Adenosine deaminase deficiency with normal immune function. An acidic enzyme mutation.

In most instances, marked deficiency of the purine catabolic enzyme adenosine deaminase results in lymphopenia and severe combined immunodeficiency disease. Over a 2-yr period, we studied a white male child with markedly deficient erythrocyte and lymphocyte adenosine deaminase activity and normal immune function. We have documented that (a) adenosine deaminase activity and immunoreactive protein are undetectable in erythrocytes, 0.9% of normal in lymphocytes, 4% in cultured lymphoblasts, and 14% in skin fibroblasts; (b) plasma adenosine and deoxyadenosine levels are undetectable and deoxy ATP levels are only slightly elevated in lymphocytes and in erythrocytes; (c) no defect in deoxyadenosine metabolism is present in the proband's cultured lymphoblasts; (d) lymphoblast adenosine deaminase has normal enzyme kinetics, absolute specific activity, S20,w, pH optimum, and heat stability; and (e) the proband's adenosine deaminase exhibits a normal apparent subunit molecular weight but an abnormal isoelectric pH. In contrast to the three other adenosine deaminase-deficient healthy subjects who have been described, the proband is unique in demonstrating an acidic, heat-stable protein mutation of the enzyme that is associated with less than 1% lymphocyte adenosine deaminase activity. Residual adenosine deaminase activity in tissues other than lymphocytes may suffice to metabolize the otherwise lymphotoxic enzyme substrate(s) and account for the preservation of normal immune function.     

Bone marrow transplantation only partially restores purine metabolites to normal in adenosine deaminase-deficient patients.

To delineate the extent to which bone marrow transplantation provides "enzyme replacement therapy", we have determined metabolite concentrations in two patients with adenosine deaminase (ADA) deficiency treated with bone marrow transplants and rendered immunologically normal. 10 yr after engraftment of lymphoid cells, erythrocyte deoxy ATP was markedly decreased compared to the marked elevations of deoxy ATP observed in untreated patients, but was still significantly elevated (62 and 90 vs. normal of 6.0 +/- 6.0 nmol/ml packed erythrocytes). Similarly, deoxyadenosine and adenosine excretion were both markedly diminished compared to that of untreated patients but deoxyadenosine excretion was still clearly increased (20.1 and 38.6 vs. normal of less than 0.2 nmol/mg creatinine) while adenosine excretion was in the upper range of normal (7.0 and 8.1 vs. normal of 5.6 +/- 3.6 nmol/mg creatinine). Mononuclear cell deoxy ATP content was also elevated compared to normal (5.25 and 14.4 vs. 1.2 +/- 0.3). Separated mononuclear cells of bone marrow transplanted patients contain both donor lymphocytes and recipient monocytes. When mononuclear cells were depleted of the cells enriched for donor lymphocytes (i.e. monocyte depleted) was lower than that of the mixed mononuclear cells (2.2 vs. 5.26). Surprisingly, plasma adenosine was as high as in untreated ADA-deficient patients (3.2 and 1.5 vs. untreated of 0.3-3 microM). Consistent with the elevated plasma adenosine and urinary deoxyadenosine, erythrocyte S-adenosyl homocysteine hydrolase activity was diminished (0.88 and 1.02 vs. normal of 5.64 +/- 0.25). Thus, bone marrow transplantation of ADA-deficient patients not only provides lymphoid stem cells, but also partially, albeit incompletely, clears abnormally increased metabolites from nonlymphoid body compartments.     

Myoadenylate deaminase deficiency. Functional and metabolic abnormalities associated with disruption of the purine nucleotide cycle.

To assess the role of the purine nucleotide cycle in human skeletal muscle function, we evaluated 10 patients with AMP deaminase deficiency (myoadenylate deaminase deficiency; MDD). 4 MDD and 19 non-MDD controls participated in an exercise protocol. The latter group was composed of a patient cohort (n = 8) exhibiting a constellation of symptoms similar to those of the MDD patients, i.e., postexertional aches, cramps, and pains; as well as a cohort of normal, unconditioned volunteers (n = 11). The individuals with MDD fatigued after performing only 28% as much work as their non-MDD counterparts. Muscle biopsies were obtained from the four MDD patients and the eight non-MDD patients at rest and following exercise to the point of fatigue. Creatine phosphate content fell to a comparable extent in the MDD (69%) and non-MDD (52%) patients at the onset of fatigue. Following exercise the 34% decrease in ATP content of muscle from the non-MDD subjects was significantly greater than the 6% decrease in ATP noted in muscle from the MDD patients (P = 0.048). Only one of four MDD patients had a measurable drop in ATP compared with seven of eight non-MDD patients. At end-exercise the muscle content of inosine 5'-monophosphate (IMP), a product of AMP deaminase, was 13-fold greater in the non-MDD patients than that observed in the MDD group (P = 0.008). Adenosine content of muscle from the MDD patients increased 16-fold following exercise, while there was only a twofold increase in adenosine content of muscle from the non-MDD patients (P = 0.028). Those non-MDD patients in whom the decrease in ATP content following exercise was measurable exhibited a stoichiometric increase in IMP, and total purine content of the muscle did not change significantly. The one MDD patient in whom the decrease in ATP was measurable, did not exhibit a stoichiometric increase in IMP. Although the adenosine content increased 13-fold in this patient, only 48% of the ATP catabolized could be accounted for by the combined increases of adenosine, inosine, hypoxanthine, and IMP. Studies performed in vitro with muscle samples from seven MDD and seven non-MDD subjects demonstrated that ATP catabolism was associated with a fivefold greater increase in IMP in non-MDD muscle. There were significant increases in AMP and ADP content of the muscle from MDD patients following ATP catabolism in vitro, while there was no detectable increase in AMP or ADP in non-MDD muscle. Adenosine content of MDD muscle increased following ATP catabolism, but there was no detectable increase in adenosine content of non-MDD muscle following ATP catabolism in vitro. These studies demonstrate that AMP deaminase deficiency leads to reduced entry of adenine nucleotides into the purine nucleotide cycle during exercise. We postulate that the resultant disruption of the purine nucleotide cycle accounts for the muscle dysfunction observed in these patients.     

Radioimmunochemical quantitation of human adenosine deaminase.

Markedly reduced or absent adenosine deaminase activity in man is associated with an autosomal recesive form of severe conbined immunodeficiency disease. To further define the genetic nature of this enzyme defect, we have quantitated immunologically active adenosine deaminase (CRM) in the hemolysate of homozygous deficient patients and their heterozygous parents. A highly specific radioimmunoassay was developed capable of detecting 0.05% of normal erythrocyte adenosine deaminase. Hemolysates from nine heterozygotes (five families) showed a wide range in CRM (32--100% of normal) and variable absolute specific activities with several being at least 1 SD BELOW THE NORMAL MEAN. Hemolysates from four unrelated patients showed less than 0.09% adenosine deaminase activity with CRM ranging from less than 0.06 to 5.6% of the normal mean. In conclusion, heterozygote and homozygote hemolysates from five of the eight families analyzed revealed variable levels of CRM suggesting heterogeneous genetic alteration or expression of the silent or defective allele(s) of adenosine deaminase.     

The activity and expression of NTPDase is altered in lymphocytes of multiple sclerosis patients

BackgroundMultiple sclerosis (MS) is a demyelinating neurological disease, which is presumed to be a consequence of infiltrating lymphocytes that are autoreactive to myelin proteins. ATP and adenosine contribute to fine-tuning immune responses and NTPDase (CD39) and adenosine deaminase (ADA) are important enzymes in the control of the extracellular levels of these molecules at the site of inflammation. We evaluated the activity and expression of NTPDase and adenosine deaminase (ADA) activity in lymphocytes from patients with the relapsing–remitting form of MS (RRMS).MethodsThis study involved 22 patients with RRMS and 22 healthy subjects as a control group. The lymphocytes were isolated from blood and separated on Ficoll density gradients and after isolation the NTPDase and ADA activities were determined.ResultsThe NTPDase activity and expression were increased in lymphocytes from RRMS patients when compared with the control group (p < 0.05). In addition, a decrease in ADA activity was observed in lymphocytes from these patients when compared to the control group (p < 0.05).ConclusionsThe regulation of ATP and adenosine levels by NTPDase and ADA activities may be important to preserve cellular integrity and to modulate the immune response in MS.     

Biochemical and Functional Abnormalities in Lymphocytes from an Adenosine Deaminase-deficient Patient during Enzyme Replacement Therapy

Biochemical and immunological properties of lymphocytes were measured repetitively over a period of 40 mo during enzyme replacement by transfusion in a child with adenosine deaminase (ADA) deficiency and severe combined immunodeficiency disease. Catalytically defective ADA protein is present in the child's cells. ADA activity in his lymphocytes is 7 nmol/min per 10⁸ cells with 51 ng of ADA protein/10⁸ cells by radioimmunoassay. ADA activities in normal cord and adult lymphocytes average 193 and 92 nmol/min per 10⁸ cells, respectively, with 429 and 223 ng of ADA protein/10⁸ cells. Deoxy(d)ATP accumulates in the patient's erythrocytes and lymphocytes. Transfusion of irradiated packed erythrocytes partially corrects the metabolic defects. Frank metabolic relapse occurs if transfusions are discontinued for several months. The amounts of dATP in erythrocytes and lymphocytes averaged 13 and 2 times normal, respectively, during periods when transfusions were administered every 2-4 wk. Deoxyguanosine triphosphate and deoxycytidine triphosphate in lymphocytes were normal on 11 occasions, but deoxyribosylthymine triphosphate was ninefold increased. On 11 occasions dATP was measured in lymphocytes and erythrocytes isolated simultaneously. There was a positive, but statistically insignificant, correlation between amounts of dATP in the two types of cells (r = 0.25,P > 0.1). The absolute peripheral lymphocyte count was correlated with the activity of ADA in circulating erythrocytes and with the response of lymphocytes to phytohemagglutinin (r = 0.64, P < 0.01; r = 0.49, P < 0.05). Response of lymphocytes to stimulation by phytohemagglutinin in vitro and absolute peripheral lymphocyte counts were not significantly correlated with levels of dATP in the erythrocyte or lymphocyte during periods of intensive therapy. Although there was objective improvement during enzyme replacement, the child remained immunodeficient and biochemically abnormal.     

Heterogeneity of b lymphocyte differentiation in severe combined immunodeficiency disease.

Pokeweed mitogen-induced B lymphocyte differentiation in vitro into antibody secreting plaque-forming cells (PFC) was investigated in nine patients with severe combined immunodeficiency having variable proportions of circulating B lymphocytes. When cultured by themselves, the peripheral blood mononuclear cells did not respond to stimulation with pokeweed mitogen in any patient. In the presence of irradiated allogeneic T cells as helpers, however, PFC responses were elicited in lymphocyte cultures from peripheral blood and/or bone marrow in some patients. In one of these patients, results of allogeneic co-culture experiments were suggestive of genetically restricted suppressor cells. In a single patient with deficiency of the enzyme adenosine deaminase, PFC were generated in bone marrow lymphocyte cultures only when they were supplemented with exogenous adenosine deaminase and allogeneic helper cells. A parallel study of T lymphocyte differentiation in vitro performed in fractionated bone marrow cells was suggestive of arrested differentiation at different steps along the differentiation pathway. In two patients with evidence of functional B cell precursors, deficiencies of helper T cell function could be attributed to differentiation defects at the level of the stem cells in one and the thymus in the other. The findings reported here further substantiate the heterogeneity of the severe combined immunodeficiency disease syndromes.     

Acquired secretion defect in platelets after cryopreservation in dimethyl sulfoxide

Despite the use of preservatives, platelets are severely damaged during cryopreservation and, following freezing, function poorly in a number of in vitro tests. We report here that cryopreserved platelets show diminished aggregation in response to collagen. This may be a consequence of a secretion defect as evidenced by a 20 to 30 percent loss of dense- and alpha-granule content (p less than 0.05) and an impaired secretion mechanism. Analysis of adenine nucleotides confirmed the defect in dense granule adenosine triphosphate (ATP) and adenosine diphosphate (ADP) content (storage pool), but in addition revealed a 50 percent fall in cytosolic ATP (metabolic pool). In contrast, the adenylate energy charge, (ATP + 1/2 ADP)/(ATP + ADP + adenosine monophosphate), was normal. We concluded that platelet cryopreservation leads to a secretion defect, probably as a result of activation during freezing and thawing procedures.     

Modulation of erythropoietin production by adenosine

Adenosine is an important regulatory molecule that increases in hypoxic and ischemic tissues and has been proposed to mediate blood flow in response to oxygen availability. The current study ascribes another oxygen-responsive role to adenosine, that of regulating synthesis of the erythropoiesis-stimulating hormone, erythropoietin. When perfused through isolated rat kidneys, exogenous adenosine in nanomolar concentrations increased erythropoietin production, whereas inosine, the deaminated nucleoside, had no effect. In addition, an adenosine antagonist, and adenosine deaminase, diminished erythropoietin titers in renal perfusates. In intact rats, adenosine deaminase injections followed by a hypoxic stimulus slightly reduced erythropoietin serum concentrations, whereas an adenosine deaminase inhibitor sharply increased erythropoietin titers. The results suggest that adenosine may function as a mediator to link oxygen supply with erythropoietin production.     

Mechanisms whereby exogenous adenine nucleotides improve rabbit renal proximal function during and after anoxia.

When a suspension of rabbit proximal tubules is subjected to anoxia, ATP falls by 80-90% during 40 min of anoxia, and upon reoxygenation (reox) the cells only recover 25-50% of their initial ATP. Addition of Mg-ATP (magnesium chloride-treated ATP), Mg-ADP, or Mg-AMP (five aliquots of 200 nmol/ml added 10 min apart) during anoxia causes complete recovery of ATP levels, and respiratory and transport function after 40 min of reox. Similar additions of adenosine (ADO), or inosine (INO), or Mg-ATP only during reox are less effective. Lactate dehydrogenase (LDH) release after 40 min of anoxia is 30-40% under control conditions, only 10-15% when adenine nucleotides or ADO are added during anoxia, and 20% when INO is added, suggesting that these additions may stabilize the plasma membrane during anoxia and help preserve cellular integrity. During reox, recovery may depend on the entry of ATP precursors and, therefore, we explored the mechanism whereby exogenous ATP increases the intracellular ATP content. Additions of Mg-ATP, Mg-ADP, or Mg-AMP to continuously oxygenated tubules increase cellular ATP content three- to fourfold in 1 h. The added ATP and ADP are rapidly degraded to AMP, and more slowly to ADO, INO, and hypoxanthine. Furthermore, the ATP-induced increase in cellular ATP is abolished by the exogenous addition of adenosine deaminase, which converts extracellular ADO to INO. These results suggest that the increase in cellular ATP requires extracellular ADO. The ADO obtained from the breakdown of AMP may be preferentially transported into the renal cells to be resynthesized into cellular AMP and ATP.     

Disruption of the Purine Nucleotide Cycle: A POTENTIAL EXPLANATION FOR MUSCLE DYSFUNCTION IN MYOADENYLATE DEAMINASE DEFICIENCY

A patient with symptoms of easy fatigability, postexercise myalgias, and delayed recovery of muscle strength after activity is described. Skeletal muscle from this patient had <1.0% normal myoadenylate deaminase activity and NH₃ was not released from muscle after ischemic exercise. In association with this enzyme deficiency, exercise led to a >90% reduction in muscle content of adenine nucleotides. No inosine monophosphate accumulated after exercise and total purine content of the muscle fell to 21% of control. Repletion of the adenine nucleotide pool in this patient was delayed compared to controls, and ATP content had only returned to 68% of control at 165 min after exercise. These studies demonstrate that disruption of the purine nucleotide cycle as a consequence of myoadenylate deaminase deficiency results in marked alterations in ATP content of muscle, and potentially, these changes in ATP content could account for muscle dysfunction in this patient.     

An abnormal form of purine nucleoside phosphorylase in a family with a child with severe defective T-cell-and normal B-cell immunity.

1. Purine nucleoside phosphorylase and adenosine deaminase (ADA) were studied in normal red blood cells and lymphocytes and in the cells of a family with a child with a defective T-cell-and normal B-cell immunity. 2. In the propositus no purine nucleoside phosphorylase (NP) activity could be detected in her red cells and lymphocytes, while the ADA activity was somewhat increased. The NP activities of the father, mother and brother of the propositus are in the heterozygote range. The decreased activity of NP was not only found for the substrate inosine but also when guanosine or xanthosine were used as substrate. The mode of inheritance is autosomal recessive. 3. With starch gel electrophoresis no NP activity could be detected in the patient's haemolysate. The electrophoretic patterns of NP from the father, mother and brother of the patient seem to be the same as for normal NP with six bands of NP activity. 4. The nucleoside phosphorylases of the father, mother and brother of the patient were characterized by an increased KM for the substrate inosine, normal pH optimum and a decreased heat stability.     

Dosage de l'activité adénosine désaminasique dans les érythrocytes et les lymphocytes humains

A radiochromatographic method is described for measuring enzymatic activity of adenosine deaminase in human erythrocytes and lymphocytes. [8-¹⁴C]-adenosine is converted into inosine and hypoxanthine; after Chromatographie separation of the products, the radioactivity is determined.The kinetic properties of the enzyme have been studied. The K_m values for the erythrocyte and lymphocyte enzymes are higher as compared with purified deaminase. Optimum conditions for substrate concentration for assay were established.The mean normal activity (± S.E. of mean) is: for erythrocytes, 494 ± 61; nmol min^-1 ml^-1; for lymphocytes, 147 ± 0.18 nmol min^?1 10⁶ cellules.The mean values are higher than that given by other methods working at a lower (non-staurating) substrate concentration.     

Adenosine metabolism in phytohemagglutinin-stimulated human lymphocytes.

The association of a human genetic deficiency of adenosine deaminase activity with combined immunodeficiency prompted a study of the effects of adenosine and of inhibition of adenosine deaminase activity on human lymphocyte transformation and a detailed study of adenosine metabolism throughout phytohemagglutinin-induced blastogenesis. The adenosine deaminase inhibitor, coformycin, at a concentration that inhibited adenosine deaminase activity more than 95%, or 50 muM adenosine, did not prevent blastogenesis by criteria of morphology or thymidine incorporation into acid-precipitable material. The combination of coformycin and adenosine, however, substantially reduced both the viable cell count and the incorporation of thymidine into DNA in phytohemagglutinin-stimulated lymphocytes. Incubation of lymphocytes with phytohemagglutinin for 72 h produced a 12-fold increase in the rate of deamination and a 6-fold increase in phosphorylation of adenosine by intact lymphocytes. There was no change in the apparent affinity for adenosine with either deamination or phosphorylation. The increased rates of metabolism, apparent as early as 3 h after addition of mitogen, may be due to increased entry of the nucleoside into stimulated lymphocytes. Increased adenosine metabolism was not due to changes in total enzyme activity; after 72 h in culture, the ratios of specific activities in extracts of stimulated to unstimulated lymphocytes were essentially unchanged for adenosine kinase, 0.92, and decreased for adenosine deaminase, 0.44. As much as 38% of the initial lymphocyte adenosine deaminase activity accumulated extracellularly after a 72-h culture with phytohemagglutinin. In phytohemagglutinin-stimulated lymphocytes, the principal route of adenosine metabolism was phosphorylation at less than 5 muM adenosine, and deamination at concentrations greater than 5 muM. In unstimulated lymphocytes, deamination was the principal route of adenosine metabolism over the range of adenosine concentrations studied (0.5-250 muM). These studies demonstrate the dependence of both the unstimulated and stimulated lymphocyte on adenosine and may account for the observed sensitivity of mitogen-stimulated lymphocytes to the toxic effects of exogenously supplied adenosine in the presence of the adenosine deaminase inhibitor coformycin. A single case of immunodeficiency disease has been reported in association with purine nucleoside phosphorylase deficiency. The catabolism of guanosine was also found to be enhanced in stimulated normal lymphocytes; phosphorolysis of guanosine to guanine by intact lymphocytes increased six fold after 72-h culture with phytohemagglutinin. The specific activity of purine nucleoside phosphorylase in extracts, with guanosine as substrate, was essentially the same in stimulated and unstimulated lymphocytes after 72 h of culture.     

Biochemical study of a case of hemolytic anemia with increased (85-fold) red cell adenosine deaminase

An increased red cell adenosine deaminase (ADA) activity (85-fold) was found in a 10-year-old male child suffering from severe hemolytic disease. Evidence is given that the excessive ADA activity in erythrocytes is due to an abnormal amount of a catalytically and immunologically normal enzyme. Metabolic studies with the patient's erythrocytes show that the low ATP concentration in these cells (64% of comparably reticulocyte-rich blood) is due both to a diminished synthesis of adenylic nucleotides from adenosine, and to an excessive catabolism of AMP.     

The erythrocyte as instigator of inflammation. Generation of amidated C3 by erythrocyte adenosine deaminase.

Myocardial ischemia is characterized by the liberation of adenosine and by complement-mediated inflammation. We have reported that amidated C3, formed when ammonia (NH3) disrupts the thiolester bond of C3, serves as an alternative pathway convertase, generates C5b-9, and stimulates phagocytic oxidative metabolism. We investigated whether the deamination of adenosine by adenosine deaminase in hematopoietic cells might liberate sufficient ammonia to form amidated C3 and thereby trigger complement-mediated inflammation at ischemic sites. In the presence of 4 mM adenosine, NH3 production per erythrocyte (RBC) was equal to that per neutrophil (PMN) (3.3 X 10(-15) mol/cell per h). Because RBC outnumber PMN in normal blood by a thousandfold, RBC are the major source of NH3 production in the presence of adenosine. NH3 production derived only from the deamination of adenosine by the enzyme adenosine deaminase and was abolished by 0.4 microM 2'-deoxycoformycin, a specific inhibitor of adenosine deaminase. When purified human C3 was incubated with 5 X 10(8) human RBC in the presence of adenosine, disruption of the C3 thiolester increased more than twofold over that measured in C3 incubated with buffer, or in C3 incubated with RBC (P less than 0.05). The formation of amidated C3 was abolished by the preincubation of RBC with 2'-deoxycoformycin (P less than 0.001). Amidated C3 elicited statistically significant release of superoxide, myeloperoxidase, and lactoferrin from PMN. Thus, the formation of amidated C3 by RBC deamination of adenosine triggers a cascade of complement-mediated inflammatory reactions.     

Erythrocyte adenosine deaminase, purine nucleoside phosphorylase and phosphoribosyltransferase activity in patients with Down's syndrome

The erythrocyte adenosine deaminase, nucleoside phosphorylase, hypoxanthineguanine phosphoribosyltransferase and adenine phosphoribosyltransferase activities and plasma urate concentrations were measured in 20 cases of Down's syndrome and in 20 age- and sex-matched control subjects. The mean erythrocyte adenosine deaminase and adenine phosphoribosyltransferase activities and plasma urate concentrations were significantly higher in Down's syndrome subjects than in controls (p less than 0.001, p less than 0.01 and p less than 0.001, respectively). In all subjects studied there was a positive correlation between the erythrocyte adenosine deaminase activity and plasma urate concentration (r = 0.488, p less than 0.005). The concentrations of the erythrocyte adenine nucleotides, AMP, ADP and ATP, did not differ in Down's syndrome (n = 10) from those of control subjects (n = 10). The results suggest that the increase of plasma urate concentrations is a consequence of the increase in adenosine deaminase activity in Down's syndrome patients.     

Amplification of the cyclic AMP response to forskolin in pheochromocytoma PC12 cells through adenosine A(2A) purinoceptors.

In this study, we present evidence on the ability of endogenous adenosine to modulate adenylyl cyclase activity in intact PC12 cells. The adenosine receptor antagonists PD 115199, xanthine amine congener, 8-cyclopentyl-1,3-dipropylxanthine, 8-(p-sulfophenyl)theophylline, and 3,7-dimethyl-1-propargylxanthine inhibited 10 microM forskolin-induced cyclic AMP (cAMP) accumulation, with IC(50) values of 2.76 +/- 1.16 nM, 17.4 +/- 1.08 nM, 443 +/- 1. 03 nM, 2.00 +/- 1.01 microM, and 2.25 +/- 1.05 microM, respectively. Inhibition by 2.5 nM PD 115199 was only partially reversed by increasing forskolin concentrations up to 100 microM. The addition of PD 115199 with or 60 min after forskolin caused a comparable inhibition of forskolin effect over the next hour. Both exogenous adenosine (0.1 microM) and its precursor, AMP (10 and 100 microM), significantly enhanced forskolin-induced cAMP accumulation, whereas inosine was ineffective. Forskolin activity was also potentiated by the hydrolysis-resistant adenosine receptor agonists 5'-N-ethylcarboxamido adenosine and CGS 21680 (8.9- and 12.2-fold increase, respectively). Adenosine deaminase (1 U/ml) and 8-SPT (25 microM), which nearly abolished the response to 1 microM adenosine, also reduced cAMP accumulation caused by AMP (-78 and -54%, respectively). These results demonstrate that in PC12 cells, activation of adenylyl cyclase by forskolin is highly dependent on the occupancy of A(2A) adenosine receptors and that AMP potentially contributes to the amplification of forskolin response.     

ATP inhibits glutamate synaptic release by acting at P2Y receptors in pyramidal neurons of hippocampal slices

It has been proposed that extracellular ATP inhibits synaptic release of glutamate from hippocampal CA1 synapses after its catabolism to adenosine. We investigated the possibility that at least part of this effect is mediated by ATP itself acting on P2Y receptors. ATP and various analogs decreased the amplitude and duration of glutamate-mediated excitatory postsynaptic potentials in all tested neurons. This effect was reversible and concentration-dependent and had the following rank order of agonist potency: AMP = ATP = adenosine-5'-O-(3-thio)triphosphate > adenosine = ADP. alpha,beta-Methylene ATP, beta,gamma-methylene ATP, 2-methylthioadenosine 5'-triphosphate, GTP, and UTP induced only a partial response. The depolarization induced by exogenous glutamate was not affected by ATP, indicating that this nucleotide acts presynaptically to inhibit glutamate-mediated excitatory postsynaptic potentials. Neither inhibition of ectonucleotidase activity with alpha,beta-methylene ADP, suramin, or pyridaxalphosphate-6-azophenyl-2',4'-disulfonic acid 4-sodium nor removal of extracellular adenosine (with adenosine deaminase) altered ATP effects. 8-Cyclopentyltheophylline competitively inhibited ATP effects, whereas P2 receptor antagonists (pyridaxalphosphate-6-azophenyl-2',4'-disulfonic acid 4-sodium, suramin, and reactive blue 2) were ineffective. ATP effects were by far more sensitive to pertussis toxin (PTX) than those of adenosine. After PTX, adenosine-5'-O-(3-thio)triphosphate induced only a partial response, and ATP concentration-response curve was biphasic. The second phase of this curve was blocked by adenosine deaminase, implying that it is mediated by adenosine as a result of ATP catabolism. Under control conditions, however, catabolism of ATP is not required to explain its actions. In conclusion, ATP inhibits synaptic release of glutamate by direct activation of P2Y receptors that are PTX- and 8-cyclopentyltheophylline-sensitive.     

Adenine nucleotide metabolism in human blood – important roles for leukocytes and erythrocytes

Adenosine diphosphate (ADP) released into blood induces platelet aggregation and contributes to hemostasis and thrombosis. Released ATP can also induce platelet aggregation and there is evidence that blood leukocytes and also erythrocytes play important roles in this. Rapid metabolism of ADP and ATP by endothelial cells is important in protecting platelets from their effects. Here we have performed a systematic investigation of adenine nucleotide metabolism in human blood and the involvement of blood cells. Conversion of ATP to ADP in blood was due almost exclusively to the presence of leukocytes; plasma, platelets and erythrocytes made little or no contribution. Mononuclear leukocytes (MNLs) and polymorphonuclear leukocytes (PMNLs) were equally effective. Conversion of ADP to AMP was also promoted by leukocytes, with no involvement of platelets or erythrocytes. Some ADP was also converted to ATP in blood, apparently via an enzyme present in plasma, but ATP was then rapidly removed by the leukocytes. Conversion of AMP to adenosine occurred via a plasma enzyme with little or no contribution from any cellular element. As expected, in blood the adenosine produced was removed very rapidly by erythrocytes and then converted to inosine and then hypoxanthine. In the absence of erythrocytes plasma supported only a slow conversion of adenosine to inosine and hypoxanthine, which was not influenced by platelets or leukocytes. This study has demonstrated that leukocytes and erythrocytes play a major role in adenine nucleotide metabolism in blood and that these cells, as well as endothelial cells, may be important determinants of the effects of ATP and ADP on platelets.