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

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

Enhanced NTPDase and 5′-nucleotidase activities in diabetes mellitus and iron-overload model

The activity of the enzymes NTPDase and 5′-nucleotidase was studied in both diabetes mellitus and an associated model of iron-overload. Rats were divided in five groups: citrate (CC), saline (S), diabetic (D), iron-overload (IO), and diabetic iron-overload (DIO). Diabetes was induced with alloxan (150 mg/kg), and iron-overload was induced with iron-dextran (10 intramuscular applications of ±80 mg/kg). The enzymatic activities were evaluated in the platelets. The results demonstrated an increase in the activity of NTPDase with substrates ATP and ADP (60% and 120%, respectively; P < 0.001), and 5′-nucleotidase (60%, P < 0.001). This increase was more intense in the IO and DIO groups. The results obtained in vitro showed an activation in ATP, ADP, and AMP hydrolysis between 1 μM and 1,000 μM ferric nitrate concentrations, being more pronounced at 100 μM and decreasing at 1,000 μM. We concluded that diabetes mellitus in association with iron-overload increased the hydrolysis of adenine nucleotides in platelets, contributing to the abnormalities found in these pathological conditions.     

Characterization of NTPDase (NTPDase1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5) activity in human lymphocytes

Human lymphocytes contain NTPDase (NTPDase-1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5), a cation-dependent enzyme that hydrolyzes ATP and ADP and also other di- and triphosphate nucleosides, acting at an optimum pH of 8.0. A significant inhibition of ATP and ADP hydrolysis (P<0.05) was observed in the presence of 20 mM sodium azide. NTPDase inhibitors, 20 mM sodium fluoride, 0.2 mM trifluoperazine and 0.3 mM suramin, significantly decreased ATP and ADP hydrolysis (P<0.05) and ADP hydrolysis was only inhibited by 0.5 mM orthovanadate (P<0.05). ATP and ADP hydrolysis was not inhibited in the presence of 0.01 mM Ap5A (P1,P5-di(adenosine-5')pentaphosphate), 0.1 mM ouabain, 1 mM levamisole, 2 microg/mL oligomycin, 0.1 mM N-ethylmaleimide (NEM), or 5 mM sodium azide. With respect to kinetic behavior, apparent K(m) values of 77.6+/-10.2 and 106.8+/-21.0 microM, and V(max) values of 68.9+/-8.1 and 99.4+/-8.5 (mean+/-S.E., n=3) nmol Pi/min/mg protein were obtained for ATP and ADP, respectively. A Chevilard plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. The presence of CD39 was determined by flow cytometry, showing a low density of 2.72+/-0.24% (mean+/-S.E.; n=30) in human peripheral lymphocytes. The study of NTPDase activity in human lymphocytes may be important to determine the immune response status against infectious agents related to ATP and ADP hydrolysis.     

Human Homologue of Seta Binding Protein 1 Interacts with Cathepsin B and Participates in TNF-Induced Apoptosis in Ovarian Cancer Cells

Extracellular adenine nucleotide hydrolysis in the circulation is mediated by the action of an NTPDase (CD39, apyrase) and of a 5′-nucleotidase (CD73), presenting as a final product, adenosine. Among other properties described for adenine nucleotides, an anti-cancer activity is suggested, since ATP is considered a cytotoxic molecule in several tumour cell systems. Conversely, some studies demonstrate that adenosine presents a tumour-promoting activity. In this study, we evaluated the pattern of adenine nucleotide hydrolysis by serum and platelets from rats submitted to the Walker 256 tumour model. Extracellular adenine nucleotide hydrolysis by blood serum and platelets obtained from rats at, 6, 10 and 15 days after the subcutaneous Walker 256 tumour inoculation, was evaluated. Our results demonstrate a significant reduction in ATP, ADP and AMP hydrolysis in blood serum at 6, 10 and 15 days after tumour induction. In platelets, a significant reduction in ATP and AMP hydrolysis was observed at 10 and 15 days after tumour induction, while an inhibition of ADP hydrolysis was observed at all times studied. Based on these results, it is possible to suggest a physiologic protection mechanism against the tumoral process in circulation. The inhibition in nucleotide hydrolysis observed probably maintains ATP levels elevated (cytotoxic compound) and, at the same time, reduces the adenosine production (tumoor-promoting molecule) in the circulation.     

Kinetic characterization of ATP diphosphohydrolase and 5'-nucleotidase activities in cells cultured from submandibular salivary glands of rats

The participation of ecto-ATP diphosphohydrolase (CD39; ecto-NTPDase) and ecto-5'-nucleotidase (CD73) activities in the nucleotide hydrolysis by salivary gland cells from rats was evaluated. We investigated the biochemical characteristics of these ectoenzymes in cells cultured from submandibular salivary glands of rats. The V(max) for the hydrolysis of ATP, ADP and AMP were 2275+/-153 (mean+/-SEM, n = 4), 941+/-96 (mean+/-SEM, n = 5) and 175+/-5 (mean+/-SEM, n = 5) nmol Pi liberated per min per mg of protein, respectively. The K(m) values for ATP, ADP and AMP were 224+/-8 microM (mean+/-SEM, n = 4), 163+/-15 microM (mean+/-SEM, n = 5) and 117+/-5 microM (mean+/-SEM, n = 5), respectively. The competition plot showed that ATP and ADP were hydrolyzed at the same active site on the enzyme. It may be postulated that the physiological role for this ecto-enzyme cascade is to terminate the action of the co-transmitter ATP, generating adenosine.     

E-NTPDases and ecto-5'-nucleotidase expression profile in rat heart left ventricle and the extracellular nucleotide hydrolysis by their nerve terminal endings

In this study, we have identified the E-NTPDase family members and ecto-5'-nucleotidase/CD73 in rat heart left ventricle. Moreover, we characterize the biochemical properties and enzyme activities from synaptosomes of the nerve terminal endings of heart left ventricle. We observe divalent cation-dependent enzymes that presented optimum pH of 8.0 for ATP and ADP hydrolysis, and 9.5 for AMP hydrolysis. The apparent K(M) values are 40 microM, 90 microM and 39 microM and apparent V(max) values are 537, 219 and 111 nmol Pi released/min/mg of protein for ATP, ADP and AMP hydrolysis, respectively. Ouabain, orthovanadate, NEM, lanthanum and levamisole do not affect ATP and ADP hydrolysis in rat cardiac synaptosomes. Oligomycin (2 microg/mL) and sodium azide (0.1 mM), both mitochondrial ATPase inhibitors, inhibit only the ATP hydrolysis. High concentrations of sodium azide and gadolinium chloride show an inhibition on both, ATP and ADP hydrolysis. Suramin inhibit more strongly ATP hydrolysis than ADP hydrolysis whereas Evans blue almost abolish both hydrolysis. AMP hydrolysis is not affected by levamisole and tetramisole, whereas 0.1 mM ammonium molybdate practically abolish the ecto-5'-nucleotidase activity. RT-PCR analysis from left ventricle tissue demonstrate different levels of expression of Entpd1 (Cd39), Entpd2 (Cd39L1), Entpd3 (Cd39L3), Entpd5 (Cd39L4) Entpd6, (Cd39L2) and 5'-NT/CD73. By quantitative real-time PCR we identify the Entpd2 as the enzyme with the highest expression in rat left ventricle. Our results contribute to the understanding about the control of the extracellular nucleotide levels in and cardiac system.     

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

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

Characterization of an ATP diphosphohydrolase activity (APYRASE,EC 3.6.1.5) in rat blood platelets

In the present report we describe an apyrase (ATP diphosphohydrolase, EC 3.6.1.5) in rat blood platelets. The enzyme hydrolyses almost identically quite different nucleoside di- and triphosphates. The calcium dependence and pH requirement were the same for the hydrolysis of ATP and ADP and the apparent Km values were similar for both Ca²⁺-ATP and Ca²⁺-ADP as substrates. Ca²⁺-ATP and Ca²⁺-ADP hydrolysis could not be attributed to the combined action of different enzymes because adenylate kinase, inorganic pyrophosphatase and nonspecific phosphatases were not detected under our assay conditions. The Ca²⁺-ATPase and Ca²⁺-ADPase activity was insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors, thus excluding these enzymes as contaminants. The results demonstrate that rat blood platelets contain an ATP diphosphohydrolase involved in the hydrolysis of ATP and ADP which are vasoactive and platelet active adenine nucleotides.     

Characterization of an ectonucleoside triphosphate diphosphohydrolase 1 activity in alkaline phosphatase-depleted rat osseous plate membranes: possible functional involvement in the calcification process

An ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) activity present in alkaline phosphatase-depleted rat osseous plate membranes, obtained 14 days after implantation of demineralized bone particles in the subcutaneous tissue of Wistar rats, was characterized. At pH 7.5, NTPDase1 hydrolyzed nucleotide triphosphates at rates 2.4-fold higher than those of nucleotide diphosphates, while the hydrolysis of nucleotide monophosphates and non-nucleotide phosphates was negligible. NTPDase 1 hydrolyzed ATP and ADP following Michaelis-Menten kinetics with V=1278.7+/-38.4 nmol Pi/min/mg and K(M)=83.3+/-2.5 microM and V=473.9+/-18.9 nmol Pi/min/mg and K(M)=150.6+/-6.0 microM, respectively, but in the absence of magnesium and calcium ions, ATP or ADP hydrolysis was negligible. The stimulation of the NTPDase1 by calcium (V=1084.7+/-32.5 nmol Pi/min/mg; and K(M)=377.8+/-11.3 microM) and magnesium (V=1367.2+/-41.0 nmol Pi/min/mg and K(M)=595.3+/-17.8 microM) ions suggested that each ion could replace the other during the catalytic cycle of the enzyme. Oligomycin, ouabain, bafilomycin A(1), theophylline, thapsigargin, ethacrynic acid, P(1),P(5)-(adenosine-5')-pentaphosphate and omeprazole had negligible effects on the hydrolysis of ATP and ADP by NTPDase1. However, suramin and sodium azide were effective inhibitors of ATP and ADP hydrolysis.To our knowledge this is the first report suggesting the presence of NTPDase1 in rat osseous plate membranes. Considering that the ectonucleoside triphosphate diphosphohydrolase family of enzymes participates in many regulatory functions, such as response to hormones, growth control, and cell differentiation, the present observations raise interesting questions about the participation of this activity in the calcification process.     

Regulation of respiration in brain mitochondria and synaptosomes: restrictions of ADP diffusion in situ, roles of tubulin, and mitochondrial creatine kinase

The role of ubiquitous mitochondrial creatine kinase (uMtCK) reaction in regulation of mitochondrial respiration was studied in purified preparations of rat brain synaptosomes and mitochondria. In permeabilized synaptosomes, apparent Km for exogenous ADP, Km (ADP), in regulation of respiration in situ was rather high (110 +/- 11 microM) in comparison with isolated brain mitochondria (9 +/- 1 microM). This apparent Km for ADP observed in isolated mitochondria in vitro dramatically increased to 169 +/- 52 microM after their incubation with 1 muM of dimeric tubulin showing that in rat brain, particularly in synaptosomes, mitochondrial outer membrane permeability for ADP, and ATP may be restricted by tubulin binding to voltage dependent anion channel (VDAC). On the other hand, in synaptosomes apparent Km (ADP) decreased to 25 +/- 1 microM in the presence of 20 mM creatine. To fully understand this effect of creatine on kinetics of respiration regulation, complete kinetic analysis of uMtCK reaction in isolated brain mitochondria was carried out. This showed that oxidative phosphorylation specifically altered only the dissociation constants for MgATP, by decreasing that from ternary complex MtCK.Cr.MgATP (K (a)) from 0.13 +/- 0.02 to 0.018 +/- 0.007 mM and that from binary complex MtCK.MgATP (K (ia)) from 1.1 +/- 0.29 mM to 0.17 +/- 0.07 mM. Apparent decrease of dissociation constants for MgATP reflects effective cycling of ATP and ADP between uMtCK and adenine nucleotide translocase (ANT). These results emphasize important role and various pathophysiological implications of the phosphocreatine-creatine kinase system in energy transfer in brain cells, including synaptosomes.     

Characterization of nucleoside triphosphate diphosphohydrolase activity in Trichomonas gallinae and the influence of penicillin and streptomycin in extracellular nucleotide hydrolysis

Here we described an nucleoside triphosphate diphosphohydrolase (NTPDase) activity in living trophozoites of Trichomonas gallinae. The enzyme hydrolyzes a variety of purine and pyrimidine nucleoside di- and triphosphates in an optimum pH range of 6.0-8.0. This enzyme activity was activated by high concentrations of divalent cations, such as calcium and magnesium. Contaminant activities were ruled out because the enzyme was not inhibited by classical inhibitors of ATPases (ouabain, 5.0 mM sodium azide, oligomycin) and alkaline phosphatases (levamisole). A significant inhibition of ATP hydrolysis (38%) was observed in the presence of 20 mM sodium azide. Sodium orthovanadate inhibited ATP and ADP hydrolysis (24% and 78%), respectively. The apparent K(M) (Michaelis constant) values were 667.62+/-13 microM for ATP and 125+/-5.3 microM for ADP. V(max) (maximum velocity) values were 0.44+/-0.007 nmol Pi min(-1) per 10(6) trichomonads and 0.91+/-0.12 nmol Pi min(-1) per 10(6) trichomonads for ATP and ADP, respectively. Moreover, we showed a marked decrease in ATP, ADP and AMP hydrolysis when the parasites were grown in the presence of penicillin and streptomycin. The existence of an NTPDase activity in T. gallinae may be involved in pathogenicity, protecting the parasite from the cytolytic effects of the extracellular nucleotides.     

Activities of enzymes that hydrolyze adenine nucleotides in platelets from rats experimentally demyelinated with ethidium bromide and treated with interferon-beta

The activities of the enzymes NTPDase (EC 3.6.1.5, apyrase, CD39) and 5'-nucleotidase (EC 3.1.3.5, CD73) were analyzed in platelets from rats submitted to demyelination by ethidium bromide (EB) and treated with interferon beta (IFN-beta). The following groups were studied: I - control (saline), II - (saline and IFN-beta), III - (EB) and IV - (EB and IFN-beta). After 7, 15 and 30 days, the animals (n=7) were sacrificed and the platelets were separated by the method of Lunkes et al. [Lunkes, G., Lunkes D., Morsch, V., Mazzanti, C., Morsch, A., Miron, V., Schetinger, M.R.C., 2004. NTPDase and 5'-nucleotidase in rats alloxan- induced diabetes. Diabetes Research and Clinical Practice 65, 1-6]. NTPDase activity for ATP and ADP substrates was significantly lower in groups II and III after seven days, when compared to control (p<0.001). At fifteen days, ATP hydrolysis was significantly lower in group III and IV and higher in group II (p<0.001), while there was an activation of ADP hydrolysis in group II (p<0.001), when compared with the control. 5'-nucleotidase activity was significantly higher in group IV (p<0.001) after seven days, and lower in the groups III and IV (p<0.001) after fifteen days in relation to the control. No significant differences were observed in NTPDase and 5'-nucleotidase activities after thirty days. In conclusion, our study demonstrated that the hydrolysis of adenine nucleotides is modified in platelets of rats demyelinated and treated with IFN-beta.     

Characterisation of an ATP diphosphohydrolase (Apyrase, EC 3.6.1.5) activity in Trichomonas vaginalis

In the present report the enzymatic properties of an ATP diphosphohydrolase (apyrase, EC 3.6.1.5) in Trichomonas vaginalis were determined. The enzyme hydrolyses purine and pyrimidine nucleoside 5'-di- and 5'-triphosphates in an optimum pH range of 6.0--8.0. It is Ca(2+)-dependent and is insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (5 mM). A significant inhibition of ADP hydrolysis (37%) was observed in the presence of 20 mM sodium azide, an inhibitor of ATP diphosphohydrolase. Levamisole, a specific inhibitor of alkaline phosphatase, and P(1), P(5)-di (adenosine 5'-) pentaphosphate, a specific inhibitor of adenylate kinase, did not inhibit the enzyme activity. The enzyme has apparent K(m) (Michaelis Constant) values of 49.2+/-2.8 and 49.9+/-10.4 microM and V(max) (maximum velocity) values of 49.4+/-7.1 and 48.3+/-6.9 nmol of inorganic phosphate x min(-1) x mg of protein(-1) for ATP and ADP, respectively. The parallel behaviour of ATPase and ADPase activities and the competition plot suggest that ATP and ADP hydrolysis occur at the same active site. The presence of an ATP diphosphohydrolase activity in T. vaginalis may be important for the modulation of nucleotide concentration in the extracellular space, protecting the parasite from the cytolytic effects of the nucleotides, mainly ATP.     

Enzymes that hydrolyze adenine nucleotides in platelets from breast cancer patients

The activities of NTPDase (EC 3.6.1.5, apyrase, CD39) and 5'-nucleotidase (EC 3.1.3.5, CD73) enzymes were analyzed in platelets from breast cancer patients. Initially, patients were compared in terms of length (years) of tamoxifen use. The following groups were studied: breast cancer patients who did not use tamoxifen, patients using tamoxifen for 1-48 months, patients using tamoxifen for 49-84 months, and controls (healthy subjects). Results demonstrated that adenosine triphosphate (ATP) hydrolysis was enhanced (F(3,114)=8.53; P<0.001) and adenosine diphosphate (ADP) hydrolysis was reduced (F(3,106)=5.09, P=0.002) as a function of tamoxifen use, while adenosine monophosphate (AMP) hydrolysis was unchanged. Next, patients were compared statistically according to disease stage, determined by the tumor-node-metastasis (TNM) staging system for classifying breast tumor. ATP hydrolysis was significantly elevated in patients with stage I and II breast cancer (F(4,113)=4.35; P=0.003), but was normal in patients with stage III and IV cancer. ADP hydrolysis was reduced in stages II to IV (F(4,105)=3.88, P=0.006) and AMP hydrolysis was elevated in stage II (F(4,105)=3.45 P=0.01), but was normal in stages III and IV. Platelet aggregation time was similar in all patients regardless of tamoxifen use or disease stage. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were also within the normal range and similar among all groups. Similarly, fibrinogen and fibrin degradation product (FDP) were unchanged in all groups. In conclusion, our study demonstrated for the first time that hydrolysis of adenine nucleotides is modified in platelets from breast cancer patients taking tamoxifen.     

Previous treatment with ebselen and vitamin E alters adenine nucleotide hydrolysis in platelets from adult rats experimentally demyelinated with ethidium bromide

Many aspects of the relationship between the demyelinating pathology and platelet function need to be elucidated. Thus, the activity of NTPDase and 5'-nucleotidase enzymes was analyzed in platelets from rats demyelinated with ethidium bromide (EB) and previously treated with ebselen (Ebs) and vitamin E (Vit. E). The animals were divided into four groups: for ebselen, the groups were: I-control (saline), II-(saline and Ebs), III-(EB) and IV-(EB and Ebs); and for vitamin E, the groups were: I - control (saline), II-(saline and Vit. E), III-(EB) and IV-(EB and Vit. E). After 3 and 21 days, the blood was collected and the platelets were separated for enzymatic assays. For the treatment with Ebs, the NTPDase activity for ATP substrate was significantly lower in groups II, III and IV (p < 0.05) after 3 days, while after 21 days, a reduction was observed in group III (p < 0.05). ADP hydrolysis was reduced in group II (p < 0.05) and increased in group IV (p < 0.05) after 3 days, while after 21 days there was an increase in group IV (p < 0.05). In the treatment with Vit. E, ATP hydrolysis was lower in groups II, III and IV (p < 0.05) after 3 and 21 days. ADP hydrolysis was increased in group II (p < 0.05) after 3 days, and in group IV (p < 0.05) after 21 days. However, 5'-nucleotidase activity was not altered by the treatments. These findings demonstrate that NTPDase activity in platelets is diminished in demyelinating events and the treatments with Ebs and Vit. E modulated adenine nucleotide hydrolysis.     

Evaluation of the antimicrobial activity of ebselen: role of the yeast plasma membrane H+-ATPase

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a selenium-containing antioxidant demonstrating anti-inflammatory and cytoprotective properties in mammalian cells and cytotoxicity in lower organisms. The mechanism underlying the antimicrobial activity of ebselen remains unclear. It has recently been proposed that, in lower organisms like yeast, the plasma membrane H+-ATPase (Pma1p) could serve as a potential target for this synthetic organoselenium compound. Using yeast and bacteria, the present study found ebselen to inhibit microbial growth in a concentration- and time-dependent manner, and yeast and Gram-positive bacteria to be more sensitive to this action (IC50 approximately 2-5 microM) than Gram-negative bacteria (IC50 < 80 microM). Washout experiments and scanning electron microscopic analysis revealed ebselen to possess fungicidal activity. In addition, ebselen was found to inhibit medium acidification by PMA1-proficient haploid yeast in a concentration-dependent manner. Additional studies comparing PMA1 (+/-) and PMA1 (+/+) diploid yeast cells revealed the mutant to be more sensitive to treatment with ebselen than the wild type. Ebselen also inhibited the ATPase activity of Pma1p from S. cerevisiae in a concentration-dependent manner. The interaction of ebselen with the sulfhydryl-containing compounds L-cysteine and reduced glutathione resulted in the complete and partial prevention, respectively, of the inhibition of Pma1p ATPase activity by ebselen. Taken together, these results suggest that the fungicidal action of ebselen is due, at least in part, to interference with both the proton-translocating function and the ATPase activity of the plasma membrane H+-ATPase.     

Characterization of ATP-diphosphohydrolase activities in the intima and media of the bovine aorta: evidence for a regulatory role in platelet activation in vitro.

The inner layer of the aorta contains the enzyme ATP diphosphohydrolase (ATPDase: EC 3.6.1.5) which catalyzes the sequential phosphorolysis of ATP----ADP----AMP. Two zones of the inner layer, the intima and media, were separated and both were shown to contain ATPDase activity of similar specific activity (0.08 and 0.10 U/mg protein, respectively). However, the media exhibited about 100-times more enzyme activity than the intima. Both preparations were virtually identical with respect to pH optima (7.5), migration patterns after electrophoresis under non-denaturing conditions, relative rates of ATP and ADP hydrolysis and potency to inhibit ADP-induced platelet aggregation in both human platelet-rich plasma and whole blood. The IC50 values for ADP (2 microM)-induced aggregation were 6.8 and 12.9 mU/ml in platelet-rich plasma and whole blood, respectively. Addition of ATPDase to platelets pre-aggregated with ADP resulted in a dose-dependent disaggregation in platelet-rich plasma (IC50 4.9 mU/ml), but not in whole blood. When both ATPDase (5.6-58.7 mU/ml) and ATP (0.5-10 microM) were added to platelet-rich plasma, there was an immediate dose-dependent aggregation of platelets followed by a slowly developing disaggregation. These data show that ATPDase is present in both the intima and media layers of bovine aorta and suggest a dual role for this enzyme in platelet activation. By converting ATP released from damaged cells into ADP, the enzyme could facilitate platelet aggregation at the site of vascular injury, whereas the subsequent conversion of ADP to AMP could inhibit or reverse platelet aggregation. The consequence of these activities would be to control the growth of a platelet thrombus.     

Extracellular adenosine nucleotides stimulate protein kinase C activity and human neutrophil activation

We studied the effect of adenosine nucleotides on several aspects of the functional activation of human peripheral blood polymorphonuclear leukocytes (PMN). Radiolabeled ATP bound to PMN in a manner suggesting the existence of specific binding sites because: 1) binding was reversed (92 +/- 6%) by 100-fold excess concentrations of unlabeled ATP but minimally by either ADP (43 +/- 12%) or GTP (37 +/- 8%); and 2) binding saturation was achieved (i.e., specific binding did not increase) above 250 microM ATP. Binding studies revealed that significant ATP hydrolysis occurred, even at low temperatures and in the presence of phosphatase inhibitors. Adenosine nucleotides activated signal transduction mechanisms in PMN because: 1) 1 to 100 microM ATP and 5'-adenylylimidodiphosphate (AMP-PNP) stimulated increased production of 1,2-diacylglycerols; 2) ATP (0.5 to 500 microM) and ADP (0.1 to 10 mM) induced increased insoluble protein kinase (PKC) activity in a dose-dependent manner when used at concentrations greater than 50 microM; 3) ATP (greater than or equal to 50 microM) induced a shift in the solubility of phorbol receptors from mostly soluble (89% in untreated cells) to mostly insoluble (68%), whereas ADP, GTP, and GDP were effective at higher concentrations; and 4) greater than or equal to 50 microM ATP stimulated increased phosphorylation of endogenous PMN proteins. AMP-PNP induced PKC activity and phosphoprotein changes that were qualitatively similar to those observed when PMN were treated with ATP, suggesting that extracellular ATP hydrolysis is not required for signal transduction to activate PKC. Functionally, ATP stimulated the secretion of specific (but not azurophil) granules because vitamin B12-binding protein and low levels of lysozyme, but not beta-glucuronidase, were released; qualitatively similar results were obtained by using AMP-PNP. These results suggest that certain adenosine nucleotides employed at physiologically relevant concentrations stimulate increased 1,2-diacylglycerol production, PKC activity, granule secretion, and endogenous phosphoprotein formation in a manner that is independent of extracellular ATP hydrolysis.     

Dual regulation of arachidonic acid release by P2U purinergic receptors in dibutyryl cyclic AMP-differentiated HL60 cells.

ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neutrophil-like HL60 cells: stimulation in the micromolar range (EC50 = 3.2 +/- 0.9 microM) and inhibition at higher concentrations (EC50 = 90 +/- 11 microM). ATP also inhibited UTP- and platelet activating factor-stimulated AA release. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca2+, cell permeabilization, or changes in the rise of [Ca2+]i induced by agonist. The inhibition was rapid, being detected within 5-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desensitized by pretreatment of the cells with 2 mM ATP, but not 20 microM ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on generation of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with P1 purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP), only UTP and ATP gamma S displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EC50 = 60-300 microM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of P2U receptors in HL60 cells.