Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

Overexpression of an ADP-ribose pyrophosphatase, AtNUDX2, confers enhanced tolerance to oxidative stress in Arabidopsis plants

Mutant pqr-216 from an Arabidopsis activation-tagged line showed a phenotype of increased tolerance to oxidative stress after treatment with 3 μ m paraquat (PQ). Based on the phenotype of transgenic plants overexpressing the genes flanking the T-DNA insert, it was clear that enhanced expression of a Nudix (nucleoside diphosphates linked to some moiety X) hydrolase gene, AtNUDX2 (At5g47650), was responsible for the tolerance. It has been reported that the AtNUDX2 protein has pyrophosphatase activities towards both ADP-ribose and NADH ( Ogawa et al ., 2005 ). Interestingly, the pyrophosphatase activity toward ADP-ribose, but not NADH, was increased in pqr-216 and Pro _35S :AtNUDX2 plants compared with control plants. The amount of free ADP-ribose was lower in the Pro _35S :AtNUDX2 plants, while the level of NADH was similar to those in control plants under both normal conditions and oxidative stress. Depletion of NAD⁺ and ATP resulting from activation of poly(ADP-ribosyl)ation under oxidative stress was observed in the control Arabidopsis plants. Such alterations in the levels of these molecules were significantly suppressed in the Pro _35S :AtNUDX2 plants. The results indicate that overexpression of AtNUDX2 , encoding ADP-ribose pyrophosphatase, confers enhanced tolerance of oxidative stress on Arabidopsis plants, resulting from maintenance of NAD⁺ and ATP levels by nucleotide recycling from free ADP-ribose molecules under stress conditions.     

Biochemistry of an Olfactory Purinergic System: Dephosphorylation of Excitatory Nucleotides and Uptake of Adenosine

Abstract: The olfactory organ of the spiny lobster, Panu-lirus argus , is composed of chemosensory sensilla containing the dendrites of primary chemosensory neurons. Receptors on these dendrites are activated by the nucleotides AMP, ADP, and ATP but not by the nucleoside adenosine. It is shown here that the lobster chemosensory sensilla contain enzymes that dephosphorylate excitatory nucleotides and an uptake system that internalizes the nonexcitatory dephosphorylated product adenosine. The uptake of [³H]-adenosine is saturable with increasing concentration, linear with time for up to 3h, sodium dependent, insensitive to moderate pH changes and has a K _m of 7.1 μ M and a V_max of 5.2 fmol/sensillum/min (573 fmol/μg of protein/min). Double-label experiments show that sensilla dephosphorylate nucleotides extracellularly; ³H from adenine-labeled AMP or ATP is internalized, whereas ³²P from phosphate-labeled nucleotides is not. The dephosphorylation of AMP is very rapid; ³H from AMP is internalized at the same rate as ³H from adenosine. Sensillar 5'-ectonucleotidase activity is inhibited by ADP and the ADP analog α,β-methylene ADP. Collectively, these results indicate that the enizymes and the uptake system whereby chemosensory sensilla of the lobster inactivate excitatory nucleotides and clear adenosine from extracellular spaces are very similar to those present in the internal tissues of vertebrates, where nucleotides have many neuroactive effects.     

Comparative assessment of inorganic pyrophosphate and pyrophosphatase levels of Escherichia coli, Clostridium pasteurianum, and Clostridium thermoaceticum

Abstract Pyrophosphatase (PP_iase) specific activities were much higher in anaerobic cultures of Escherichia coli (0.54 units) than in Clostridium pasteurianum (0.067 units) and Clostridium thermoaceticum (0.017 units) (1 unit = 1 μ mole PP_i hydrolyzed/min per mg cell dry wt.), and were fairly constant throughout the growth of all three organisms. Conversely, intracellular levels of pyrophosphate (PP_i) were very low and constant in E. coli throughout growth (0.3 mM), while those of C. pasteurianum and C. thermoaceticum were higher (1.44 and 0.8 mM, respectively) and peaked sharply during mid log-phase of growth. PP_iase and intracellular PP_i remained relatively constant in E. coli when grown aerobically or anaerobically, and when growth was in medium containing PP_i as the sole source of supplemental phosphorus.     

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF HUMAN CEREBROSPINAL FLUID

Abstract— Cyclic 3',5'-AMP (cAMP) and cyclic 3',5'–GMP (cGMP) phosphodiesterase activities were found in human cerebrospinal fluid (CSF) using low substrate concentration (0.4μM). More rapid hydrolysis of cGMP than that of cAMP was observed in human CSF. However, cGMP hydrolytic activity of CSF was very much lower (0.3 pmol/min/ml CSF) than that of human cerebral cortex (33.7 nmol/min/g wet cortex). The pH optimum was found to be 8.0 (cGMP phosphodiesterase) and 7.5 (cAMP phosphodiesterase). The maximum stimulation of both cAMP and cGMP phosphodiesterase was achieved at 4 mM-MgCl₂. Cyclic AMP had relatively little effect on the hydrolysis of cGMP in CSF and the cortex, while cGMP inhibited hydrolysis of cAMP in both tissues. Snake venom was found to stimulate cAMP and cGMP phosphodiesterase activity of CSF, by 60% and 110% respectively. This stimulation by snake venom was also observed in the cortex phosphodiesterase, but was not observed in human plasma or thyroid phosphodiesterase. When CSF was applied to Sepharose 6B column, cGMP phosphodiesterase was separated into three different molecular forms. A plot of activity against substrate concentration using peak I (largest molecular size) revealed a high affinity ( K _m= 2.6μM) and a low affinity ( K _m= 100μM) for cAMP suggesting the existence of at least two molecular forms of the enzyme. On the other hand, using a cGMP as substrate the only one K _m value (1.90 μm) was obtained. These K _m values of CSF enzymes described above were close to those obtained from human cerebral cortex preparations. The enzyme under peak I corresponded to the cortex enzyme when judged from its molecular size and stimulation by snake venom. It seems likely from our results that at least a part of CSF phosphodiesterase originates from the central nervous system.     

Effects of ATP analogs on the proton pumping by the vacuolar H+-ATPase from maize roots

Understanding the regulatory properties of the activities of the V-type adenosine triphosphatase (ATPase) on tonoplast membranes is important in determining the mechanisms by which this enzyme controls cytoplasmic and vacuolar pH. The possible existence of a regulatory site for adenine nucleotides was examined by comparing the effects of ADP, adenylylimidodiphosphate (AMP-PNP) and 3'- o -(4-benzoyl) benzoyladenine 5'-triphosphate (BzATP) to those of the 2',3'-dialdehyde derivative of AMP (oAMP) and ATP by using highly purified tonoplast vesicles from maize ( Zea mays L. cv. FRB 73) roots. The addition of either AMP-PNP or BzATP reversibly inhibited the initial rate of proton transport catalyzed by the H⁺-ATPase in a concentration-dependent manner. Less than 20 μ M AMP-PNP or 50 μ M BzATP was sufficient to inhibit half the initial rate of proton transport in the presence of 2 m M ATP and an excess of Mg. Both analogs increased the K_m for ATP and reduced the maximum enzyme velocity. The presence of ADP also inhibited proton transport. The characteristics of ADP-induced inhibition were similar to those of BzATP and AMP-PNP. The addition of the periodated derivative of AMP (oAMP) irreversibly inhibited the ATPase in a concentration and time-dependent manner similar to that reported previously (Chow et al. 1992, Plant Physiology 98: 44–52). Irreversible inhibition by oAMP reduced the maximum velocity of the tonoplast ATPase and was prevented by the addition of ATP. The presence of ADP, AMP-PNP or BzATP had no effect on irreversible inhibition by oAMP. The effects of ADP, AMP-PNP and BzATP on the kinetics of ATP utilization and the lack of protection against inhibition by oAMP argue in favor of at least two types of nucleotide binding sites on the V-type ATPase from maize root tonoplast membranes.     

In vivo effects of pyrophosphate in Escherichia coli

Abstract Pyrophosphate (PP_i), a noncompetitive inhibitor of Rho poly(C)-dependent ATPase activity in vitro has been shown to relieve polarity in the lac operon. This indicates that PP_i could inhibit Rho activity in vivo too. An additional effect of PP_i on adenosine 3',5'-cyclic monophosphate (cAMP) synthesis during stationary phase of growth is also described.     

Characterization of the pyrophosphate-dependent proton transport in microsomal membranes from maize roots

Cleared maize ( Zea mays L. cv. LG 11) root homogenates were prepared and layered on the top of sucrose step gradients (10, 35 and 45%). The ATP- and pyrophosphate (PP_i)-dependent proton-pumping activities were recovered almost completely at the 10%/35% interface, corresponding to the microsomal fraction (Golgi, tonoplast and endoplasmic reticulum). The PP_i-dependent proton pump was characterized by the fluorescence quenching of quenching of quinacrine. The pH optimum was 7 to 8. The H⁺-PPase was Mg²⁺-dependent and the K_m for PP_i (in the presence of 3 m M MgSO₄) was 28 μ M . The pump was electrogenic, K⁺-dependent and a permeant anion was necessary to dissipate the membrane potential (NO₃⁻= I⁻ >Br⁻ > Cl⁻). No activity was detected in the presence of electroneutral proton inonophores or, when valinomycin was added, with electrogenic ionophores. The H⁺-PPase was insensitive to vanadate, oligomycin and molybdate. -Diethylstilbestrol (DES) and N,N'-dicyclohexylcarbodiimide (DCCD) were strongly inhibitory at 100 μ M .     

Effect of plasma components on the feeding response of the mosquito Aedes aegypti L. to adenine nucleotides

ABSTRACT. The gorging response of Aedes aegypti to the ATP dissolved in platelet-poor plasma is greater than that of ATP dissolved in 0.15 m NaCl. The plasma components NaHCO₃ and albumin account for the full effect of the potentiation. Phosphate or tris buffers do not duplicate the bicarbonate effect. In 0.15 m NaCl with bicarbonate but lacking albumin the concentrations inducing 50% feeding are 58 μM ATP, 140 μM ADP, 460 μM AMP and 1500 μM cAMP. Non-adenine nucleotides such as ITP and GTP and phytic acid, and 2,3-diphosphoglyceric acid had no activity.     

Failure to corroborate claims that the developing endosperm of wheat (Triticum aestivum) contains significant activities of fructose-1,6-bisphosphatase

This work was done to test claims (Sangwan and Singh, Physiol. Plant. 73: 21–26) that the developing endosperm of wheat ( Triticum aestivum L.) contains a cytosolic and a plastidic fructose- 1,6-bisphosphatase (EC 3.1.3.11; FBPase). Repetition of the procedure of Sangwan and Singh with extracts of developing endosperm of Triticum aestivum cv. Mercia produced two peaks of apparent FBPase activity on elution from DEAE-cellulose. Both peaks showed high activity of pyrophosphate:fructose-6-phos-phate 1-phosphotransferase [EC 2.7.1.90; PFK(PP_i)]. The apparent FBPase activity in both peaks was stimulated by 20 μ M fructose-2,6-bisphosphate and inhibited by antibodies to PFK(PP_i). Antibody to plastidic FBPase did not react positively in an immunoblot analysis with any protein of M_r comparable to that of known FBPase in either peak. It is argued that the ability of each peak to convert fructose-1,6-bisphosphate to fructose-6-phosphate was due to PFK(PP_i). and that there remains no substantiated evidence for the presence of a plastidic FBPase in the developing endosperm of wheat.     

Acetyl-coenzyme A synthetase in the thermophilic, acetate-utilizing methanogen Methanothrix sp. strain CALS-1

Abstract There is considerable evidence that acetyl-CoA synthetase (acetate thiokinase, ACS, EC 6.2.1) is responsible for acetate activation in the mesophilic acetotrophic methanogen Methanothrix soehngenii . If the pyrophosphate produced by ACS is simply cleaved, two high-energy phosphodiester bonds are expended in acetate activation. Hi High ACS activity (2–4 μmol min⁻¹ mg protein⁻¹) was present in cell-free extracts of the thermophile Methanothrix sp. strain CALS-1. The 23-fold purified enzyme had a molecular mass near 165 kDa and a subunit molecular mass near 78 kDa, suggesting that the enzyme is a homodimer. The temperature optimum for ACS was near 70°C and the apparent K _m values were 2–4 mM for acetate and 5.5 mM for MgATP. Coenzyme A at concentrations greater than 0.2 mM inhibited ACS, while acetyl-CoA was not inhibitory. AMP and pyrophosphate inhibited ACS with K _i values of 5 mM and 1.5 mM respectively. Other divalent cations could replace Mg²⁺, with Mn²⁺ showing the highest activity. Activity with ITP was 20% of that with ATP, and other nucleotides tested were considerably less active. Since Methanothrix sp. strain CALS-1 has an active soluble pyrophosphatase, it appears that it uses the same energetically costly method for acetate activation as M. soehngenii .     

H+/PPi stoichiometry of a membrane‐bound pyrophosphatase of plant mitochondria

The H⁺/PP_i stoichiometry of the mitochondrial H⁺‐PP_iase from pea ( Pisum sativum L.) stem was determined by two kinetic approaches, and compared with the H⁺/substrate stoichiometries of the mitochondrial H⁺‐ATPase, and the vacuolar H⁺‐PP_iase and H⁺‐ATPase. Using sub‐mitochondrial particles or preparations enriched in vacuolar membranes, the rates of substrate‐dependent H⁺‐transport were evaluated: by a mathematical model, describing the time‐course of H⁺‐gradient (ΔpH) formation; or by determining the rate of H⁺‐leakage following H⁺‐pumping inhibition by EDTA at the steady‐state ΔpH. When the H⁺‐transport rates were divided by those of PP_i or ATP hydrolysis, measured under identical conditions, apparent stoichiometries of ca 2 were determined for the mitochondrial H⁺‐PP_iase and H⁺‐ATPase, and for the vacuolar H⁺‐ATPase. The stoichiometry of the vacuolar H⁺‐PP_iase was found to be ca 1. From these results, it is suggested that the mitochondrial H⁺‐PP_iase may, in theory, function as a primary H⁺‐pump poised towards synthesis of PP_i and, therefore, acting in parallel with the main H⁺‐ATPase.     

Feeding responses of the horsefly, Tabanus nigrovittatus, to physical factors, ATP analogues and blood fractions

ABSTRACT. Wild-caught female horseflies, Tabanus nigrovittatus Macq. (Diptera: Tabanidae), were presented solutions of seven analogues of ATP in 0.15 m NaCl, or various blood fractions, either as free liquids at 22 or 38C or covered with a Parafilm M membrane at 38C. Warming the diet, so that it can stimulate the insects' heat receptors, or presenting it warmed and covered with a membrane, which the flies can pierce and thus deploy their mouthparts as they would when blood-feeding, enhances the response to gorging stimulants. ADP (ED₅₀ 45 μM) was the most potent chemical phagostimulant. There were no significant differences between the potencies of AMP, A(TETRA)P, AMP-PCP, AMP-PNP or AMP-S, which were 3.5-5 times less potent than ADP. Cyclic AMP had no phagostimulatory activity at concentrations of 400 or 1000 μM. The ED₅₀ for washed red blood cells (RBC) in saline was 4.5% (one tenth the concentration found in blood). RBC-free plasma caused only 10% gorging but plasma with 0.5% RBC caused 61% gorging, indicating synergism between RBC and plasma.     

Proton pumping pyrophosphatase from higher plant mitochondria

In higher plant cells, there are some enzymes capable of utilizing pyrophosphate (PP_i) as an energy donor. Among these, membrane-bound proton pumping pyrophosphatases (H⁺-PP_iase) have been identified. In addition to the well-known vacuolar H⁺-PP_iase (V-PP_iase), there is evidence for the presence of a mitochondrial H⁺-PP_iase. This enzyme is localized on the inner surface of the inner membrane and catalyzes the specific hydrolysis of PP_i, coupled to proton transport, with a H⁺/PP_i stoichiometry of ca 2. This activity is Mg²⁺-requiring, is stimulated by monovalent cations, and is inhibited by Ca²⁺, F⁻ and diphosphonates. The H⁺-PP_iase contains a catalytic head which is constituted by a 35-kDa protein which is loosely bound to the inner membrane. This protein exhibits a PP_iase activity, stimulated by phospholipids, with characteristics very similar to the membrane-bound enzyme. The mitochondrial PP_iase is distinct from the V-PP_iase, because an antibody raised against the 35-kDa protein does not react with tonoplast membranes. The mitochondrial H⁺-PP_iase seems to have an F-type structure, similar to the F-ATP synthase and the membrane-bound PP_iases from mammalian and yeast mitochondria. It is suggested that, beside synthesizing PP_i, this enzyme may act as a buffer for the electrochemical proton gradient, by hydrolyzing PP_i, during conditions of oxygen deprivation.     

Effects of pH on proton transport by vacuolar pumps from maize roots

Protons pumps of the tonoplast may be involved in the regulation of cytosolic pH, but the effects of pH on the coupled activities of these transporters are poorly understood. The effects of pH on the activities of the H⁺-translocating pyrophosphatase (PP_iase) and vacuolar-type H⁺-translocating adenosine triphosphatase (H⁺-ATPase) from maize ( Zea mays L. cv. FRB 73) root membranes were assessed by model that simultaneously considers proton transport by the pump and those processes that reduce net transport. The addition of either pyrophosphate or ATP to either microsomal or tonoplast membranes generated a pH gradient. The pH gradient generated in the presence of both substrates was not the sum of the gradients produced by the two substrates added separately. When membranes were separated by sucrose density gradient centrifugation, pyrophosphate (PP_i)-dependent proton transport was associated with light density membranes having tonoplast H⁺-ATPase activity. These results indicate that some portion of the PP_iase was located on the same membrane system as the tonoplast ATPase; however, tonoplast vesicles may be heterogeneous, differing slightly in the ratio of ATP- to PP_i-dependent transport. Proton transport by both the PP_iase and ATPase had maximal activity at pH 7.0 to 8.0 Decreases in proton transport by the ATPase at pH above the optimum were associated with increases in the processes that reduce net transport. Such an association was not observed at pH values below the optimum. These results are discussed in terms of in situ regulation of cytoplasmic pH by the two pumps.     

Investigations on Myelination In Vitro: Regulation of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase by Thyroid Hormone in Cultures of Dissociated Brain Cells from Embryonic Mice

Abstract: The direct influence of l -3,3',5-triiodothyronine (T₃) on the development of 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37, CNPase) is demonstrated by using an in vitro culture system of dissociated embryonic mouse brain cells. Serum from a thyroidectomized calf, which contained low levels of T₃ (31 ng/100 ml), and thyroxine, T₄ (<1 μg/ml), was used in the culture medium in place of normal calf serum (T₃, 103 ng/100 ml; T₄, 5.7 μg/ml) to render the culture responsive to exogenously added T₃. The lower levels of enzyme activity observed in the presence of such a deficient medium could be restored to normal values by T₃ supplementation. Half-maximal effect was obtained with 2.5 ± 10⁻⁹ m -T₃. Three days of hormone treatment resulted in the maximal stimulation of CNPase. T₄ was less effective in inducing CNPase activity and the inactive analog of the hormone, reverse T₃ (3,3',5'-T₃) was ineffective. The morphological appearance of the cells was characterized by deformed (smaller size and less in number) reaggregates in the cultures, lacking hormone.     

Gorging response of Glossina palpalis palpalis to ATP analogues

ABSTRACT. The potencies of seventeen analogues of ATP as gorging inducers for Glossina palpalis palpalis were evaluated. The ranking for effective dose that induced half the flies to gorge (ED₅₀) was: A tetra P 5 ATP=2'd ATP ADP=2'd ADP > AMP-PNP > 3'd ATP 2'3'dd ATP > AMP-PCP > adenosine 5' triphosphate 2',3'dialdehyde AMP-CPP >> AMP. Females detect ATP and its analogues better than males. The ED₅₀ of ATP was 5 × 10^-7 M for teneral females and 1.5 × 10^-6 M for males. According to the potency order of the ATP analogues, the G.p.palpalis gustatory receptors recognizing ATP can be classified as P_2y purinoceptors.     

Feeding responses of the horsefly, Tabanus nigrovittatus, to phagostimulants

ABSTRACT. Wild caught horseflies, Tabanus nigrovittatus Macq. (Diptera, Tabanidae), were presented solutions of 0.15 MNaCl at 37°C containing various concentrations of ATP, ADP, AMP, adenosine, phytic acid or 2,3-diphosphoglycerate in an artificial feeding apparatus. The insects fed upward through a Para-film M® membrane. ADP (ED₅₀ 35 μM) was more potent than ATP (ED₅₀ 112 μM) and AMP (ED₅₀ 382 mUM). All of these diets were deposited in the midgut, an indication that the flies were in the 'blood feeding' mode. Adenosine caused only 23% gorging at 1 mM. Phytic acid caused only 10% gorging at 1 mM and 2,3-diphosphoglyceric acid had no activity at 0.6 mM. Flies would feed only in highly reflective cages under high levels of light intensity (1200–1500 lux) at the membrane surface.     

Phosphorylation of the α and β polypeptides of the light-harvesting complex I (B870) of Rhodobacter capsulatus in an in vitro translation system

Abstract Sporozoites and unsporulated oocysts of Eimeria tenella were shown to contain a pyrophosphate-dependent phosphofructokinase (PP_i-PFK) but apparently lack an ATP-specific activity. The PP_i-PFK resembles those that occur in a number of other protists in being reversible and not subject to metabolic control. In contrast, the ADP-utilising pyruvate kinase, present in two developmental stages of the parasite, exhibited strong positive cooperativity with respect to its substrate, phosphoenolpyruvate, and was shown to be allostetically activated by glucose 6-phosphate, fructose 6-phosphate and AMP. It is suggested that the PP_i-PFK represents an adaptation of the parasite towards life in an environment containing only low concentrations of oxygen and that the unusual allosteric regulation of pyruvate kinase evolved to compensate for glycolysis not being controlled at the PP_i-PFK step.     

Beta,gamma-methylene ATP-induced cAMP formation in C6Bu-1 cells: involvement of local metabolism and subsequent stimulation of adenosine A2B receptor

The mechanism underlying beta,gamma-methylene ATP (beta,gamma-MeATP)-induced cAMP elevation was investigated in rat glioma C6Bu-1 cells. Beta,gamma-MeATP increased forskolin-stimulated cAMP formation in a manner sensitive to both the P1 antagonist xanthine amine congener (XAC) and the P2 antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Adenosine deaminase (ADA; 1 U/mL), which abolished the adenosine-induced response, did not eliminate the beta,gamma-MeATP-induced response. However, combination of ADA with alpha,beta-methylene ADP (alpha,beta-MeADP), an ecto-5'-nucleotidase inhibitor, blocked the beta,gamma-MeATP-induced response. AMP, the substrate for ecto-5'-nucleotidase, also induced cAMP formation in a manner sensitive to XAC and alpha,beta-MeADP inhibition. However, the AMP-induced response was not blocked by PPADS. HPLC analyses revealed that adenosine was generated from beta,gamma-MeATP and AMP. In addition, alpha,beta-MeADP inhibited the conversion of beta,gamma-MeATP and AMP to adenosine, whereas PPADS blocked adenosine formation from beta,gamma-MeATP but not from AMP. [3H]Adenosine generated from [3H]AMP was preserved on the cell surface environment even in the presence of ADA. The mRNAs for ecto-phosphodiesterase/pyrophosphatase 1 (EC 3.1.4.1), ecto-5'-nucleotidase (EC 3.1.3.5) and adenosine A2B receptor were detected by RT-PCR. These results suggest that C6Bu-1 cells possess ecto-enzymes converting beta,gamma-MeATP to adenosine, and the locally accumulated adenosine in this mechanism efficiently stimulates A2B receptors in a manner resistant to exogenous ADA.