Fluoxetine and nortriptyline affect NTPDase and 5'-nucleotidase activities in rat blood serum

Depression is a serious condition associated with considerable morbidity and mortality. Selective serotonin reuptake inhibitors and tricyclic antidepressants, such as fluoxetine and nortriptyline, respectively, were commonly used in treatment for depression. Selective serotonin reuptake inhibitors have been associated with increased risk of bleeding complications, possibly as a result of inhibition of platelet aggregation. ATP, ADP and adenosine are signaling molecules in the vascular system and nucleotidases activities are considered an important thromboregulatory system which functions in the maintenance of blood fluidity. Therefore, here we investigate the effect of in vivo (acute and chronic) and in vitro treatments with the antidepressant drugs on nucleotidases activities in rat blood serum. In acute treatment, nortriptyline decreased ATP hydrolysis (41%), but not altered ADP and AMP hydrolysis. In contrast, fluoxetine did not alter NTPDase and ecto-5'-nucleotidase activities. A significant inhibition of ATP, ADP, and AMP hydrolysis were observed in chronic treatment with fluoxetine (60%, 32%, and 42% for ATP, ADP, and AMP hydrolysis, respectively). Similar effects were shown in chronic treatment with nortriptyline (37%, 41%, and 30% for ATP, ADP, and AMP hydrolysis, respectively). In addition, there were no significant changes in NTPDase and ecto-5'-nucleotidase activities when fluoxetine and nortriptyline (100, 250, and 500 microM) were tested in vitro. Our results have shown that fluoxetine and nortriptyline changed the nucleotide catabolism, suggesting that homeostasis of vascular system can be altered by antidepressant treatments.     

Enzymes that hydrolyze adenine nucleotides in chronic renal failure: relationship between hemostatic defects and renal failure severity

The activities of the enzymes NTPDase (E.C.3.6.1.5, apyrase, ATP diphosphohydrolase, ecto-CD 39) and 5'-nucleotidase (E.C.3.1.3.5, CD 73) were analyzed in platelets from patients with chronic renal failure (CRF), both undergoing hemodialysis treatment (HD) and not undergoing hemodialysis (ND), as well as from a control group. The results showed an increase in platelet NTPDase activity in CRF patients on HD treatment (52.88%) with ATP as substrate (P<0.0001). ADP hydrolysis was decreased (33.68% and 39.75%) in HD and ND patients, respectively. In addition, 5'-nucleotidase activity was elevated in the HD (160%) and ND (81.49%) groups when compared to the control (P<0.0001). Significant correlation was found among ATP, ADP and AMP hydrolysis and plasma creatinine and urea levels (P<0.0001). Patients were compared statistically according the time of hemodialysis treatment. We found enhanced NTPDase and 5'-nucleotidase activities between 49 and 72 months on HD patients. Our result suggests the existence of alterations in nucleotide hydrolysis in platelets of CRF patients. Possibly, this altered nucleotide hydrolysis could contribute to hemostasis abnormalities found in CRF.     

NTPDase and 5′-Nucleotidase Activities in Synaptosomes of Hippocampus and Serum of Rats Subjected to Homocysteine Administration

Patients with homocystinuria, an inborn error of metabolism, present neurological dysfunction and commonly experience frequent thromboembolic complications. The nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase enzymes regulate the nucleotide/nucleoside ratio in the central nervous system and in the circulation and are thought to be involved in these events. Thus, the current study investigated the effect of homocysteine administration on NTPDase and 5'-nucleotidase activities, in the synaptosomal fraction of rat hippocampus, and on nucleotidase activities in rat serum. Twenty-nine-day-old Wistar rats were divided in two groups: group I (control), animals received 0.9% saline; group II (homocysteine-treated), animals received one single subcutaneous injection of homocysteine (0.6 micromol/g). Rats were killed 1 h after the injection. NTPDase and 5'-nucleotidase activities from brain and serum were significantly increased in the homocysteine-treated group. Results show that, in hippocampus, ATP and ADP hydrolysis increased by 20.5% and 20%, respectively, and AMP hydrolysis increased by 48%, when compared to controls. In serum, ATP and ADP hydrolysis increased 136% and 107%, respectively, and AMP hydrolysis increased 95%, in comparison to controls. The current data strongly indicate that in vivo homocysteine administration alters the activities of the enzymes involved in nucleotide hydrolysis, both in the central nervous system and in the serum of adult rats.     

Heparin and chondroitin sulfate inhibit adenine nucleotide hydrolysis in liver and kidney membrane enriched fractions

The inhibition of adenine nucleotide hydrolysis by heparin and chondroitin sulfate (sulfated polysaccharides) was studied in membrane preparations from liver and kidney of adult rats. Hydrolysis was measured by the activity of NTPDase and 5′-nucleotidase. The inhibition of NTPDase by heparin was observed at three different pH values (6.0, 8.0 and 10.0). In liver, the maximal inhibition observed for ATP and ADP hydrolysis was about 80% at pH 8.0 and 70% at pH 6.0 and 10.0. Similarly to the effect observed in liver, heparin caused inhibition of ATP and ADP hydrolysis that reached a maximum of 70% in kidney (pH 8.0). Na⁺, K⁺ and Rb⁺ changed the inhibitory potency of heparin, suggesting that its effects may be related to charge interaction. In addition to heparin, chondroitin sulfate also caused a dose-dependent inhibition in liver and kidney membranes. The maximal inhibition observed for ATP and ADP hydrolysis was about 60 and 50%, respectively. In addition, the hepatic and renal activity of 5′-nucleotidase was inhibited by heparin and chondroitin sulfate, except for kidney membranes where chondroitin sulfate did not alter AMP hydrolysis. On this basis, the findings indicate that glycosaminoglycans have a potential role as inhibitors of adenine nucleotide hydrolysis on the surface of liver and kidney cell membranes in vitro.     

The effect of ebselen on adenine nucleotide hydrolysis by platelets from adult rats

The extracellular hydrolysis of adenine nucleotides by intact rat blood platelets occurs by the action of a cascade of enzymes constituted by an NTPDase 3 (CD39, EC 3.6.1.5, apyrase) and a 5'-nucleotidase (CD73, EC 3.5.7.3), whose final product is adenosine. Ebselen is a seleno-organic compound that possesses low toxicity and exhibits antioxidant, anti-inflammatory, anti-atherosclerotic, and cytoprotective properties. The main objective of this study was to evaluate if the anti-inflammatory drug ebselen can modulate the extracellular adenine nucleotide hydrolysis by platelets from rats. Our results showed that ebselen, at final concentrations of 30 and 100 microM, inhibits in vitro ATP extracellular hydrolysis by 48 and 60%, respectively. Ebselen, at final concentrations of 100 and 130 microM, also inhibited the in vitro extracellular hydrolysis of ADP by 28 and 35%, respectively. However, this drug did not alter AMP hydrolysis by platelets in the appropriate assay conditions. Kinetic analysis showed that the inhibition of ADP and ATP hydrolysis by ebselen, in rat platelets, is of the uncompetitive type. The IC50 calculated from the results were 99 +/- 10 and 186 +/- 47 microM (mean +/- S.D., n = 3) for ATP and ADP hydrolysis, respectively.     

ATP and ADP hydrolysis in brain membranes of zebrafish (Danio rerio)

Nucleotides, e.g. ATP and ADP, are important signaling molecules, which elicit several biological responses. The degradation of nucleotides is catalyzed by a family of enzymes called NTPDases (nucleoside triphosphate diphosphohydrolases). The present study reports the enzymatic properties of a NTPDase (CD39, apyrase, ATP diphosphohydrolase) in brain membranes of zebrafish (Danio rerio). This enzyme was cation-dependent, with a maximal rate for ATP and ADP hydrolysis in a pH range of 7.5-8.0 in the presence of Ca(2+) (5 mM). The enzyme displayed a maximal activity for ATP and ADP hydrolysis at 37 degrees C. It was able to hydrolyze purine and pyrimidine nucleosides 5'-di and triphosphates, being insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (0.1 mM). A significant inhibition of ATP and ADP hydrolysis (68% and 34%, respectively) was observed in the presence of 20 mM sodium azide, used as a possible inhibitor of ATP diphosphohydrolase. Levamisole (1 mM) and tetramisole (1 mM), specific inhibitors of alkaline phosphatase and P1, P(5)-di (adenosine 5'-) pentaphosphate, an inhibitor of adenylate kinase did not alter the enzyme activity. The presence of a NTPDase in brain membranes of zebrafish may be important for the modulation of nucleotide and nucleoside levels, controlling their actions on specific purinoceptors in central nervous system of this specie.     

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.     

NTPDase and 5′-nucleotidase Activities of Synaptosomes from Hippocampus of Rats Subjected to Hyperargininemia

ATP is an important excitatory neurotransmitter and adenosine acts as a neuromodulatory structure inhibiting neurotransmitters release in the central nervous system. Since the ecto-nucleotidase cascade that hydrolyzes ATP to adenosine is involved in the control of brain functions and previous studies realized in our laboratory have recently reported that acute administration of Arg decreases the NTPDase and 5′-nucleotidase activities of rat blood serum, in the present study we investigated the effect of arginine administration on NTPDase and 5′-nucleotidase activities by synaptosomes from hippocampus of rats. First, sixty-days-old rats were treated with a single or a triple intraperitoneal injection of arginine (0.8 g/Kg) or an equivalent volume of 0.9% saline solution (control) and were killed 1 h later. Second, rats received an intracerebroventricular injection of 1.5 mM arginine solution or saline (5 μL) and were killed 1 h later. We also tested the in vitro effect of arginine (0.1–1.5 mM) on nucleotide hydrolysis in synaptosomes from rat hippocampus. Results showed that intraperitoneal arginine administration did not alter nucleotide hydrolysis. On the other hand, arginine administered intracerebroventricularly reduced ATP (32%), ADP (30%) and AMP (21%) hydrolysis, respectively. In addition, arginine added to the incubation medium, provoked a decrease on ATP (19%), ADP (17%) and AMP (23%) hydrolysis, respectively. Furthermore, kinetic studies showed that the inhibitory effect of arginine was uncompetitive in relation to ATP, ADP and AMP. In conclusion, according to our results it seems reasonable to postulate that arginine alters the cascade involved in the extracellular degradation of ATP to adenosine.     

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.     

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.     

The C-terminal cysteine-rich region dictates specific catalytic properties in chimeras of the ectonucleotidases NTPDase1 and NTPDase2.

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) comprise a novel family of ectonucleotidases that are important in the hydrolysis of extracellular nucleotides. The related NTPDase1 (ecto-apyrase) and NTPDase2 (ecto-ATPase) share a common membrane topography with a transmembrane domain at both the N- and C-terminus, an extensive extracellular loop with five 'apyrase conserved regions' (ACR1 to ACR5), and a cysteine-rich C-terminal region. Whereas NTPDase1 expressed in CHO cells hydrolyzes ATP and ADP equivalently, NTPDase2 has a high preference for the hydrolysis of ATP over ADP. In addition recombinant NTPDase1 hydrolyzes ATP to AMP with the formation of only minor amounts of free ADP. In contrast, ADP appears as the major free product when ATP is hydrolyzed by NTPDase2. In order to determine molecular domains responsible for these differences in catalytic properties, chimeric cDNAs were constructed in which N-terminal sequences of increasing length of NTPDase1 were substituted by the corresponding sequences of NTPDase2 and vice versa. The turnover points were contained within ACR1 to ACR5. Chimeric cDNAs were expressed in CHO cells and surface expression was verified by immunocytochemistry. ATP and ADP hydrolysis rates and ADP and AMP product formation were determined using HPLC. Amino-acid residues between ACR3 and ACR5 and in particular the cysteine-rich region between ACR4 and ACR5 conferred a phenotype to the chimeric enzymes that corresponded to the respective wild-type enzyme. Protein structure rather than the conserved ACRs may be of major relevance for determining differences in the catalytic properties between the related wild-type enzymes.     

Up-regulation of ectonucleotidase activity after cortical stab injury in rats

The objective of this study was to examine the changes in the activity and expression of ectonucleotidase enzymes in the model of unilateral cortical stab injury (CSI) in rat. The activities of ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) and ecto 5'-nucleotidase were assessed by measuring the levels of ATP, ADP and AMP hydrolysis in the crude membrane preparations obtained from injured left cortex, right cortex, left and right caudate nucleus, whole hippocampus and cerebellum. Significant increase in NTPDase and ecto 5'-nucleotidase activities was observed in the injured cortex following CSI, whereas in other brain areas only an increase in ecto 5'-nucleotidase activity was seen. Immunohistochemical analysis performed using antibodies specific to NTPDase 1 and ecto 5'-nucleotidase demonstrated that CSI induced significant changes in enzyme expression around the injury site. Immunoreactivity patterns obtained for NTPDase 1 and ecto 5'-nucleotidase were compared with those obtained for glial fibrillary acidic protein, as a marker of astrocytes and complement receptor type 3 (OX42), as a marker of microglia. Results suggest that up-regulation of ectonucleotidase after CSI is catalyzed by cells that activate in response to injury, i.e. cells immunopositive for NTPDase 1 were predominantly microglial cells, whereas cells immunopositive for ecto 5'-nucleotidase were predominantly astrocytes.     

Genomic-independent action of thyroid hormones on NTPDase activities in Sertoli cell cultures from congenital hypothyroid rats

The Sertoli cells play an essential role in the maintenance and control of spermatogenesis. The ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) and 5'-nucleotidase activities can modulate the extracellular adenine nucleotide levels, controlling nucleotide-mediated signaling events in Sertoli cells. Since thyroid hormones (TH) and adenine nucleotides and nucleosides play important modulatory roles in Sertoli cell proliferation and differentiation, the aim of our study was to investigate the effect of hypothyroidism upon the NTPDase and 5'-nucleotidase activities in Sertoli cell cultures, as well as to verify whether these effects may be reversed by short and long-term supplementation with TH. Congenital hypothyroidism was induced by adding 0.02% methimazole in the drinking water from day 9 of gestation and continually until 18 days of age. Hypothyroidism significantly decreased the extracellular ATP and ADP hydrolysis and this effect was significantly reversed when cell cultures were supplemented with 1 microM T3 or 0.1 microM T4 for 30 min. In contrast, AMP hydrolysis was not altered by hypothyroidism, but was increased by T4 supplementation for 24 h. The presence of the enzymes NTPDase 1, 2 and 3 was detected by RT-PCR in Sertoli cell cultures, however, hypothyroidism was not able to alter the expression of these enzymes. These findings demonstrate that TH modify NTPDase activities in hypothyroid Sertoli cells, probably via nongenomic mechanisms and, consequently, may influence the reproductive function throughout development.     

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.     

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.     

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.     

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.     

Characterization of the NTPDase activities in the mesentery pre- and post-capillary circuits of the guinea pig

NTPDase is one of the principal enzymes involved in the sequential hydrolysis of ATP. In the present study, the presence and functionality of NTPDase in the mesenteric vein and artery were examined. Adenosine triphosphate (ATP) (0.01-1000 pmol) induces a dose-dependent vasodilation in the isolated arterial and venous mesenteric vasculatures of the guinea pig. Adenosine diphosphate (ADP) (0.01-1000 pmol) but not adenosine monophosphate (AMP) (0.01-1000 pmol) induces a similar response in the mesenteric vascular circuit. L-NAME, a nitric oxide synthase inhibitor (200 microM, 30 min), significantly reduces the arterial dilatory effect of ATP and abolishes the responses to ADP and AMP. Complete removal of the endothelium with 3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate (CHAPS) (20 mM, 2 x 45 s) abolishes ATP-induced responses. Infusion of ATP in the vascular circuit generated detectable amounts of ADP and AMP, as measured by HPLC. CHAPS treatment significantly reduced the level of ATP and the production of AMP in the arterial mesenteric circuit. In contrast to the arterial mesenteric vasculature, endothelium removal in the venous circuit triggered a marked potentiation of ADP release and, interestingly, a marked reduction in the release of AMP. Moreover, a specific inhibitor of NTP diphosphohydrolase, 1-hydroxynaphthlene-3,6-disulfonic acid BGO 136 (10 mM for 20 min), significatively reduced AMP production in both vascular preparations. These results confirm that the endothelium contributes to the vasoactive properties of ATP, ADP, and AMP. Our data also demonstrated a significant role of endothelium in NTPDase activity on ADP and AMP production prior to exogenous administration of ATP. The activity of this particular enzyme appears to be different from the reaction products viewpoint (i.e., the production of ADP) in the pre- and post-mesenteric circuits, suggesting two different isoforms with different substrate specificities.