A Low-Km 5'-Nucleotidase from Rat Brain Cytosolic Fraction: Purification, Kinetic Properties, and Description of Regulation by a Novel Factor that Increases Sensitivity to Inhibition by ATP and ADP

Abstract: A readily soluble 5'-nucleotidase was purified 1,800-fold from rat brain 105,000- g supernatant. The enzyme showed similarity to the 5'-nucleotidase ectoenzyme of plasma membranes. It exhibited a low K _m for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by α,β-methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol-specific phospholipase C increased its hydrophilicity. A single subunit of M_r = 54,300 ± 800 was observed, which is appreciably smaller than published values for the 5'-nucleotidase ectoenzyme or for other low- K _m"soluble" 5'-nucleotidases. The soluble 5'-nucleotidase showed an elution profile on AMP-Sepharose affinity chromatography or on Mono Q ion-exchange chromatography different from that of the brain ectoenzyme. Forty-two percent of the soluble 5'-nucleotidase in brain 105,000- g supernatant did not bind to a Mono Q ion-exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A-Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5'-nucleotidase toward the inhibitor ATP by 20-fold. This factor was also able to increase the inhibition of brain 5'-nucleotidase ectoenzyme by ATP.     

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.     

Absolute rates of adenosine formation during ischaemia in rat and pigeon hearts.

1. The activities of ecto- and cytosolic 5'-nucleotidase (EC 3.1.3.5), adenosine kinase (EC 2.7.1.20), adenosine deaminase (EC 3.5.4.4) and AMP deaminase (EC 3.5.4.6) were compared in ventricular myocardium from man, rats, rabbits, guinea pigs, pigeons and turtles. The most striking variation was in the activity of the ecto-5'-nucleotidase, which was 20 times less active in rabbit heart and 300 times less active in pigeon heart than in rat heart. The cytochemical distribution of ecto-5'-nucleotidase was also highly variable between species. 2. Adenosine formation was quantified in pigeon and rat ventricular myocardium in the presence of inhibitors of adenosine kinase and adenosine deaminase. 3. Both adenosine formation rates and the proportion of ATP catabolized to adenosine were greatest during the first 2 min of total ischaemia at 37 degrees C. Adenosine formation rates were 410 +/- 40 nmol/min per g wet wt. in pigeon hearts and 470 +/- 60 nmol/min per g wet wt. in rat hearts. Formation of adenosine accounted for 46% of ATP plus ADP broken down in pigeon hearts and 88% in rat hearts. 4. The data show that, in both pigeon and rat hearts, adenosine is the major catabolite of ATP in the early stages of normothermic myocardial ischaemia. The activity of ecto-5'-nucleotidase in pigeon ventricle (16 +/- 4 nmol/min per g wet wt.) was insufficient to account for adenosine formation, indicating the existence of an alternative catabolic pathway.     

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.     

Effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis

Here we report the effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis was 5- to 7-fold higher for the fresh clinical strain, when compared with the ATCC (American Type Culture Collection) strain. ATP hydrolysis was activated in the presence of metronidazole in the ATCC strain, whilst it was inhibited 33% by 50 microM tinidazole in a fresh clinical isolate. The treatment of cells in the presence of metronidazole for 2 h inhibited ATP and ADP hydrolysis, whilst treatment with tinidazole inhibited ATP and ADP hydrolysis only in the fresh clinical isolate. The drugs did not change the ecto-5'-nucleotidase activity for both strains. Our results suggest that the modulation of extracellular ATP and ADP levels during treatment with these drugs could be a parasitic defence strategy as a survival mechanism in an adverse environment.     

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.     

Adenosine production inside rat polymorphonuclear leucocytes.

Adenosine synthesis was studied during 2-deoxyglucose-induced ATP catabolism in intact rat polymorphonuclear leucocytes. When both adenosine kinase (EC 2.7.1.20) and adenosine deaminase (EC 3.5.4.4) were selectively inhibited, adenosine accumulated. Adenosine formation took place inside the intact cells by a metabolic pathway independent of the ecto-5'-nucleotidase (EC 3.1.3.5). Distinct metabolic pathways are proposed for adenosine production from intracellular or extracellular nucleotides.     

An Ecto-Nucleotide Pyrophosphatase Is One of the Main Enzymes Involved in the Extracellular Metabolism of ATP in Rat C6 Glioma

Abstract : The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the α-and γ-phosphates. The enzyme degraded ATP into AMP and PP_i and, depending on the ATP concentration, accounted for ~50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 μ M abolished the degradation of ATP into AMP and PP_i. The nucleotide pyrophosphatase has an alkaline pH optimum and a K _m for ATP of 17 ± 5 μ M . The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.     

Adenosine Formation and Release by Embryonic Chick Neurons and Glia in Cell Culture

Adenosine formation and release were studied in 48-h-old cultured ciliary ganglia and confluent peripheral and CNS glial cultures from embryonic chicks. Metabolic poisoning induced by 30 mM 2-deoxyglucose and 2 micrograms/ml oligomycin reduced ATP concentration by 90%. An increase in adenosine accounted for 15-40% of the fall in ATP. Dilazep (3 X 10(-6) M), a nucleoside transport inhibitor, decreased both incorporation of adenosine (an index of nucleoside transport) and release of adenosine by 80-90%. Dilazep trapped the newly formed adenosine intracellularly. A concentration of alpha, beta-methylene ADP that inhibited ecto-5'-nucleotidase by 80-90% did not alter the concentration of adenosine or AMP in neurons, glia, or medium. The results demonstrate that adenosine is formed intracellularly and exported out of the cell via the nucleoside transporter. The participation of ecto-5'-nucleotidase was excluded.     

Phospholipase C axis is the preferential pathway leading to PKC activation following PTH or PTHrP stimulation in human term placenta

In the present report we describe an NTPDase 1 (ATP diphosphohydrolase; ecto-apyrase; EC 3.6.1.5) in rat hippocampal slices. The effect of glutamate on the ATPase and ADPase activities in rat hippocampal slices of different ages was also studied since adenosine, the final product of an enzymatic chain that includes NTPDase 1 and 5'-nucleotidase, can act upon A1 receptors in turn decreasing the release of glutamate. Hippocampal slices from 7, 14, 20-23 and 60 day-old rats were prepared and ATPase and ADPase activities were measured. The parallelism of ATPase and ADPase activities in all parameters tested indicated the presence of an ATP diphosphohydrolase. In addition, a Chevillard plot indicated that ATP and ADP are hydrolyzed at the same active site on the enzyme. ATPase activity was significantly activated by glutamate in 20-23 and 60 day-old rats, but ADPase activity was not activated. These results could indicate distinct behavior of the ATPase and ADPase activities of NTPDase 1 in relation to glutamate or the simultaneous action of the ecto-ATPase. Activation of ATPase activity by glutamate may constitute an important role in this developmental period, possibly protecting against the neurotoxicity induced by ATP, as well as producing high levels of ADP, by increasing adenosine production, a neuroprotective compound.     

ATP hydrolysis pathways and their contributions to pial arteriolar dilation in rats

ATP is thought to be released to the extracellular compartment by neurons and astrocytes during neural activation. We examined whether ATP exerts its effect of promoting pial arteriolar dilation (PAD) directly or upon conversion (via ecto-nucleotidase action) to AMP and adenosine. Blockade of extracellular direct ATP to AMP conversion, with ARL-67156, significantly reduced sciatic nerve stimulation-evoked PADs by 68%. We then monitored PADs during suffusions of ATP, ADP, AMP, and adenosine in the presence and absence of the following: 1) the ecto-5'-nucleotidase inhibitor α,β-methylene adenosine 5'-diphosphate (AOPCP), 2) the A(2) receptor blocker ZM 241385, 3) the ADP P2Y(1) receptor antagonist MRS 2179, and 4) ARL-67156. Vasodilations induced by 1 and 10 μM, but not 100 μM, ATP were markedly attenuated by ZM 241385, AOPCP, and ARL-67156. Substantial loss of reactivity to 100 μM ATP required coapplications of ZM 241385 and MRS 2179. Dilations induced by ADP were blocked by MRS 2179 but were not affected by either ZM 241385 or AOPCP. AMP-elicited dilation was partially inhibited by AOPCP and completely abolished by ZM 241385. Collectively, these and previous results indicate that extracellular ATP-derived adenosine and AMP, via A(2) receptors, play key roles in neural activation-evoked PAD. However, at high extracellular ATP levels, some conversion to ADP may occur and contribute to PAD through P2Y(1) activation.     

Nature of Extrasynaptosomal Accumulation of Endogenous Adenosine Evoked by K+ and Veratridine

When rat brain synaptosomes were incubated for 10 min at 37 degrees C, basal accumulation of adenosine in the medium was 66 pmol/mg of protein. An elevated K+ level (24 mM) evoked an additional accumulation of 200 pmol/mg of protein, and 50 microM veratridine evoked 583 pmol of adenosine accumulation/mg of protein. K+- and veratridine-evoked accumulation of adenosine did not arise from microsomal or mitochondrial contaminants of the synaptosomal preparation, because purified microsomes and mitochondria did not exhibit evoked accumulation of adenosine in the medium. K+-evoked accumulation of extrasynaptosomal adenosine was Ca2+-dependent, whereas veratridine-evoked accumulation of adenosine was increased in Ca2+-free medium. In the presence of alpha,beta-methylene ADP and GMP, which inhibit ecto-5'-nucleotidase, conversion of added ATP and AMP to adenosine was inhibited by 90% in synaptosomal suspensions. However, inhibition of ecto-5'-nucleotidase only reduced basal extrasynaptosomal accumulation of adenosine by 74%, veratridine-evoked accumulation of adenosine by 46%, and K+-evoked accumulation by 33%. Most of the basal accumulation of extrasynaptosomal adenosine appears to be derived from released nucleotide, probably ATP, but about half of the veratridine-evoked accumulation of adenosine and most of the K+-evoked accumulation may arise from adenosine released in its own right, rather than from a released nucleotide.     

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.     

N-Methyl-d-Aspartate- and Non-N-Methyl-d-Aspartate-Evoked Adenosine Release from Rat Cortical Slices: Distinct Purinergic Sources and Mechanisms of Release

Abstract: Excitatory amino acids, acting at both N methyl- d -aspartate (NMDA) and non-NMDA receptors, release the inhibitory neuromodulator adenosine from superfused rat cortical slices. This study was initiated to investigate the possible purinergic sources and mechanisms of release for the adenosine release evoked by NMDA and non-NMDA receptor activation. Inhibition of the bidirectional nucleo-side transporter with dipyridamole greatly enhanced adenosine release evoked by glutamate, NMDA, kainate, and ( RS -α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Inhibition of ecto -5'-nucleotidase with α,β-methylene ADP and GMP had no effect on either kainateor AMPA-evoked adenosine release, but it decreased glutamate- and NMDA-evoked adenosine release by 23 and 68%, respectively. A similar inhibition of NMDA-evoked adenosine release was observed with α,β-methylene ADP alone, indicating that the inhibitory effect was not due to the reported competitive inhibition of NMDA receptors by GMP. Finally, NMDA-evoked adenosine release, but not kainate- or AMPA-evoked release, was Ca²⁺ dependent. These results indicate that activation of non-NMDA receptors releases adenosine per se in a Ca²⁺-independent manner. In contrast, NMDA receptor activation releases primarily a nucleotide that is subsequently converted extracellularly to adenosine; in this case, release is Ca²⁺ dependent. Although neither NMDA- nor non-NMDA-evoked adenosine release occurs via the nucleoside transporter, this transporter does appear to be a major route for removal of adenosine from the extracellular space.     

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.     

Maintenance of oxidative phosphorylation protects cells from Actinobacillus actinomycetemcomitans leukotoxin-induced apoptosis

Subnanomolar concentrations (3 × 10⁻¹⁰ M) of Actinobacillus actinomycetemcomitans leukotoxin (Ltx) trigger apoptosis of JY cells, as shown by a decrease in mitochondrial transmembrane potential (ΔΨ_m), hyperproduction of reactive oxygen species (ROS) and release of cytochrome c from the intermembrane space. When compared with heat-inactivated leukotoxin (ΔI Ltx) controls, ATP levels in Ltx-treated JY cells continued to decrease during a 24 h experiment while cytoplasmic ADP concentrations were increasing. These results suggest that a blockage occurred in ATP/ADP exchange. To maintain ATP/ADP exchange, JY cells were transfected with bcl -2 and bcl -x_L and incubated with Ltx. ATP levels of the transfected cells decreased to 67% (JY/ bcl -2) and 73% (JY/ bcl-x _L) after the experiment. Furthermore, cytochrome c remained localized to the mitochondrial fraction of Ltx-treated JY/ bcl -2 and JY/ bcl-x _L cells, whereas its presence in the cytoplasmic fraction of JY/ gen cells suggests an uncoupling of electron transport. Expression of bcl -2 and bcl-x _L in cells inhibited downstream apoptotic events such as cleavage of poly(ADP-ribose) polymerase, DNA fragmentation and activation of a family of caspases. The results indicate that Ltx induces apoptosis through a mitochondrial pathway that involves decreased levels of the ADP in the mitochondrial matrix, a lack of substrate for ATP synthetase and arrest of oxidative phosphorylation.     

Ecto-5''-Nucleotidase Is Associated with Cholinergic Nerve Terminals in the Hippocampus but Not in the Cerebral Cortex of the Rat

The extracellular catabolism of exogenously added AMP was studied in immunopurified cholinergic nerve terminals and in slices of the hippocampus and cerebral cortex of the rat. AMP (10 microM) was catabolized into adenosine and inosine in hippocampal cholinergic nerve terminals and in hippocampal slices, as well as in cortical slices. IMP formation from extracellular AMP was not detected. alpha, beta-Methylene ADP (100 microM) inhibited almost completely the extracellular catabolism of AMP in these preparations. The relative rate of catabolism of AMP was greater in hippocampal slices than in cortical slices. AMP was virtually not catabolized when added to immunopurified cortical cholinergic nerve terminals, although ATP could be catabolized extracellularly under identical conditions. The comparison of the relative rates of catabolism of exogenously added AMP, calculated from the amount of AMP catabolized after 5 min, in hippocampal cholinergic nerve terminals and in hippocampal slices revealed a nearly 50-fold enrichment in the specific activity of ecto-5'-nucleotidase upon immunopurification of the cholinergic nerve terminals from the hippocampus. The results suggest that there is a regional variation in the subcellular distribution of ecto-5'-nucleotidase activity in the rat brain, the ecto-5'-nucleotidase in the hippocampus being closely associated with the cholinergic nerve terminals, whereas in the cerebral cortex ecto-5'-nucleotidase activity seems to be located preferentially outside the cholinergic nerve terminals.     

The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface

The time course of the extracellular reaction sequence ATP----ADP----AMP----adenosine has been examined during recirculation of substrate solutions over cultured pig aortic endothelial cells attached to polystyrene beads. This permits the study of reactions at volume to cell surface ratios approaching those of small blood vessels. When endothelial cells were presented with an initial bolus of ATP, high concentrations of the intermediates ADP and AMP developed before significant conversion of AMP to adenosine occurred. Further, the higher the initial ATP concentration, the slower the conversion of AMP to adenosine. Kinetic constants for each reaction were estimated by fitting simulated reaction curves to observed time courses. Apparent Km values estimated in this way agreed well with those reported for initial velocity measurements (ATPase = 300 microM; ADPase = 240 microM; and 5'-nucleotidase = 26 microM). The ratio of maximum velocities was ATPase:ADPase:AMPase = 6:1.5:1, with absolute values varying among cell batches. The data could only be fitted if the model incorporated inhibition of 5'-nucleotidase by ATP or ADP, and satisfactory fitting was achieved with a Ki value for ADP of 5 microM. These kinetic properties maximize the time separation of the intermediate pools. In vivo, at sites of platelet degranulation, they would create a time gap proportional to the size of the initial release between release of ADP (a proaggregatory milieu) and the appearance of adenosine (an anti-aggregatory milieu).     

Adenosine production and its interaction with protection of ischemic and reperfusion injury of the myocardium

Adenosine exerts cardioprotective effects on the ischemic myocardium. A flexibly mounted microdialysis probe was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase (a key enzyme responsible for adenosine production) in in vivo rat hearts. The level of adenosine during perfusion of adenosine 5'-adenosine monophosphate (AMP) was given as an index of the activity of ecto-5'-nucleotidase in the tissue. Endogenous norepinephrine (NE) activates both alpha(1)-adrenoceptors and protein kinase C (PKC), which, in turn, activates ecto-5'-nucleotidase via phosphorylation thereby enhancing the production of interstitial adenosine. Histamine-release NE activates PKC, which increased ecto-5'-nucleotidase activity and augmented release of adenosine. Opening of cardiac ATP sensitive K(+) (K(ATP)) channels may cause hydroxyl radical (.OH) generation through NE release. Lysophosphatidylcholine (LPC), an endogenous amphiphiphilic lipid metabolite, also increases the concentration of interstitial adenosine in rat hearts, through the PKC-mediated activation of endogenous ecto-5'-nucleotidase. Nitric oxide (NO) facilitates the production of interstitial adenosine, via guanosine 3',5'-cyclic monophosphate (cGMP)-mediated activation of ecto-5'-nucleotidase as another pathway. These mechanisms play an important role in high sensitivity of the cardiac adenosine system. Adenosine plays an important role as a modulator of ischemic reperfusion injury, and that the production and mechanism of action of adenosine are linked with NE release.