NTPDase and 5' ecto-nucleotidase expression profiles and the pattern of extracellular ATP metabolism in the Walker 256 tumor

In this study, we evaluated the NTPDases and ecto-5'-nucleotidase (CD73) expression profiles and the pattern of adenine nucleotide hydrolysis in rats submitted to the Walker 256 tumor model, 6, 10 and 15 days after the subcutaneous inoculation. Using RT-PCR analysis, we identified mRNA for all of the members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated and a 5'-nucleotidase. By quantitative real-time PCR, Entpd1 (Cd39) and Entpd2 (Cd39L1) and CD73 were identified as the dominant genes expressed by the Walker 256 tumor, at all times studied. Extracellular adenine nucleotide hydrolysis by the Walker 256 tumor was estimated by HPLC analysis. Rapid hydrolysis of extracellular ATP by the tumor cells was observed, leading to the formation of adenosine and inosine in cells obtained from solid tumors at 6 and 10 days after inoculation. Cells obtained from solid tumors at 15 days of growth presented high levels of AMP and presented adenosine as a final product after 90 min of incubation. Results demonstrate that the presence of NTPDases and 5'-nucleotidase enzymes in Walker 256 tumor cells may be important for regulation of the extracellular adenine nucleotides/adenine nucleoside ratio, therefore leading to tumor growth.     

NTPDase and 5′ ecto-nucleotidase expression profiles and the pattern of extracellular ATP metabolism in the Walker 256 tumor

In this study, we evaluated the NTPDases and ecto-5′-nucleotidase (CD73) expression profiles and the pattern of adenine nucleotide hydrolysis in rats submitted to the Walker 256 tumor model, 6, 10 and 15 days after the subcutaneous inoculation. Using RT-PCR analysis, we identified mRNA for all of the members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated and a 5′-nucleotidase. By quantitative real-time PCR, Entpd1 (Cd39) and Entpd2 (Cd39L1) and CD73 were identified as the dominant genes expressed by the Walker 256 tumor, at all times studied. Extracellular adenine nucleotide hydrolysis by the Walker 256 tumor was estimated by HPLC analysis. Rapid hydrolysis of extracellular ATP by the tumor cells was observed, leading to the formation of adenosine and inosine in cells obtained from solid tumors at 6 and 10 days after inoculation. Cells obtained from solid tumors at 15 days of growth presented high levels of AMP and presented adenosine as a final product after 90 min of incubation. Results demonstrate that the presence of NTPDases and 5′-nucleotidase enzymes in Walker 256 tumor cells may be important for regulation of the extracellular adenine nucleotides/adenine nucleoside ratio, therefore leading to tumor growth.     

In vitro effect of homocysteine on nucleotide hydrolysis by blood serum from adult rats

During the past few years, elevated blood levels of homocysteine (Hcy) have been linked to increased risk of premature coronary artery disease, stroke and thromboembolism. These processes can be also related to the ratio adenine nucleotide/adenosine, since extracellularly these nucleotides are associated with modulation of processes such as platelet aggregation, vasodilatation and coronary flow. Furthermore, there are some studies that suggest a relationship between Hcy and plasma adenosine concentrations. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes one of the systems for rapid inactivation of circulating adenine nucleotides. Thus, the main objective of this study was to evaluate if Hcy can participate in the modulation of the extracellular adenine nucleotide hydrolysis by rat blood serum. Our results showed that Hcy, at final concentrations of 5.0 mM, inhibits in vitro ATP, ADP and AMP hydrolysis by 26, 21 and 16%, respectively. Also Hcy, at final concentrations of 8.0mM, inhibited the in vitro hydrolysis of ATP, ADP and AMP by 46, 44 and 44%, respectively. Kinetic analysis showed that the inhibitions of the three adenine nucleotide hydrolyses in the presence of Hcy, by serum of adult rats, is of the uncompetitive type. The IC50 calculated from the results obtained were 6.52+/-1.75 mM (n = 4), 5.18 +/- 0.64 mM (n = 3) and 5.16 +/- 1.22 mM (n = 3) for ATP, ADP and AMP hydrolysis, respectively.     

The effect of ebselen on adenine nucleotide hydrolysis by platelets from adult rats

The extracellular hydrolysis of adenine nucleotides by intact rat blood platelets occurs by the action of a cascade of enzymes constituted by an NTPDase 3 (CD39, EC 3.6.1.5, apyrase) and a 5'-nucleotidase (CD73, EC 3.5.7.3), whose final product is adenosine. Ebselen is a seleno-organic compound that possesses low toxicity and exhibits antioxidant, anti-inflammatory, anti-atherosclerotic, and cytoprotective properties. The main objective of this study was to evaluate if the anti-inflammatory drug ebselen can modulate the extracellular adenine nucleotide hydrolysis by platelets from rats. Our results showed that ebselen, at final concentrations of 30 and 100 microM, inhibits in vitro ATP extracellular hydrolysis by 48 and 60%, respectively. Ebselen, at final concentrations of 100 and 130 microM, also inhibited the in vitro extracellular hydrolysis of ADP by 28 and 35%, respectively. However, this drug did not alter AMP hydrolysis by platelets in the appropriate assay conditions. Kinetic analysis showed that the inhibition of ADP and ATP hydrolysis by ebselen, in rat platelets, is of the uncompetitive type. The IC50 calculated from the results were 99 +/- 10 and 186 +/- 47 microM (mean +/- S.D., n = 3) for ATP and ADP hydrolysis, respectively.     

Relation of tegumentary phosphohydrolase to purine and pyrimidine transport in Schistosoma mansoni.

Inhibition of the saturable influx of 0.05 mM 14C-labeled adenine or adenosine by AMP in adult Schistosoma mansoni in vitro suggested hydrolysis of this nucleotide at the tegumental surface of the parasite. Adenosine liberated as a result of AMP hydrolysis was the inhibitor of uptake of labeled adenine or adenosine. Inhibition of adenosine uptake by AMP or ATP was relieved by paranitrophenyl phosphate or ammonium molybdate supporting the hypothesis of nucleotide hydrolysis at the tengumental surface. Addition of glucose-1-phosphate, glucose-6-phosphate, NaF, or cysteine did not relieve AMP inhibition of adenosine uptake indicating substrate and inhibitor specificity for the surface enzyme(s). AMP, ATP, UMP, and p-nitrophenyl are hydrolyzed, at least in part, by the same enzyme(s). Apparent absorption of labeled AMP was preceded by hydrolysis, with labeled adenosine as the actual compound absorbed, although there was a small diffusion component for absorption of intact AMP. The site of nucleotide hydrolysis in close proximity to absorption sites provides a kinetic advantage for uptake of products of adenine nucleotide hydrolysis but not for products of uracil nucleotide hydrolysis.     

Extracellular metabolism of nucleotides in neuroblastoma x glioma NG108-15 cells determined by capillary electrophoresis

1. The metabolism of extracellular nucleotides in NG108-15 cells, a neuroblastoma × glioma hybrid cell line, was studied by means of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC).2. In NG108-15 cells ATP, ADP, AMP, UTP, UDP, and UMP were hydrolyzed to the nucleosides adenosine and uridine indicating the presence of ecto-nucleotidases and ecto-phosphatases. The hydrolysis of the purine nucleotides ATP and ADP was significantly faster than the hydrolysis of the pyrimidine nucleotides UTP and UDP.3. ATP and UTP breakdown appeared to be mainly due to an ecto-nucleotide- diphosphohydrolase. ADP, but not UDP, was initially also phosphorylated to some extent to the corresponding triphosphate, indicating the presence of an adenylate kinase on NG108-15 cells. The alkaline phosphatase (ALP) inhibitor levamisole did not only inhibit the hydrolysis of AMP to adenosine and of UMP to uridine, but also the degradation of ADP and to a larger extent that of UDP. ATP and UTP degradation was only slightly inhibited by levamisole.4. These results underscore the important role of ecto-alkaline phosphatase in the metabolism of adenine as well as uracil nucleotides in NG108-15 cells. Dipyridamole, a potent inhibitor of nucleotide breakdown in superior cervical ganglion cells, had no effect on nucleotide degradation in NG108-15 cells.5. Dipyridamole, which is a therapeutically used nucleoside reuptake inhibitor in humans, reduced the extracellular adenosine accumulation possibly by allosteric enhancement of adenosine reuptake into the cells.     

Thyroid hormones alter the adenine nucleotide hydrolysis in adult rat blood serum

The effects of ATP, ADP, and adenosine in the processes of platelet aggregation, vasodilatation, and coronary flow have been known for many years. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes the main system for rapid inactivation of circulating adenine nucleotides. Thyroid disorders affect a number of biological factors including adenosine levels in different fractions. Then, we intend to investigate if the soluble nucleotidases responsible for the ATP, ADP, and AMP hydrolysis are affected by variations in the thyroid hormone levels in blood serum from adult rats. Hyperthyroidism was induced by daily intraperitoneal injections of L-thyroxine (T4) (2.5 and 10.0 μg/100 g body weight, respectively) for 7 or 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) added to their drinking water during 7 or 14 days. The treatments efficacy was confirmed by determination of hemodynamic parameters and cardiac hypertrophy evaluation. T4 treatment predominantly inhibited, and hypothyroidism (14 days after thyroidectomy) predominantly increased the ATP, ADP, and AMP hydrolysis in rat blood serum. These results suggest that both excess and deficiency of thyroid hormones can modulate the ATP diphosphohydrolase and 5'-nucleotidase activities in rat blood serum and consequently modulate the effects mediated by these enzymes and their products in vascular system.     

Hyperthyroidism changes nociceptive response and ecto-nucleotidase activities in synaptosomes from spinal cord of rats in different phases of development

Changes in transport, receptors and production of extracellular adenosine have been observed after induction of hyperthyroidism. Adenosine is associated with inhibitory actions such as reduction in release of excitatory neurotransmitters and antinociception at spinal site. In contrast, ATP acts as an excitatory neurotransmitter and produces pronociceptive actions. ATP may be completely hydrolyzed to adenosine by an enzyme chain constituted by an ATP diphosphohydrolase and an ecto-5'-nucleotidase, as previously described in the spinal cord. Thus, we now investigated the effects of the hyperthyroidism on adenine nucleotide hydrolysis in the spinal cord and verified the nociceptive response in this pathology during different phases of development. Hyperthyroidism was induced in male Wistar rats, aged 5, 60 and 330 days by daily intraperitoneal injections of L-thyroxine (T4) for 14 days. Nociception was assessed with a tail-flick apparatus. Rats starting the treatment aged 5 days demonstrated a significant increase in ADP and AMP hydrolysis and increased tail-flick latency (TFL). In contrast, in the spinal cord from hyperthyroid rats aged 60 and 330 days old, the hydrolysis of ATP, ADP and AMP were significantly decreased. Accordingly, the tail-flick latency was decreased, indicating a hyperalgesic response. These results suggest the involvement of ecto-nucleotidases in the control of the hyperthyroidism-induced nociceptive response in rats at distinct developmental stages.     

Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.     

Nucleotide metabolizing ecto-enzymes in Walker 256 tumor cells: molecular identification, kinetic characterization and biochemical properties

In this study we describe the molecular identification, kinetic characterization and biochemical properties of an E-NTPDase and an 5'-nucleotidase in Walker 256 cells. For the ATP, ADP and AMP hydrolysis there were optimum pH in the range 6.5-8.0, and absolute requirement for divalent cations (Mg(2+)>Ca(2+)). A significant inhibition of ATP and ADP hydrolysis was observed in the presence of high concentrations of sodium azide and 0.5 mM of Gadolinium chloride. These activities were insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors. The K(m) values were 464.2+/-86.6 microM (mean+/-SEM, n=4), 137.0+/-31 microM (mean+/-SEM, n=5) and 44.8+/-10.2 microM (mean+/-SEM, n=4), and V(max) values were 655.0+/-94.6 (mean+/-SEM, n=4), 236.3+/-27.2 (mean+/-SEM, n=5) and 177.6+/-13.8 (mean+/-SEM, n=5) nmol of inorganic phosphate min(-1) mg of protein(-1) for ATP, ADP and AMP, respectively. Using RT-PCR analysis we identified the mRNA of two members of the ecto-nucleoside triphosphate diphosphohydrolase family (NTPDase 2 and 5) and a 5'-nucleotidase. The presence of NTPDases and 5'-nucleotidase enzymes in Walker 256 tumor cells may be important to regulate the ratio adenine nucleotides/adenine nucleoside extracellularly, therefore motivating tumor growth.     

Increase of Adenine Nucleotide Hydrolysis in Rat Hippocampal Slices after Seizures Induced by Quinolinic Acid

Quinolinic acid (QUIN), an endogenous convulsant compound, overstimulates the glutamatergic system stimulating N-methyl-D-aspartate receptors, enhancing glutamate release and inhibiting glutamate uptake. Glutamate releases the neuroprotector adenosine, which in turn reduces glutamate release and depresses the neuronal activity. Additionally, adenine nucleotides are an important source of adenosine, by action of ecto-nucleotidases. Here we evaluated the adenine nucleotide hydrolysis in hippocampal slices of adult rats in different times after seizures induced by QUIN. After 45 min, there was an increase of ATP and ADP hydrolysis. After 5 h, there was an increase of ATP, ADP and AMP hydrolysis. After 12 h, there was an increase only of ATP hydrolysis. After 24 h, all hydrolysis returned to control levels. As slice preparations maintain tissue integrity, this study indicates, more than previously observed with synaptosomal preparations, that the extracellular production of the neuroprotector adenosine may be involved in brain responses to seizures.     

Effects of phenylalanine and phenylpyruvate on ATP-ADP hydrolysis by rat blood serum

The nucleotide (ATP-ADP)/nucleoside (adenosine) ratio in the circulation can modulate the processes of vasoconstriction, vasodilatation and platelet aggregation. The main objective of the present study with rat blood serum was to evaluate the possibility of changes in nucleotide hydrolysis by phenylalanine (Phe) and phenylpyruvate (PP), the levels of which could increase in the circulation of individuals with phenylketonuria. Results demonstrated that Phe in the range 1.0-5.0 mM inhibited the ADP hydrolysis by rat serum. The effect of inhibition by Phe on ATP hydrolysis appeared only at a concentration of 5.0 mM. PP had no significant effect upon nucleotide hydrolysis. Kinetic analysis indicated that the inhibition of ADP and ATP hydrolysis by Phe in rat blood serum is uncompetitive. Conversely, Phe and PP did not affect the hydrolysis of p-nitrophenyl-5'-TMP by rat serum.     

Role of membrane-bound 5''-nucleotidase in nucleotide uptake by the moderate halophile Vibrio costicola

Intact cells of Vibrio costicola hydrolyzed ATP, ADP, and AMP. The membrane-bound 5'-nucleotidase (C. Bengis-Garber and D. J. Kushner, J. Bacteriol. 146:24-32, 1981) was solely responsible for these activities, as shown by experiments with anti-5'-nucleotidase serum and with the ATP analog, adenosine 5'-(beta gamma-imido)-diphosphate. Fresh cell suspensions rapidly accumulated 8-14C-labeled adenine 5'-nucleotides and adenosine. The uptake of ATP, ADP, and AMP (but not the adenosine uptake) was inhibited by adenosine 5'-(beta gamma-imido)-diphosphate similarly to the inhibition of the 5'-nucleotidase. Furthermore, the uptake of nucleotides had Mg2+ requirements similar to those of the 5'-nucleotidase. The uptake of ATP was competitively inhibited by unlabeled adenosine and vice versa; inhibition of the adenosine uptake by ATP occurred only in the presence of Mg2+. These experiments indicated that nucleotides were dephosphorylated to adenosine before uptake. The hydrolysis of [alpha-32P]ATP as well as the uptake of free adenosine followed Michaelis-Menten kinetics. The kinetics of uptake of ATP, ADP, and AMP also each appeared to be a saturable carrier-mediated transport. The kinetic properties of the uptake of ATP were compared with those of the ATP hydrolysis and the uptake of adenosine. It was concluded that the adenosine moiety of ATP was taken up via a specific adenosine transport system after dephosphorylation by the 5'-nucleotidase.     

P1 and P2 purinergic receptor signal transduction in rat type II pneumocytes

Extracellular ATP is a potent agonist of surfactant phosphatidylcholine (PC) exocytosis from type II pneumocytes in culture. We studied P1 and P2 receptor signal transduction in type II pneumocytes. The EC50 for ATP on PC exocytosis was 10(-6) M, whereas the EC50 for ADP, AMP, adenosine, and the nonmetabolizable ATP analogue alpha,beta-methylene ATP was 10(-4) M. The rank order of agonists for PC exocytosis was ATP greater than ADP greater than AMP greater than adenosine greater than alpha,beta-methylene ATP. The rank order of agonists for phosphatidylinositol (PI) hydrolysis was ATP greater than ADP, whereas AMP, adenosine, and alpha,beta-methylene ATP did not stimulate PI hydrolysis. ATP (10(-4) M) caused a 15-fold increase in adenosine 3',5'-cyclic monophosphate (cAMP) production, and the nonmetabolizable adenosine analogue 5'-N-ethylcarboxyamidoadenosine (10(-6) M) increased cAMP production threefold. The effects of both these agonists on cAMP production were completely inhibited by the adenosine antagonist 8-phenyltheophylline (10(-5) M). The effects of ATP (10(-4) M) on PC exocytosis were inhibited 38% by 10(-5) M 8-phenyltheophylline. Thus, ATP regulates PC exocytosis by activating P2 receptors, which stimulate PI hydrolysis to inositol phosphate, as well as by activating P1 receptors, which stimulate cAMP production. Interactions between the P1 and P2 pathways may explain the high potency of extracellular ATP as an agonist of PC exocytosis.     

Influence of Trypanosoma evansi in adenine nucleotides and nucleoside concentration in serum and cerebral cortex of infected rats

This study aimed to evaluate the adenine nucleotides and nucleoside concentration in serum and cerebral cortex of rats infected with Trypanosma evansi. Each rat was intraperitoneally infected with 1 × 10(6) trypomastigotes suspended in cryopreserved blood (Group A; n = 18). Twelve animals were used as controls (Group B). The infected animals were monitored daily by blood smears. At days 4 and 20 post-infection (PI) it was collected serum and cerebral cortex to measure the levels of ATP, ADP, AMP and adenosine by high performance liquid chromatography (HPLC). In serum there was a significant (P < 0.05) increase in the ATP, AMP and adenosine concentrations at days 4 and 20 PI in infected rats when compared to not-infected. Furthermore, in the cerebral cortex it was observed a significant (P < 0.05) increase in the concentrations of ATP, AMP and decreased adenosine levels at day 4 PI. At day 20 PI it was only observed an increase in the AMP and adenosine concentrations in cerebral cortex of infected rats when compared to not-infected. It was not observed any difference in ADP concentration in serum and brain at days 4 and 20 PI. No change was observed histologically in the cerebral cortex of infected animals. The results allow us to conclude that infection with T. evansi in rats causes an increase in the concentrations of ATP, AMP and adenosine in serum and cerebral cortex the time periods evaluated. These alterations occurred as a result of T. evansi infection which involves neurotransmission, neuromodulation and immune response impairment confirm the importance of the purinergic system in this pathology.     

Extracellular Adenosine, Inosine, Hypoxanthine, and Xanthine in Relation to Tissue Nucleotides and Purines in Rat Striatum During Transient Ischemia

Extracellular (EC) adenosine, hypoxanthine, xanthine, and inosine concentrations were monitored in vivo in the striatum during steady state, 15 min of complete brain ischemia, and 4 h of reflow and compared with purine and nucleotide levels in the tissue. Ischemia was induced by three-vessel occlusion combined with hypotension (50 mm Hg) in male Sprague-Dawley rats. EC purines were sampled by microdialysis, and tissue adenine nucleotides and purine catabolites were extracted from the in situ frozen brain at the end of the experiment. ATP, ADP, and AMP were analyzed with enzymatic fluorometric techniques, and adenosine, hypoxanthine, xanthine, and inosine with a modified HPLC system. Ischemia depleted tissue ATP, whereas AMP, adenosine, hypoxanthine, and inosine accumulated. In parallel, adenosine, hypoxanthine, and inosine levels increased in the EC compartment. Adenosine reached an EC concentration of 40 microM after 15 min of ischemia. Levels of tissue nucleotides and purines normalized on reflow. However, xanthine levels increased transiently (sevenfold). In the EC compartment, adenosine, inosine, and hypoxanthine contents normalized slowly on reflow, whereas the xanthine content increased. The high EC levels of adenosine during ischemia may turn off spontaneous neuronal firing, counteract excitotoxicity, and inhibit ischemic calcium uptake, thereby exerting neuroprotective effects.     

Studies on adenine nucleotide exchange in mitochondria from the muscle tissue of Ascaris lumbricoides (Nematoda)

Adenine nucleotide exchange between the intra- and extramitochondrial compartments of mitochondria isolated from the muscle tissue of Ascaris lumbricoides was investigated. The exchange was specific for ATP and ADP, AMP, adenosine and non-adenine nucleotides were not exchanged at significant rates. All combinations of counter exchange were found to be possible between intra- and extramitochondrial ATP and ADP. Adenine nucleotide exchange in Ascaris muscle mitochondria was inhibited by atractyloside; was strongly temperature dependent; activated by potassium and magnesium and only slightly activated by calcium. The K_m for adenine nucleotide exchange in Ascaris mitochondria was 4·1 and 2·85 μm for ATP and ADP respectively. The properties of adenine nucleotide exchange in Ascaris muscle mitochondria are thus similar in general features to the adenine nucleotide translocase system of mammalian mitochondria.     

Effects of an inhibitor of adenosine deaminase,deoxycoformycin, and of nucleoside transport,propentofylline, on post-ischemic recovery of adenine nucleotides in rat brain

The effects of an adenosine deaminase inhibitor (deoxycoformycin, 500 μg/kg) and of an inhibitor of nucleoside transport (propentofylline, 10 mg/kg) on adenosine and adenine nucleotide levels in the ischemic rat brain were investigated. The brains of the rats were microwaved before, at the end of a 20 min period of cerebral ischemia (4 vessel occlusion+hypotension), or after 5, 10, 45, and 90 min of reperfusion. Deoxycoformycin increased brain adenosine levels during both ischemia and the initial phases of reperfusion. AMP levels were elevated during ischemia and after 5 min of reperfusion. ATP levels were elevated above those in the non-treated animals after 10 and 45 min of reperfusion. ADP levels were elevated above the non-drug controls at 90 min. These increases in ATP, ADP and AMP resulted in significant increases in total adenylates during ischemia, and after 10 min and 90 min of reperfusion. Propentofylline administration resulted in enhanced AMP levels during ischemia but did not alter adenosine or adenine nucleotide levels during reperfusion in comparison with non-treated controls.     

Nucleotide release provides a mechanism for airway surface liquid homeostasis

Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca(2+) -and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A(2b) adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N(6)-ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolndogenayers that eously express a luminal A(2b) adenosine receptor, we found that basal as well asforskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A(2b) receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis.