Effects of adenosine receptor agonists on volume-activated ion transport in pig red cells.

Swelling of pig red cells leads to an increase in a chloride-dependent K flux which can be potentiated by cAMP, whereas cell shrinking causes a selective increase in Na movement which is mediated by a Na/H exchanger. We examined the influence of adenosine and adenosine receptor agonists on the volume-sensitive, ouabain-resistant, chloride-dependent K flux, referred to as Rb flux and volume-activated Na/H exchange pathway. It was found that adenosine and adenosine receptor agonists inhibited the Rb flux. N6-cyclohexyl adenosine (CHA) has been found to be the most potent inhibitor with EC50 of approximately 4.5 microM followed by 2-chloroadenosine (Cl-ado) with EC50 of approximately 27 microM and 5'-(N-ethyl)-carboxamido-adenosine (NECA) with EC50 of approximately 185 microM. CHA also inhibits the cAMP-stimulated Rb flux. However, CHA does not alter the basal intracellular cAMP level nor the intracellular cAMP content raised by exogenously added cAMP. In contrast to the adenosine agonist action on the Rb flux, Na/H exchange, which is activated upon cell shrinkage, exhibits a slight stimulation in response to CHA. These findings suggest that the presence of A1 adenosine receptors on the surface of red cells influences the regulation of volume-activated ion transport.     

Properties of rat heart adenosine kinase.

Adenosine kinase was purified 870-fold from rat heart by a combination of gel filtration and affinity chromatography. The preparation was free of purine-metabolizing enzymes that could interfere in the assay of the kinase. A study of the properties of the purified enzyme showed that it is activated by Na+ and K+, it possesses a broad pH optimum between 6 and 8, MgATP is the nucleotide substrate, free Mg2+ is an inhibitor with respect to both MgATP and adenosine, and the enzyme is subject to substrate inhibition by adenosine. The severity of this inhibition increases as the concentration of free Mg2+ increase. The Km for MgATP was calculated to be 0.8 mM and that for adenosine, at likely physiological concentrations of MgATP and free MgCl2, was about 0.2 microM. In vivo the enzyme is likely to be saturated with both MgATP and adenosine. Indeed, the adenosine concentration in rat heart in vivo is probably sufficient to cause substrate inhibition, and this would be increased by an increase in free Mg2+ concentration. Changes in the concentrations of adenosine and free Mg2+ may play a role in modifying the activity of the enzyme in vivo.     

Na+/adenosine co-transport in Vibrio parahaemolyticus

Adenosine transport in Vibrio parahaemolyticus was studied. Na+ greatly stimulated adenosine uptake. Addition of adenosine to a cell suspension under anaerobic conditions elicited Na+ uptake, and the Na+ uptake was inhibited by monensin, an Na+ ionophore. Imposition of an electrochemical potential of Na+ or a membrane potential in energy-depleted cells elicited adenosine uptake. Therefore, adenosine transport in this organism was concluded to proceed by an Na+/adenosine co-transport mechanism. The Na+/adenosine co-transport system was induced when cells were grown in the presence of adenosine, and repressed by glucose. Although Na+ uptake elicited by adenosine was reduced by glucose, it was enhanced by methyl alpha-glucoside, which reduced the intracellular ATP level. Thus, the effects of glucose and the glucoside on the Na+/adenosine co-transport system did not seem to be due to inducer exclusion, but to be related to the intracellular ATP level.     

The localization and properties of membrane adenosine triphosphatases in the guinea-pig placenta.

The distribution and properties of cytochemically demonstrable phosphatases in the near-term guinea-pig placenta were examined using a strontium capture technique for sodium- and potassium-dependent adenosine triphosphatase (Na+, K+-ATPase) and a lead capture technique for magnesium-dependent adenosine triphosphatase (Mg2+-ATPase). Localizations with the strontium technique in the presence of an alkaline phosphatase inhibitor were mainly on the syncytiotrophoblast plasma membranes; the reaction was potassium-dependent and ouabain-sensitive. Reaction product using the lead capture method was found on both trophoblast and endothelial cell plasma membranes and was independent of magnesium and insensitive to p-hydroxymercuribenzoate (POHMB), an inhibitor of membrane ATPases. However, a very large proportion of this reaction could be blocked by an alkaline phosphatase inhibitor. It is concluded that the strontium capture technique gave a reliable localization for Na+, K+-ATPase. However, the lead capture method mainly demonstrated alkaline phosphatase, and does not offer a useful approach to specific ATPase studies in this particular system.     

Adenosine reduces the reverse mode of the Na+/Ca(2+) exchanger in ferret cardiac fibres

The aim of this study was to investigate the effects of adenosine on reverse mode Na+/Ca(2+) exchange. In intact ferret cardiac trabeculae, Na+-free contractures were investigated after treating preparations with ryanodine, a sarcoplasmic reticulum Ca(2+) -channel inhibitor, and thapsigargin, a sarcoplasmic reticulum Ca(2+) -pump inhibitor added to suppress the sarcoplasmic reticulum function. The effects of adenosine (50-100 nmol/L), adenosine deaminase (ADA, 0.1-0.5 U/L), the A1 and A2A receptor agonists CCPA (3-100 nmol/L) and CGS 21680 (25-100 nmol/L), and the A1 and A2A receptor antagonists DPCPX (25 nmol/L) and ZM 241385 (25 nmol/L) were tested on Na+-free contractures. The application of adenosine (50-100 nmol/L) had no significant effect on the characteristics of the Na+-free contractures. However, the results show that treatment with ADA (0.3 U/L), adenosine (> or =50 nmol/L) and CCPA, a specific A1 receptor agonist (3-100 nmol/L), all reduced the Na+-free contracture amplitude. In the presence of ADA, the effects of adenosine and CCPA were also reduced by a specific antagonist of A1 receptors (DPCPX, 25 nmol/L). Furthermore, adenosine, ADA, and CCPA did not affect the properties of the contractile apparatus in Triton-skinned fibres. It is therefore proposed that endogenous adenosine reduced the reverse mode of the Na+/Ca(2+) exchanger by acting on A1 receptors present in the sarcolemmal membrane.     

Inhibition of the soluble adenosine triphosphatase from mitochondria by adenylyl imidodiphosphate

1. Adenylyl imidodiphosphate is an inhibitor with high affinity for the soluble ATPase (adenosine triphosphatase) from mitochondria. 2. The reaction of the inhibitor with the ATPase is slow and estimates for the association and dissociation reaction rate constants are given. 3. The number of binding sites for the inhibitor appears to be doubled in the presence of 2,4-dinitrophenol. 4. Adenylyl imidodiphosphate is less effective as an inhibitor of the ATPase activity of this enzyme than of the inosine triphosphatase activity. It is also less effective on the ATPase of frozen-thawed or intact mitochondria and did not inhibit ADP-stimulated respiration by intact mitochondria.     

The mode of inhibition by calcium of cell-membrane adenosine-triphosphatase activity

1. The mechanism of the inhibition of Na(+)-plus-K(+)-activated adenosine triphosphatase by calcium was investigated with an enzyme preparation from rabbit kidney cortex and with membranes of human erythrocytes. 2. CaATP, rather than ionic Ca(2+), acts as a competitive inhibitor, competing with MgATP in the Na(+)-plus-K(+)-activated adenosine-triphosphatase reaction. 3. There appears to be no competition between calcium and Na(+) for the activation of adenosine triphosphatase. 4. The inhibition of Na(+)-plus-K(+)-activated adenosine triphosphatase of cell membranes by low concentrations of CaATP and the consequent need of intact cells to keep the cytoplasmic concentration of calcium low relative to that of magnesium suggests a raison d'être for the mitochondrial calcium pump.     

A novel mechanism of cation/substrate cotransport: Na+/H+/adenosine cotransport in Vibrio parahaemolyticus

Adenosine is actively transported with Na+ in Vibrio parahaemolyticus (Sakai, Y., Tsuda, M., Tsuchiya, T. (1987) Biochim, Biophys. Acta 893, 43-48). The proton conductor carbonylcyanide m-chlorophenylhydrazone, CCCP, strongly inhibited active transport of adenosine at pH 8.5 as well as at pH 7.0. This seemed peculiar because the driving force, an electrochemical potential of Na+, is established by the Na(+)-extruding respiratory chain at pH 8.5 in this organism, although it is established by the function of the Na+/H+ antiporter at pH 7.0. This suggested that H+ might be involved in the adenosine transport. We detected H+ uptake induced by adenosine influx in V. parahaemolyticus cells in the presence of Na+, but not in its absence, suggesting the occurrence of Na+/H+/adenosine cotransport. We isolated formycin A-resistant mutants which showed defective adenosine transport. The mutation resulted in simultaneous losses of Na+ uptake and H+ uptake induced by adenosine. In revertants from these mutants the Na+ uptake and H+ uptake were restored simultaneously. The frequencies of reversion were in the order of 10(-7), indicating that the mutations were single mutations; namely that Na+/adenosine cotransport and H+/adenosine cotransport took place via the same carrier. Thus, we conclude that adenosine is transported by the novel mechanism of Na+/H+/adenosine cotransport in V. parahaemolyticus.     

Role of S-adenosylhomocysteine hydrolase in adenosine metabolism in mammalian heart.

S-Adenosylhomocysteine hydrolase of mammalian hearts from different species is exclusively a cytosolic enzyme. The apparent Km for the guinea-pig enzyme was 2.9 microM (synthesis) and 0.39 microM (hydrolysis). Perfusion of isolated guinea-pig hearts for 120 min with L-homocysteine thiolactone (0.23 mM) and adenosine (0.1 mM), in the presence of erythro-9-(2-hydroxynon-3-yl)adenine to inhibit adenosine deaminase, caused tissue contents of S-adenosylhomocysteine to increase from 3.5 to 3600 nmol/g. When endogenous adenosine production was accelerated by perfusion of hearts with hypoxic medium (30% O2), L-homocysteine thiolactone (0.23 mM) increased S-adenosyl-homocysteine 17-fold to 64.3 nmol/g within 15 min. In the presence of 4-nitro-benzylthioinosine (5 microM), an inhibitor of adenosine transport, S-adenosylhomocysteine further increased to 150 nmol/g. L-Homocysteine thiolactone decreased the hypoxia-induced augmentation of adenosine, inosine and hypoxanthine in the tissue and the release of these purines into the coronary system by more than 50%. Our findings indicate that L-homocysteine can profoundly alter adenosine metabolism in the intact heart by conversion of adenosine into S-adenosylhomocysteine. Adenosine formed during hypoxia was most probably generated within the myocardial cell.     

Role of adenosine deaminase, ecto-(5''-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.     

The effect of caffeine, adenosine, and buffer ionic composition on the induction of cell-surface cAMP binding during starvation of Dictyostelium discoideum

We have analyzed the effects of the cAMP relay inhibitor, caffeine, and the receptor antagonist, adenosine, on the regulation of the cell-surface cAMP receptor in suspension-starved Dictyostelium discoideum cells by measuring ammonium sulfate-stabilized binding of [3-H]cAMP to intact cells. When cells were starved in fast (230 r.p.m.) shaken suspension in 10 mM Na+/5 mM K+ phosphate buffer, pH 6.5, plus 1 mM CaCl2 and 2.5 mM MgCl2, and assayed for specific cAMP binding, receptor accumulation peaked at approximately 6 hours, reaching a maximum of 1.5 pmol cAMP bound/10(7) cells (saturation binding). Neither caffeine nor adenosine inhibited the accumulation of cAMP receptors. Similar results were obtained in caffeine-treated, slow shaken (90 r.p.m.) suspension cultures. These results suggest that starvation alone is sufficient stimulus to induce the cAMP receptor. We have also tested the effects of different buffer ionic compositions on the accumulation of cAMP receptors. Elevation of the monovalent ion concentration to 30-40 mM was found to significantly inhibit the induction of cAMP receptors.     

Electrophysiological characterization of the human Na(+)/nucleoside cotransporter 1 (hCNT1) and role of adenosine on hCNT1 function

We previously reported that the human Na(+)/nucleoside transporter pyrimidine-preferring 1 (hCNT1) is electrogenic and transports gemcitabine and 5'-deoxy-5-fluorouridine, a precursor of the active drug 5-fluorouracil. Nevertheless, a complete electrophysiological characterization of the basic properties of hCNT1-mediated translocation has not been performed yet, and the exact role of adenosine in hCNT1 function has not been addressed either. In the present work we have used the two-electrode voltage clamp technique to investigate hCNT1 transport mechanism and study the kinetic properties of adenosine as an inhibitor of hCNT1. We show that hCNT1 exhibits presteady-state currents that disappear upon the addition of adenosine or uridine. Adenosine, a purine nucleoside described as a substrate of the pyrimidine-preferring transporters, is not a substrate of hCNT1 but a high affinity blocker able to inhibit uridine-induced inward currents, the Na(+)-leak currents, and the presteady-state currents, with a K(i) of 6.5 microM. The kinetic parameters for uridine, gemcitabine, and 5'-deoxy-5-fluorouridine were studied as a function of membrane potential; at -50 mV, K(0.5) was 37, 18, and 245 microM, respectively, and remained voltage-independent. I(max) for gemcitabine was voltage-independent and accounts for approximately 40% that for uridine at -50 mV. Maximal current for 5'-DFUR was voltage-dependent and was approximately 150% that for uridine at all membrane potentials. K(0.5)(Na(+)) for Na(+) was voltage-independent at hyperpolarized membrane potentials (1.2 mM at -50 mV), whereas I(max)(Na(+)) was voltage-dependent, increasing 2-fold from -50 to -150 mV. Direct measurements of (3)H-nucleoside or (22)Na fluxes with the charge-associated revealed a ratio of two positive inward charges per nucleoside and one Na(+) per positive inward charge, suggesting a stoichiometry of two Na(+)/nucleoside.     

Reaction of (Na+ + K+)-dependent adenosine triphosphatase with inorganic phosphate. Regulation by Na+, K+, and nucleotides

Effects of Na+, K+, and nucleotides on Mg2+-dependent phosphorylation of (Na+ + K+)-dependent adenosine triphosphatase by Pi were studied under equilibrium conditions. Na+ was a linear competitive inhibitor with respect to Mg2+ and a mixed inhibitor with respect to Pi. K+ was a partial inhibitor; it interacted with positive cooperativity and induced negative cooperativities in the interactions of Mg2+ and Pi with the enzyme. Adenyl-5'-yl (beta, gamma-methylene)diphosphonate, a nonhydrolyzable analog of ATP, interacted with negative cooperativity to inhibit phosphorylation in competition with Pi. ATP was also a competitive inhibitor. Na+ and K+ acted antagonistically, Na+ and nucleotides inhibited synergistically, and K+ and nucleotides were mutually exclusive. In the presence of ouabain, when nucleotides were excluded from the site inhibiting phosphorylation, a low affinity regulatory site for nucleotides became apparent, the occupation of which reduced the rate of dephosphorylation and the initial rate of phosphorylation of the enzyme without affecting the equilibrium constant of the reaction of Pi with the ouabain-complexed enzyme. The regulatory site was also detected in the absence of ouabain. The data suggest that catalytic and transport functions of the oligomeric enzyme may be regulated by homotropic and heterotropic site-site interactions, ligand-induced slow isomerizations, and distinct catalytic and regulatory sites for ATP.     

Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes

Effects of adenosine and pGlu-Glu-ProNH(2) (FPP) on the function and in vitro penetration of boar spermatozoa were examined. First, the effects of dibutyryl cAMP or agonists and antagonists of adenosine receptors (inhibitory adenosine receptors, A1AdR; stimulatory adenosine receptors, A2AdR) on freshly ejaculated spermatozoa were determined by chlortetracycline fluorescence assessment. Capacitation of spermatozoa was stimulated when they were cultured in a medium with dibutyryl cAMP, adenosine, A2AdR agonist, and adenosine plus A1AdR antagonist (CPT). However, acrosome reaction was inhibited only by adenosine. A1AdR agonist did not affect intact spermatozoa. A2AdR antagonist (DMPX) neutralized all of the effects of adenosine. Second, interaction of adenosine and FPP was examined. Gln-FPP, a competitive inhibitor of FPP, and DMPX inhibited the effects of adenosine and FPP, and CPT neutralized the inhibitory effect of FPP on acrosome reaction. Last, the effects of adenosine, FPP, and caffeine on the rate of sperm penetration were examined using frozen-thawed spermatozoa. Adenosine, FPP, and caffeine significantly enhanced the rate of sperm penetration as compared with the case of no additions. Caffeine treatment resulted in a high rate of polyspermic fertilization. In contrast, adenosine and FPP treatments resulted in an increased proportion of normal fertilization in in vitro-matured oocytes. These results suggest that boar spermatozoa can be modulated by the adenylyl cyclase/cAMP pathway via A2AdR in intact cells to induce capacitation and A1AdR in capacitated cells to inhibit spontaneous acrosome loss and that FPP receptors interact with A2AdR in intact cells and with A1AdR in capacitated cells. Furthermore, adenosine and FPP seem to be useful in reducing the incidence of polyspermic penetration.     

Complete release of adenosine deaminase from mouse lymphocytes stabilized by low-pH acetate

Complete release of adenosine deaminase from mouse lymphocytes takes place when intact cells are stabilized by low-pH acetate buffer. Both the low pH and the acetate affect the enzyme extraction markedly. At pH 5.0 all the adenosine deaminase activity detectable in the whole cell homogenates is released into the acetate buffer in very few minutes, with a total amount of 2% protein being extracted. The complete extraction of the enzyme activity is never observed when, at pH 5.0, the acetate is replaced by glutamate, citrate, succinate or maleate and only 45% and 15% of the adenosine deaminase activity is extracted by the acetate at pH 6.0 and 7.0, respectively. The breakdown of adenosine by the enzyme activity extracted from the stabilized cells is due to deamination alone, since inosine is the only product of the catalyzed reaction and its formation is completely inhibited by coformycin, a selective inhibitor of adenosine deaminase. The enzyme extracted shows a specific activity 50-times higher than that found in the crude homogenates, and a substantial purification of the enzyme extracted is achieved by a single Sephadex G-100 gel filtration.     

Substrate specificity, kinetics, and stoichiometry of sodium-dependent adenosine transport in L1210/AM mouse leukemia cells

Two equilibrative (facilitated diffusion) nucleoside transport processes and a concentrative Na(+)-dependent co-transport process contribute to zero-trans inward fluxes of nucleosides in L1210 mouse leukemia cells. Na(+)-linked inward adenosine fluxes in L1210/AM cells (a clone deficient in adenosine, deoxyadenosine, and deoxycytidine kinase activities) were measured as initial rates of [3H]adenosine influx in medium containing Na+ salts and 10 microM dipyridamole. The Na(+)-linked transporter distinguished between the D- and L-enantiomers of adenosine, the latter being a virtual nonpermeant in the initial-rate assay. Adenine arabinoside, inosine, 2'-deoxyadenosine and 2'-deoxyadenosine derivatives with halogen atoms at the purine C-2 position were recognized as substrates of the Na(+)-linked system because of their inhibition of adenosine (10 microM) fluxes under the condition of Na(+)-dependence with IC50 values ranging between 25 and 183 microM; uridine, deoxycytidine, and cytosine arabinoside (each at 400 microM) inhibited adenosine fluxes by 10-40%. Inward Na(+)-linked adenosine fluxes were saturable with respect to extracellular adenosine and Na+ concentrations [( Na+]o); Km and Vmax values for adenosine influx were 9.4 +/- 2.6 microM and 1.67 +/- 0.2 pmol/microliter cell water/s when [Na+]o was 100 mM. The stoichiometry of Na+:adenosine co-transport, determined by Hill analysis of the dependence of adenosine fluxes on [Na+]o, was 1:1. The thiol-reactive agents, N-ethylmaleimide (NEM), showdomycin and p-chloromercuriphenylsulphonate (pCMPS), inhibited Na(+)-linked adenosine fluxes with IC50 values of 40, 10, and 2 microM, respectively. This inhibition was partially reversed by the presence of adenosine in incubation media containing pCMPS, but not NEM. Thiol groups accessible to pCMPS may be involved in substrate recognition by the transporter and in the permeation step.     

Catabolism of adenine nucleotides in adenosine deaminase deficient erythrocytes

$\text{In vitro}$ incubation studies using fluoride and iodoacetate as glycolytic inhibitors have been carried out on red cells of the two subjects with adenosine deaminase deficiency. For comparison, similar studies have also been carried out on red cells from a normal subject and from a child with severe combined immunodeficiency with normal adenosine deaminase activity. The adenosine formed in the adenosine deaminase deficient red cells is a measure of adenosine 5′-phosphate breakdown initiated by 5′-nucleotidase, whereas inosine 5′-phosphate, inosine and hypoxanthine formation is a measure of adenosine 5′-phosphate breakdown initiated by adenylate deaminase. With fluoride as inhibitor, nearly all of the adenosine 5′-phosphate breakdown proceeded by way of adenylate deaminase, while with iodoacetate as inhibitor, 20–30% of the adenosine 5′-phosphate breakdown was initiated by 5′-nucleotidase acting on adenosine 5′-phosphate. In addition, significant amounts of adenine were produced in adenosine deaminase deficient red cells in the presence of the glycolytic inhibitors. Possible explanations for the findings noted in this study are discussed and related to recent studies on the properties of the pertinent purine nucleotide catabolic enzymes.     

Transient adenosine efflux in the rat caudate-putamen

Adenosine is an endogenous byproduct of metabolism that regulates cerebral blood flow and modulates neurotransmission. Four receptors, with affinities ranging from nanomolar to micromolar, mediate the effects of adenosine. Real-time measurements are needed to understand the extracellular adenosine concentrations available to activate these receptors. In this study, we measured the subsecond time course of adenosine efflux in the caudate–putamen of anesthetized rats after a 1 s, high-frequency stimulation of dopamine neurons in the substantia nigra. Fast-scan cyclic voltammetry at carbon-fiber microelectrodes was used for simultaneous detection of adenosine and dopamine, which have different oxidation potentials. While dopamine was immediately released after electrical stimulation, adenosine accumulation was slightly delayed and cleared in about 15 seconds. The concentration of adenosine measured after electrical stimulation was 0.94 ± 0.09 μM. An adenosine kinase inhibitor, adenosine transport inhibitor, and a histamine synthetic precursor were used to pharmacologically confirm the identity of the measured substance as adenosine. Adenosine efflux was also correlated with increases in oxygen, which occur because of changes in cerebral blood flow. This study shows that extracellular adenosine transiently increases after short bursts of neuronal activity in concentrations that can activate receptors.     

Characterization of human adipocyte adenosine receptors

125I-Hydroxyphenylisopropyl adenosine (125I-HPIA) was used to characterize adenosine receptors in human adipocyte plasma membranes. Steady state binding was achieved after 6 h at 37 degrees. Scatchard plots were linear, with a KD of approx. 2.5 nM, and Bmax of 360-1800 fmol/mg protein. (-)N6-phenylisopropyl adenosine (PIA) was a more potent inhibitor of binding than N-ethyl carboxamido adenosine, and (+)PIA was more than 10-fold less potent than (-)PIA, consistent with A1 adenosine receptor binding. Theophylline was a potent inhibitor of binding (IC50 approx. 10 microM). Photoaffinity cross-linking studies demonstrated that the receptor is a single subunit, Mr approx. 43 kDa. The findings demonstrate that the human adipocyte adenosine receptor is similar to the A1 adenosine receptor of rat adipocytes, although its molecular weight is higher, and its affinity for HPIA is lower than that of the rat.     

The mechanism of adenosine production in rat polymorphonuclear leucocytes.

Adenosine production in intact rat polymorphonuclear leucocytes was studied during 2-deoxyglucose-induced ATP catabolism. A cell-free system containing the cytosolic 5'-nucleotidase (EC 3.1.3.5) as the only phosphohydrolase was also studied. The rate of adenosine formation in both intact cells and the cell-free system showed a similar dependence on energy charge (([ATP] + 1/2 [ADP]/([ATP] + [ADP] + [AMP])), being maximal only at values close to 0.8. Sufficient cytosolic 5'-nucleotidase was present in intact cells to explain the observed rate of adenosine formation. We conclude that the cytosolic 5'-nucleotidase is responsible for adenosine production in rat polymorphonuclear leucocytes. This mechanism provides a direct biochemical link between the energy status of a cell and the rate of adenosine formation.