicates that all effects are dependent on the previous uptake of adenosine. The origin of free adenine in the kidney has been investigated, and it appears to come from the phosphorolysis of 5'-methylthio-adenosine. This report is the first to describe the activity of methylthioadenosine phosphorylase (E.C. 2.4.2.28) in the kidney. It is concluded that 1) extracellular adenosine improves guinea pig renal function by increasing the ATP level and the ATP/ADP ratio; and 2) there exists a functional pathway in the kidney that produces adenine and AMP coming from methionine and ATP. This latter pathway probably produces spermine and spermidine, which are likely to be important for renal function.