Adenine Nucleotide Hydrolysis Research Articles

Sec18 binds the tethering/SM complex HOPS to engage the Qc-SNARE for membrane fusion.

Membrane fusion is regulated by Rab GTPases, their tethering effectors such as HOPS, SNARE proteins on each fusion partner, SM proteins to catalyze SNARE assembly, Sec17 (SNAP), and Sec18 (NSF). Though concentrated HOPS can support fusion without Sec18, we now report that fusion falls off sharply at lower HOPS levels, where direct Sec18 binding to HOPS restores fusion. This Sec18-dependent fusion needs adenine nucleotide but neither ATP hydrolysis nor Sec17. Sec18 enhances HOPS recognition of the Qc-SNARE. With high levels of HOPS, Qc has a Km for fusion of a few nM. Either lower HOPS levels, or substitution of a synthetic tether for HOPS, strikingly increases the Km for Qc to several hundred nM. With dilute HOPS, Sec18 returns the Km for Qc to low nM. In contrast, HOPS concentration and Sec18 have no effect on Qb-SNARE recognition. Just as Qc is required for fusion but not for the initial assembly of SNAREs in trans, impaired Qc recognition by limiting HOPS without Sec18 still allows substantial trans-SNARE assembly. Thus, in addition to the known Sec18 functions of disassembling SNARE complexes, oligomerizing Sec17 for membrane association, and allowing Sec17 to drive fusion without complete SNARE zippering, we report a fourth Sec18 function, the Sec17-independent binding of Sec18 to HOPS to enhance functional Qc-SNARE engagement.

Effect of Thiazolidin-4-one Against Lipopolysaccharide-Induced Oxidative Damage, and Alterations in Adenine Nucleotide Hydrolysis and Acetylcholinesterase Activity in Cultured Astrocytes.

Astrocytes play multiple important roles in brain physiology. However, depending on the stimuli, astrocytes may exacerbate inflammatory reactions, contributing to the development and progression of neurological diseases. Therefore, therapies targeting astrocytes represent a promising area for the development of new brain drugs. Thiazolidinones are heterocyclic compounds that have a sulfur and nitrogen atom and a carbonyl group in the ring and represent a class of compounds of great scientific interest due to their pharmacological properties. The aim of this study was to investigate the effect of 3-(3-(diethylamino)propyl)-2-(4-(methylthio)phenyl)thiazolidin-4-one (DS27) on cell proliferation and morphology, oxidative stress parameters, activity of the enzymes ectonucleotidases and acetylcholinesterase (AChE) and interleukin 6 (IL-6) levels in primary astrocyte cultures treated with lipopolysaccharide (LPS), to model neuroinflammation. The astrocyte culture was exposed to LPS (10μg/ml) for 3h and subsequently treated with compound DS27 for 24 and 48h (concentrations ranging to 10-100μM). LPS induced an increase in astrocyte proliferation, AChE activity, IL-6 levels, oxidative damage, ATP and ADP and a reduction in AMP hydrolysis in rat primary astrocyte cultures. DS27 treatment was effective in reversing these alterations induced by LPS. Our findings demonstrated that DS27 is able to modulate cholinergic and purinergic signaling, redox status, and the levels of pro-inflammatory cytokines in LPS-induced astrocyte damage. These glioprotective effects of DS27 may be very important for improving neuroinflammation, which is associated with many brain diseases.

Sabicea brasiliensis Wernham: Antioxidant Activity, Proliferative Effect and Modulation of Vascular Adenine Nucleotides Metabolism

Aims: Study addressed the antioxidant activity (AA) of Sabicea brasiliensis roots crude extract (CE), ethyl acetate (EAF), and hydro-methanolic (HMF) fractions, and its impact on cell viability and adenine nucleotide hydrolysis in vascular A7r5 cells.
 Materials and Methods: AA of CE, EAF and HMF were determined by the inhibition of the DPPH and ABTS radicals. Total phenolic content was evaluated by Folin-Ciocalteau. Cell viability was determined by MTT assay at different concentrations (62.5; 125; 250 and 500 μg·mL-1) of EAF and HMF after 24, 48 and 72 h. Ectonucleotidase activities were evaluated by colorimetric methods after 48 h EAF or HMF treatment.
 Results: The highest AA was observed for CE (76%), followed by EAF (46%) and HMF (23%). Phenolic content followed the same pattern. After 48 h, EAF increased A7r5 vascular cells viability by 40%, 40%, 62% and 25% at distinct concentrations, respectively; while HMF augmented it by 50% (500 μg·mL−1). Finally, after 48 h EAF (500 μg·mL−1) decreased about 50% of ATP and ADP metabolism while HMF inhibited 56 and 59% the hydrolysis of NPP substrate (at 125 and 250 μg·mL−1).
 Conclusion: Study confirmed the high AA of S. brasiliensis, which influences vascular cells proliferation and purines metabolism, pointing to potential cellular pathways that may support the popular use of this plant.

Hydrolysis of ATP, ADP, and AMP is increased in blood plasma of prostate cancer patients.

Prostate cancer is among the major malignancies that affect men around the world. Adenine nucleotides are important signaling molecules that mediate innumerous biological functions in pathophysiological conditions, including cancer. These molecules are degraded by several ectoenzymes named ectonucleotidases that produce adenosine in the extracellular medium. Some of these ecto-enzymes can be found in soluble in the blood stream. Thus, the present study aimed to evaluate the hydrolysis of adenine nucleotides (ATP, ADP, and AMP) in the plasma blood of patients with prostate cancer. Peripheral blood samples were collected, and questionnaires were filled based on the clinical data of the medical records. The nucleotide hydrolysis was performed by Malachite Green method using ATP, ADP, and AMP as substrates. Plasma from prostate cancer patients presented an elevated hydrolysis of all nucleotides evaluated when compared to healthy individuals. NTPDase inhibitor (ARL67156) and the alkaline phosphatase inhibitor (levamisole) did not alter ATP hydrolysis. However, AMP hydrolysis was reduced by the CD73 inhibitor, APCP, and by levamisole, suggesting the action of a soluble form of CD73 and alkaline phosphatase. On microvesicles, it was observed that there was a low expression and activity of CD39 and almost absent of CD73. The correlation of ATP, ADP, and AMP hydrolysis with clinic pathological data demonstrated that patients who received radiotherapy showed a higher AMP hydrolysis than those who did not, and patients with lower clinical stage (CS-IIA) presented an elevated ATP hydrolysis when compared to those with more advanced clinical stages (CS-IIB and CS-III). Patients of all clinical stages presented an elevated AMPase activity. Therefore, we can suggest that the nucleotide hydrolysis might be attributed to soluble ecto-enzymes present in the plasma, which, in a coordinate manner, produce adenosine in the blood stream, favoring prostate cancer progression.

High levels of methionine and methionine sulfoxide: Impact on adenine nucleotide hydrolysis and redox status in platelets and serum of young rats.

We investigated acute and chronic effects administration of methionine (Met) and/or methionine sulfoxide (MetO) on ectonucleotidases and oxidative stress in platelets and serum of young rats. Wistar rats were divided into four groups: control, Met, MetO, and Met + MetO. In acute treatment, the animals received a single subcutaneous injection of amino acid(s) and were euthanized after 1 and 3 hours. In chronic protocol, Met and/or MetO were administered twice a day with an 8-hour interval from the 6th to the 28th day of life. Nucleoside triphosphate phosphohydrolase and 5'-nucleotidase activities were reduced in platelets and serum by Met, MetO, and Met + MetO after 3 hours and 21 days. Adenosine deaminase activity reduced in platelets at 3 hours after MetO and Met + MetO administration and increased after 21 days in animals treated with Met + MetO. Superoxide dismutase and catalase activities decreased in platelets in MetO and Met + MetO groups after 3 hours, while reactive oxygen species (ROS) levels increased in same groups. Catalase activity in platelets decreased in all experimental groups after chronic treatment. Met, MetO, and Met + MetO administration increased plasmatic ROS levels in acute and chronic protocols; glutathione S-transferase activity increased by MetO and Met + MetO administration at 3 hours, and ascorbic acid decreased in all experimental groups in acute and chronic protocols. Thiobarbituric acid reactive substances increased, superoxide dismutase and catalase activities reduced in the Met and/or MetO groups at 3 hours and in chronic treatment. Our data demonstrated that Met and/or MetO induced changes in adenine nucleotide hydrolysis and redox status of platelets and serum, which can be associated with platelet dysfunction in hypermethioninemia.

Purinergic system as a potential target for inflammation and toxicity induced by thymol in immune cells and tissues

Thymol is a phytochemical component present in many plants used as food additive in order to promote animal growth due to its several biological properties. However, possible side effects of thymol remain poorly known limited to few reports. In this sense, we evaluated the enzymes of the purinergic signaling such as, ectonucleoside triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA), that play an important role on toxicity induced by excessive adenosine triphosphate (ATP) content in the extracellular environment. Thus, the aim of this study was to evaluate whether purinergic signaling could be considered a potential target of thymol-induced inflammation, and the toxicity in tissues and immune cells of mice after thymol administration. NTPDase activity (ATP as substrate) in serum, spleen, and splenic lymphocytes was lower after 30 days of oral treatment at doses of 10, 20, and 40mg/kg of thymol, while ADA activity was stimulated at 20 and 40mg/kg. No differences were observed between groups regarding NTPDase (ADP as substrate) and 5'-nucleotidase activities in all evaluated tissues. Based on these evidences, adenine nucleotide hydrolysis is modified in serum, spleen, and splenic lymphocytes of mice treated with thymol, contributing to inflammation and toxicity by a reduction on ATP hydrolyses and its possible accumulation in the extracellular medium and increased Ado desamination and its possible reduction in the extracellular environment, leading to a self-sustained pro-inflammatory deleterious cycle. In summary, all tested thymol concentrations induced inflammation and toxicity in tissues and immune cells of treated mice.

Purinergic signaling as a potential target of hypoxia stress-induced impairment of the immune system in freshwater catfish Lophiosilurus alexandri

Purinergic signaling plays an important role in the immune and inflammatory responses through the hydrolysis of adenine nucleotides, such as adenosine triphosphate (ATP), and nucleosides, such as adenosine, which are involved in physiological and pathological events as pro-inflammatory and anti-inflammatory mediators. The aim of this study was to evaluate whether purinergic signaling can modulate the immune and inflammatory responses in the plasma of Lophiosilurus alexandri exposed to hypoxia. Plasma ectonucleoside triphosphate diphosphohydrolase (NTPDase) activity using ATP as a substrate decreased after 24 and 72 h of hypoxia exposure compared with the normoxia group, while no difference was observed for NTPDase (using ADP as a substrate) and 5′-nucleotidase activities. On the other hand, adenosine deaminase (ADA) activity increased after 24 and 72 h of hypoxia exposure compared with the normoxia group. Moreover, is important to emphasize that enzymatic activity of NTPDase (using ATP as a substrate) and ADA did return to control levels only after a 72 h recovery period. Also, plasmatic levels of pro-inflammatory cytokines (interleukin-1, interleukin-6 and tumor necrosis factor-alpha) and ATP increased after 24 and 72 h of hypoxia exposure compared with the normoxia group. Based on this evidence, our findings reveal that adenine nucleotide hydrolysis is not able to modulate the immune and inflammatory responses of fish exposed to hypoxia stress. Moreover, the downregulation of plasma NTPDase activity develops a pro-inflammatory profile due to the excessive ATP content in the extracellular medium elicited by interaction with the P2X7 purineceptor. In summary, purinergic signaling displays a pro-inflammatory profile in the plasma of L. alexandri exposed to hypoxia.

Purinergic signalling displays an anti-inflammatory profile in the spleen of fish experimentally infected with Aeromonas caviae: Modulation of the immune response.

Extracellular adenosine triphosphate (ATP) and its metabolite adenosine (Ado) are recognized as key mediators of immune and inflammatory responses. Depending on its concentration, ATP may act as an immunostimulant or immunodepressant, while Ado levels display an anti-inflammatory profile. The aim of this study was to evaluate whether splenic purinergic signalling is capable of modulating immune and inflammatory responses in fish experimentally infected with Aeromonas caviae. Triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase activities increased in the spleen of silver catfish (Rhamdia quelen) experimentally infected with A.caviae compared with the uninfected control group. Moreover, splenic Ado levels increased in the infected animals relative to the uninfected control group. Based on these lines of evidence, our findings revealed that adenine nucleotide hydrolysis is modified in the spleen of fish infected with A.caviae attempting to restrict the inflammatory process through the upregulation of NTPDase and 5'-nucleotidase activities, which occurs in an attempt to hydrolyse the excessive ATP in the extracellular environment and rapidly hydrolyse AMP to form Ado. In summary, purinergic signalling can modulate immune and inflammatory responses during A.caviae infection.

Aeromonas caviae alters the activities of ecto-enzymes that hydrolyze adenine nucleotides in fish thrombocytes

It is recognized that the purinergic system, through the activities of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5′-nucleotidase (E-5′-nucleotidase), and ecto-adenosine deaminase (E-ADA), is involved in the regulation and modulation of the physiological and pathological events linked to hemostasis. This occurs due to the role of adenosine diphosphate (ADP) in the activation and recruitment of platelets, and the role of adenosine (Ado) in the inhibition of platelet activation. Thus, here we aimed to evaluate whether Aeromonas caviae infection impairs the ecto-enzymes of the purinergic system in fish thrombocytes and the involvement of this system in the hemorrhagic septicemia. The total number of fish thrombocytes decreased in infected animals compared to uninfected animals. Regarding the ecto-enzymes of the purinergic system, the E-NTPDase and E-5′-nucleotidase activities increased in infected animals compared to uninfected animals, while the E-ADA activity decreased. These findings show that adenine nucleotide hydrolysis is modified in the thrombocytes of fish experimentally infected with A. caviae, which impairs the coagulation process due the excessive hydrolysis of ADP, a molecule linked with activation and recruitment of thrombocytes at the site of vascular injury, and augmentation on Ado levels, a molecule linked with inhibitory effects on platelet activation and aggregation. In summary, the purinergic system might contribute to the occurrence of hemorrhagic frames in fish infected with A. caviae.

Purinergic system displays an anti-inflammatory profile in serum of silver catfish experimentally infected with Streptococcus agalactiae: An attempt to ameliorate the inflammatory response

The purinergic system is recognized to modulate extracellular adenosine triphosphate (ATP) and related nucleotides through the activities of triphosphate diphosphohydrolase (NTPDase), 5′-nucleotidase, and adenosine deaminase (ADA), thereby playing an essential role in the immunoregulation of inflammatory and immune responses. Thus, the aim of this study was to evaluate whether the purinergic system can improve the inflammatory response in fish experimentally infected with Streptococcus agalactiae through the modulation of seric NTPDase, 5′-nucleotidase and ADA activities. Seric NTPDase (ATP as substrate) and 5′-nucleotidase activities increased in silver catfish experimentally infected with S. agalactiae compared with the uninfected control group, while seric ADA activity decreased. Based on this evidence, our findings suggest that regulation of adenine nucleotide hydrolysis occurs in an attempt to restrict the inflammatory process and improve the immune system by hydrolyzing excess extracellular ATP. On the other hand, downregulation of seric ADA activity may be an attempt to augment extracellular adenosine (a molecule with anti-inflammatory effects) levels. In summary, the purinergic system is capable of modulating the immune and inflammatory responses during fish streptococcosis.

Post-thyroidectomy hypothyroidism increases the expression and activity of ectonucleotidases in platelets: Possible involvement of reactive oxygen species

Signaling mediated by purines is a widespread mechanism of cell–cell communication related to vasomotor responses and the control of platelet function in the vascular system. However, little is known about the involvement of this signaling as well as the role of reactive oxygen species (ROS) in the development of hypothyroidism. Therefore, the present study investigates changes in the purinergic system, including enzyme activities and expression in platelets, and oxidative profiles in patients with post-thyroidectomy hypothyroidism. The nucleoside triphosphate diphosphohydrolase 1 (NTPDase/CD39) expression in patients increased by 40%, and the adenosine triphosphate (ATP) or adenosine diphosphate (ADP) hydrolyzing activity increased by 82% and 70%, respectively. The activities of ecto-5´-nucleotidase and adenosine deaminase (ADA) also significantly enhanced (39% and 52%, respectively), which correlates with a 45% decrease in adenosine concentration. Furthermore, these patients demonstrated an increased production of ROS (42%), thiobarbituric acid reactive substances (TBARS) (115%), carbonyl protein (30%) and a decreased glutathione S-transferase (GST) activity (20%). This study demonstrates that hypothyroidism interferes with adenine nucleoside and nucleotide hydrolysis and this is correlated with oxidative stress, which might be responsible for the increase in ADA activity. This increase causes rapid adenosine deamination, which can generate a decrease in their concentration in the systemic circulation, which can be associated with the development of vascular complications.

Effect of high fat diets on the NTPDase, 5′-nucleotidase and acetylcholinesterase activities in the central nervous system

High fat diets are associated with the promotion of neurological diseases, such as Alzheimer disease (AD). This study aim investigate the high fat diets role to promotion of AD using as biochemistry parameter of status of central nervous system through the NTPDase, 5′-nucleotidase and acetylcholinesterase (AChE) activities in brain of young rats. The intake of high fat diets promotes an inhibition of purinergic and cholinergic functions, mainly in the long-term exposure to saturated and saturated/unsaturated diets. The AChE activity was decreased to supernatant and synaptosomes tissues preparations obtained from cerebral cortex in average of 20%, to both groups exposed to saturated and saturated/unsaturated diets, when compared to the control group. Very similar results were found in hippocampus and cerebellum brain areas. At same time, the adenine nucleotides hydrolysis in synaptosomes of cerebral cortex were decreased to ATP, ADP and AMP after the long-term exposure to high fat diets, as saturated and saturated/unsaturated. The inhibition of ATP hydrolysis was of 26% and 39% to saturated and saturated/unsaturated diets, respectively. ADP hydrolysis was decreased in 20% to saturated diet, and AMP hydrolysis was decreased in 25% and 33% to saturated and saturated/unsaturated diets, respectively, all in comparison to the control. Thus, we can suggest that the effects of high diets on the purinergic and cholinergic nervous system may contribute to accelerate the progressive memory loss, to decline in language and other cognitive disruptions, such as AD patients presents.

Erratum to: 17β-Estradiol-Induced Synaptic Rearrangements Are Accompanied by Altered Ectonucleotidase Activities in Male Rat Hippocampal Synaptosomes.

17β-Estradiol (E2) rapidly, by binding to membrane estrogen receptors, activates cell signaling cascades which induce formation of new dendritic spines in the hippocampus of males as in females, but the interaction with other metabolic processes, such as extracellular adenine nucleotides metabolism, are currently unknown. Extracellular adenine nucleotides play significant roles, controlling excitatory glutamatergic synapses and development of neural circuits and synaptic plasticity. Their precise regulation in the synaptic cleft is tightly controlled by ecto-nucleoside triphosphate diphosphohydrolase (NTPDase)/ecto-5'-nucleotidase (eN) enzyme chain. Therefore, we sought to clarify whether a single systemic injection of E2 in male rats is accompanied by changes in the expression of the pre- and postsynaptic proteins and downstream kinases linked to E2-induced synaptic rearrangement as well as alterations in NTPDase/eN pathway in the hippocampal synaptosomes. Obtained data showed activation of mammalian target of rapamycin and upregulation of key synaptic proteins necessary for spine formation, 24 h after systemic E2 administration. In E2-mediated conditions, we found downregulation of NTPDase1 and NTPDase2 and attenuation of adenine nucleotide hydrolysis by NTPDase/eN enzyme chain, without changes in NTPDase3 properties and augmentation of synaptic tissue-nonspecific alkaline phosphatase (TNAP) activity. Despite reduced NTPDase activities, increased TNAP activity probably prevents toxic accumulation of ATP in the extracellular milieu and also hydrolyzes accumulated ADP due to unchanged NTPDase3 activity. Thus, our initial evaluation supports idea of specific roles of different ectonucleotidases and their coordinated actions in E2-mediated spine remodeling and maintenance.

17β-Estradiol-Induced Synaptic Rearrangements Are Accompanied by Altered Ectonucleotidase Activities in Male Rat Hippocampal Synaptosomes.

17β-Estradiol (E2) rapidly, by binding to membrane estrogen receptors, activates cell signaling cascades which induce formation of new dendritic spines in the hippocampus of males as in females, but the interaction with other metabolic processes, such as extracellular adenine nucleotides metabolism, are currently unknown. Extracellular adenine nucleotides play significant roles, controlling excitatory glutamatergic synapses and development of neural circuits and synaptic plasticity. Their precise regulation in the synaptic cleft is tightly controlled by ecto-nucleoside triphosphate diphosphohydrolase (NTPDase)/ecto-5'-nucleotidase (eN) enzyme chain. Therefore, we sought to clarify whether a single systemic injection of E2 in male rats is accompanied by changes in the expression of the pre- and postsynaptic proteins and downstream kinases linked to E2-induced synaptic rearrangement as well as alterations in NTPDase/eN pathway in the hippocampal synaptosomes. Obtained data showed activation of mammalian target of rapamycin and upregulation of key synaptic proteins necessary for spine formation, 24h after systemic E2 administration. In E2-mediated conditions, we found downregulation of NTPDase1 and NTPDase2 and attenuation of adenine nucleotide hydrolysis by NTPDase/eN enzyme chain, without changes in NTPDase3 properties and augmentation of synaptic tissue-nonspecific alkaline phosphatase (TNAP) activity. Despite reduced NTPDase activities, increased TNAP activity probably prevents toxic accumulation of ATP in the extracellular milieu and also hydrolyzes accumulated ADP due to unchanged NTPDase3 activity. Thus, our initial evaluation supports idea of specific roles of different ectonucleotidases and their coordinated actions in E2-mediated spine remodeling and maintenance.

Adenosine and adenosine receptors in the pathogenesis and treatment of rheumatic diseases.

Adenosine, a nucleoside derived primarily from the extracellular hydrolysis of adenine nucleotides, is a potent regulator of inflammation. Adenosine mediates its effects on inflammatory cells by engaging one or more cell-surface receptors. The expression and function of adenosine receptors on different cell types change during the course of rheumatic diseases, such as rheumatoid arthritis (RA). Targeting adenosine receptors directly for the treatment of rheumatic diseases is currently under study; however, indirect targeting of adenosine receptors by enhancing adenosine levels at inflamed sites accounts for most of the anti-inflammatory effects of methotrexate, the anchor drug for the treatment of RA. In this Review, we discuss the regulation of extracellular adenosine levels and the role of adenosine in regulating the inflammatory and immune responses in rheumatic diseases such as RA, psoriasis and other types of inflammatory arthritis. In addition, adenosine and its receptors are involved in promoting fibrous matrix production in the skin and other organs, and the role of adenosine in fibrosis and fibrosing diseases is also discussed.

Quercetin changes purinergic enzyme activities and oxidative profile in platelets of rats with hypothyroidism.

Diseases related to thyroid hormones have been extensively studied because affect a large number of individuals, and these hormones participate in the regulation of the whole organism homeostasis. However, little is known about the involvement of purinergic signaling related to oxidative stress in hypothyroidism and possible therapeutic adjuncts for treatment of this disorder. Thus, the present study investigates the effects of quercetin on NTPDase, 5'-nucleotidase and adenosine deaminase activities, platelet aggregation and oxidative profile in platelets of rats with methimazole (MMI)-induced hypothyroidism. Methimazole at a concentration of 20mg/100mL was administered for 90days. From the second month the animals received quercetin 10 or 25mg/kg for 60days. Results showed that: Ecto-5'-nucleotidase activity decreased in methimazole/water group and the treatment with quercetin 25mg/kg decreased NTPDase, 5'-nucleotidase and adenosine deaminase activities. Moreover, platelet aggregation increased in methimazole/water group. Lipid peroxidation increased while superoxide dismutase and catalase activities decreased, but, interestingly, the treatment with quercetin reversed these changes. These results demonstrated that quercetin modulates adenine nucleotide hydrolysis decreasing the ADP formation and adenosine deamination. At the same time quercetin improves the oxidative profile, as well as reduces platelet aggregation, which together with the modulation in the nucleotides levels can contribute to the prevention of platelet disorders.

Exercise Training positively modulates the Ectonucleotidase Enzymes in Lymphocytes of Metabolic Syndrome Patients.

In this study, we investigated the cardiovascular risk factors as well as ectonucleotidase activities in lymphocytes of metabolic syndrome (MetS) patients before and after an exercise intervention. 20 MetS patients, who performed regular concurrent exercise training for 30 weeks, 3 times/week, were studied. Anthropometric, biochemical, inflammatory and hepatic parameters and hydrolysis of adenine nucleotides and nucleoside in lymphocytes were collected from patients before and after 15 and 30 weeks of the exercise intervention as well as from participants of the control group. An increase in the hydrolysis of ATP and ADP, and a decrease in adenosine deamination in lymphocytes of MetS patients before the exercise intervention were observed (P<0.001). However, these alterations were reversed by exercise training after 30 weeks of intervention. Additionally, exercise training reduced the inflammatory and hepatic markers to baseline levels after 30 weeks of exercise. Our results clearly indicated alteration in ectonucleotidase enzymes in lymphocytes in the MetS, whereas regular exercise training had a protective effect on the enzymatic alterations and on inflammatory and hepatic parameters, especially if it is performed regularly and for a long period.

Effects of chlorogenic acid on adenine nucleotides hydrolyzing enzyme activities and expression in platelets of rats experimentally demyelinated with ethidium bromide.

The effects of chlorogenic acid (one of the major phenolic acid found in human diets) were investigated on the adenine nucleotides hydrolyzing enzymes; ecto-nucleotide pyrophosphatase/phophodiesterase (E-NPP), ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'- nucleotidase and ecto-adenosine deaminase (E-ADA) activities and expression in platelets of rats experimentally demyelinated with ethidium bromide. Rats were divided into four groups of eight animals each. Group I rats were control rats; injected with saline (CT), group II rats were injected with saline and treated with chlorogenic acid (AC), group III rats were injected with 0.1% ethidium bromide (EB) and group IV rats were injected with 0.1% EB and treated with chlorogenic acid (EB+AC). The activities of the enzymes were analyzed using colorimetric methods, and the gene expression of NTPDase 1, 2 and 3 were analyzed using the polymerase chain reaction (PCR). The results revealed that there was a significant (P<0.01) reduction in E-NPP activity in EB group (1.63±0.10nmol p-nitrophenol released/min/mg protein) when compared to CT group (2.33±0.14nmol p-nitrophenol released/min/mg protein). However, treatment with chlorogenic acid significantly (P<0.05) increased E-NPP activity in EB group. Furthermore, no significant (P>0.05) change was observed in the E-NPP activity of EB+AC group (2.19±0.08nmol p-nitrophenol released/min/mg protein) when compared to CT group (2.33±0.14nmol p-nitrophenol released/min/mg protein). In addition, there was a significant (P<0.05) increase in AMP hydrolysis in EB rat group when compared to CT group. No significant (P>0.05) difference was observed in AMP hydrolysis between AC, AC+EB and CT groups. Conversely, there were no significant (P>0.05) differences in ATP and ADP hydrolyses between all the groups (AC, EB, AC+EB and CT groups). Likewise, there were no significant (P>0.05) changes in E-ADA activity and percentage platelet aggregation among all groups studied. Similarly, no significant (P>0.05) change was observed in the expression of E-NTPDase 1, 2 and 3 in all the groups tested. Our study revealed that chlorogenic acid may modulate the hydrolysis of adenine nucleotides in platelets of rats demyelinated and treated with chlorogenic acid via alteration of E-NPP and ecto-5'-nucleotidase activities.

Extracellular purines' action on glomerular albumin permeability in isolated rat glomeruli: insights into the pathogenesis of albuminuria.

Purinoceptors (adrengeric receptors and P2 receptors) are expressed on the cellular components of the glomerular filtration barrier, and their activation may affect glomerular permeability to albumin, which may ultimately lead to albuminuria, a well-established risk factor for the progression of chronic kidney disease and development of cardiovascular diseases. We investigated the mechanisms underlying the in vitro and in vivo purinergic actions on glomerular filter permeability to albumin by measuring convectional albumin permeability (Palb) in a single isolated rat glomerulus based on the video microscopy method. Primary cultured rat podocytes were used for the analysis of Palb, cGMP accumulation, PKG-Iα dimerization, and immunofluorescence. In vitro, natural nucleotides (ATP, ADP, UTP, and UDP) and nonmetabolized ATP analogs (2-meSATP and ATP-γ-S) increased Palb in a time- and concentration-dependent manner. The effects were dependent on P2 receptor activation, nitric oxide synthase, and cytoplasmic guanylate cyclase. ATP analogs significantly increased Palb, cGMP accumulation, and subcortical actin reorganization in a PKG-dependent but nondimer-mediated route in cultured podocytes. In vivo, 2-meSATP and ATP-γ-S increased Palb but did not significantly affect urinary albumin excretion. Both agonists enhanced the clathrin-mediated endocytosis of albumin in podocytes. A product of adenine nucleotides hydrolysis, adenosine, increased the permeability of the glomerular barrier via adrenergic receptors in a dependent and independent manner. Our results suggest that the extracellular nucleotides that stimulate an increase of glomerular Palb involve nitric oxide synthase and cytoplasmic guanylate cyclase with actin reorganization in podocytes.

Effect of antiretroviral therapy in thromboregulation through the hydrolysis of adenine nucleotides in platelets of HIV patients.

The human immunodeficiency virus (HIV) infection results in biochemical and vascular dysfunctions. The highly active antiretroviral therapy (HAART) markedly reduces mortality and opportunistic diseases associated with acquired immunodeficiency syndrome (AIDS). This increased survival time predisposes the development of cardiovascular diseases. Platelets present purinergic system ectoenzymes such as E-NTPDase, E-5'-nucleotidase and E-ADA on its surface. In view of this, the aim of this study was to evaluate the activity of these ectoenzymes in platelets as well as the platelet aggregation and lipid profile of patients with HIV infection and also patients receiving HAART. The results showed an increase in the E-NTPDase activity for ATP hydrolysis in the HIV group compared with the control group and the HIV/HAART group. When assessing the activity E-NTPDase hydrolysis to ADP, the results revealed an increase in activity in the HIV group when compared to the control group, and a decrease in activity when in the HIV/HAART group when compared to the control and HIV groups. The activity of E-5'-nucleotidase revealed an increase in AMP hydrolysis in the HIV group, as the results from control and HIV/HAART groups showed no statistical difference. Regarding the E-ADA activity, the HIV and HIV/HAART groups revealed a decreased deamination of adenosine when compared with the control group. Furthermore, we observed an increased platelet aggregation of HIV/HAART group compared with the control group. Thus, our results suggest that antiretroviral treatment against HIV has a significant effect on the activity of purinergic system ectoenzymes demonstrating that thromboregulation is involved in the process.