RESUMO
AIMS: To investigate the role of TH (thyroid hormones) in 5'-nucleotidase activity and expression in cardiac soluble fraction (SF). MAIN METHODS: Male Wistar rats received daily injections of T4 (10, 25 or 50 µg T4/100g body weight) for 14 days to develop a hyperthyroidism condition. Thyroidectomy was performed in other animals to mimic hypothyroidism, and 14 days after surgery they were submitted to TH replacement therapy. KEY FINDINGS: T4 reduced the 5'-nucleotidase activity (T4-25, P<0.05 and T4-50, P<0.01) in the SF. Conversely, hypothyroidism significantly increased the 5'-nucleotidase activity in this fraction (P<0.001) and TH replacement therapy reversed the latter result (P<0.001 compared to hypothyroid group). The analysis of protein expression in the SF showed that 5'-nucleotidase was more expressed in hypothyroid than in the control group and that the phosphorylated state of PKC observed in this condition may contribute to a possible mechanism of 5'-nucleotidase modulation by thyroid status. SIGNIFICANCE: Taken together, these data reveal that TH can influence adenosine production by modulating 5'-nucleotidase activity and expression, which may contribute to the cardioprotective effect and the maintenance of cardiac function under TH privation.
Assuntos
5'-Nucleotidase/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Pressão Sanguínea , Peso Corporal , Regulação da Expressão Gênica , Hemodinâmica , Hipertireoidismo/metabolismo , Hipotireoidismo/metabolismo , Masculino , Modelos Biológicos , Miocárdio/metabolismo , Nucleotídeos/química , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Solubilidade , Frações Subcelulares/metabolismoRESUMO
Ectonucleotidases and the nucleotide metabolism have been implicated as important regulators of various tissue functions in diabetes disease. Here we evaluated the ectonucleotidase activities and the profile of extracellular ATP metabolism in blood serum of streptozotocin (STZ)-induced diabetic rats. We observed a raise in ATP, ADP, AMP, and 5'-TMP hydrolysis in blood serum after 30 days of diabetes induction, when compared with the citrate group. However, in serum of rats treated 6 days with insulin, the hydrolysis returned to the control levels. Extracellular ATP metabolism estimated by HPLC analysis showed a rapid hydrolysis of extracellular ATP by diabetic animals, leading to the formation of high levels of adenosine when compared with citrate and insulin groups. Since in diabetes the vascular disease is frequently present, the alterations observed are important, because these enzymes control the nucleotides/nucleosides ratio in the circulation and thus the events related to haemostasis.
Assuntos
Soro/metabolismo , Adenosina/metabolismo , Adenosina Trifosfatases , Animais , Diabetes Mellitus , Diabetes Mellitus Tipo 2 , Hidrólise , Masculino , Nucleotídeos/metabolismo , Ratos , Ratos Wistar , EstreptozocinaRESUMO
AIMS: Expression of ectoenzymes responsible for nucleotide phosphohydrolysis to form adenosine may represent a mechanism that facilitates the proliferation and spread of malignancy. In this study, we have identified and characterized the ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) family members expressed during the subcutaneous tumor growth and in the ascitic form of Walker 256 mammary tumor cells. MAIN METHODS: The biochemical characteristics in ascitic forms and expression of NPP 1, 2, and 3 in both solid and ascitic forms of Walker 256 tumor were investigated using RT-PCR and real-time PCR. KEY FINDINGS: Walker 256 tumor cells demonstrate E-NPP activities that are associated with extracellular hydrolysis of p-Nph-5'-TMP, and define the biochemical characteristics. The K(m) and maximal velocity for the hydrolysis of p-Nph-5'-TMP in the ascitic tumor cells were in accordance with the NPP reaction. The mRNA expression in the cells of the ascitic form of Walker 256 tumor revealed transcripts for NPP2 and NPP3, whereas elevated expression of NPP3 was observed in solid tumor, after 6, 10, and 15days of inoculation. The dominant gene expressed in both forms of the tumor was the NPP3 enzyme. However, this enzyme was expressed more during tumor development in vivo, when compared with the ascitic cells. SIGNIFICANCE: We have previously demonstrated that Walker 256 tumor cells express mRNA for ecto-5'-nucleotidase and E-NTPDases. Thus, coexistence with NPP3 suggests an ectonucleotidase "enzyme chain" that is responsible for the sequential hydrolysis of ATP to adenosine, which may be an important therapeutic target in anticancer therapy.
Assuntos
Carcinoma 256 de Walker/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Diester Fosfórico Hidrolases/biossíntese , Pirofosfatases/biossíntese , Animais , Ascite/enzimologia , Cátions/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Cinética , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Nucleosídeos/metabolismo , Nucleotídeos/metabolismo , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The aim of this study was to investigate the effects of intrastriatal injection of hypoxanthine on ectonucleotidase (E-NTPDases and ecto-5'-nucleotidase) activities and expressions in the striatum of rats. The effect of pre-treatment with vitamins E and C on the effects elicited by this oxypurine on enzymatic activities and on thiobarbituric reactive substances (TBARS) was also investigated. The effect of pre-incubation with hypoxanthine on nucleotide hydrolysis in striatum homogenate was also determined. Adult Wistar rats were divided into (1) control and (2) hypoxanthine-injected groups. For ectonucleotidase activity determination, the animals were sacrificed at 30 min, 24 h and 7 days after drug infusion. For the evaluation of the expression of NTPDase 1-3 and also ecto-5'-nucleotidase, TBARS assay and the influence of the pre-treatment with vitamins on ectonucleotidase activities, the animals were sacrificed 24 h after hypoxanthine infusion. Results show that hypoxanthine infusion significantly inhibited ectonucleotidase activities and increased TBARS only 24 h after administration. Pre-treatment with vitamins was able to prevent these effects. Moreover, ecto-5'-nucleotidase expression was increased (80%) at 24 h after hypoxanthine infusion. We suggest that these hypoxanthine-induced biochemical modifications could, at least in part, participate in the pathophysiology of Lesch Nyhan disease.
Assuntos
Adenosina Trifosfatases/metabolismo , Fármacos do Sistema Nervoso Central/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/enzimologia , Hipoxantina/farmacologia , 5'-Nucleotidase/metabolismo , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Ácido Ascórbico/administração & dosagem , Ácido Ascórbico/farmacologia , Fármacos do Sistema Nervoso Central/administração & dosagem , Corpo Estriado/metabolismo , Hidrólise/efeitos dos fármacos , Hipoxantina/administração & dosagem , Masculino , Nucleotídeos/metabolismo , Pirofosfatases/metabolismo , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Fatores de Tempo , Vitamina E/administração & dosagem , Vitamina E/farmacologia , Vitaminas/administração & dosagem , Vitaminas/farmacologiaRESUMO
The present study describes the enzymatic properties and molecular identification of 5'-nucleotidase in soluble and microsomal fractions from rat cardiac ventricles. Using AMP as a substrate, the results showed that the cation and the concentration required for maximal activity in the two fractions was magnesium at a final concentration of 1 mM. The pH optimum for both fractions was 9.5. The apparent K(m) (Michaelis constant) values calculated from the Eadie-Hofstee plot were 59.7+/-10.4 microM and 134.8+/-32.1 microM, with V(max) values of 6.7+/-0.4 and 143.8+/-23.8 nmol P(i)/min/mg of protein (means+/-S.D., n=4) from soluble and microsomal fractions respectively. Western blotting analysis of ecto-5'-nucleotidase revealed a 70 kDa protein in both fractions, with the major proportion present in the microsomal fraction. The presence of these enzymes in the heart probably has a physiological function in adenosine signalling. Furthermore, the presence of ecto-5'-nucleotidase in the microsomal fraction could have a role in the modulation of the excitation-contraction-coupling process through involvement of the Ca(2+) influx into the sarcoplasmic reticulum. The measurement of maximal enzyme activities in the two fractions highlights the potential capacity of the different pathways of purine metabolism in the heart.
Assuntos
5'-Nucleotidase/metabolismo , Monofosfato de Adenosina/metabolismo , Adenosina/metabolismo , Ventrículos do Coração/enzimologia , Microssomos/enzimologia , Transdução de Sinais/fisiologia , Animais , Cálcio/metabolismo , Cátions/metabolismo , Ventrículos do Coração/citologia , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Magnésio/metabolismo , Masculino , Ratos , Ratos Wistar , Retículo Sarcoplasmático/enzimologiaRESUMO
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.
Assuntos
5'-Nucleotidase/genética , Adenosina Trifosfatases/genética , Perfilação da Expressão Gênica , Miocárdio/enzimologia , Terminações Nervosas/metabolismo , Nucleotídeos/metabolismo , 5'-Nucleotidase/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Relação Dose-Resposta a Droga , Espaço Extracelular/metabolismo , Ventrículos do Coração , Concentração de Íons de Hidrogênio , Hidrólise/efeitos dos fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Masculino , Ouabaína/farmacologia , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Azida Sódica/farmacologia , Suramina/farmacologia , Sinaptossomos/metabolismoRESUMO
In the present study we investigated the in vivo (acute and chronic) and in vitro effects of proline on NTPDase and 5'-nucleotidase activities in synaptosomes obtained from cerebral cortex of rats. For acute administration, 29-day-old rats received one subcutaneous injection of proline (18.2 micromol/g body weight) or an equivalent volume of 0.9% saline solution (control) and were killed 1 h later. For chronic treatment, buffered proline was injected subcutaneously into rats twice a day at 10 h intervals from the 6th to the 28th day of age. Rats were killed 12 h after the last injection. Results showed that acute and chronic proline administration provoked a reduction (25%) of ATP hydrolysis, but did not alter ADP and AMP hydrolysis. We also verified the in vitro effect of proline (3.0 microM-1.0 mM) on nucleotide hydrolysis in synaptosomes from cerebral cortex of rats. In contrast to the in vivo studies, it was not observed any statistically significant alteration on ATP, ADP and AMP hydrolysis. In conclusion, according to our results, it seems reasonable to postulate that proline administration alters the hydrolysis of ATP and probably affects the responses mediated by adenine nucleotides in the central nervous system of proline treated rats.
Assuntos
Nucleotídeos de Adenina/metabolismo , Encefalopatias Metabólicas Congênitas/fisiopatologia , Córtex Cerebral/metabolismo , Prolina/metabolismo , Sinaptossomos/metabolismo , 5'-Nucleotidase/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Hidrólise , Masculino , Ratos , Ratos WistarRESUMO
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 microL) 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.
Assuntos
5'-Nucleotidase/metabolismo , Hipocampo/enzimologia , Hiperargininemia/enzimologia , Pirofosfatases/metabolismo , Sinaptossomos/enzimologia , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Hipocampo/citologia , Humanos , Masculino , Ratos , Ratos WistarRESUMO
The extracellular nucleotides, ATP and ADP, as well as adenosine have been implicated in a great number of physiological functions. ADP is one of the major platelet recruiting factors, whereas ATP is considered to be a competitive inhibitor of ADP-induced platelet aggregation and adenosine is able to induce vasodilatation and to inhibit platelet aggregation. The di- and triphosphate nucleosides can be hydrolyzed by members of several families of ectonucleotidases, including ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) that, together with an ecto-5'-nucleotidase, catalyze adenosine formation. The renin-angiotensin system is the most important regulator of renal and cardiovascular functions and angiotensin II induces, physiologically, platelet activation. The aim of this study was to clarify the effects of ANGII and genetic hypertension upon extracellular nucleotide hydrolysis by rat platelet ectoenzymes. ANGII, in all tested doses (5, 50, 500 and 5000 pmol), was able to increase ATP (21, 31, 44 and 27%, respectively), ADP (22, 28, 78 and 37%, respectively) and AMP (40, 64, 60 and 64%, respectively) hydrolysis by rat platelets. Furthermore, losartan, a specific antagonist of the AT1 angiotensin-receptor, prevented the nucleotide hydrolysis effects. Additionally, an increase in AMP (about 144%) hydrolysis and a decrease in p-Nph-5'TMP (about 27%) hydrolysis were observed in platelets from spontaneously hypertensive rats (SHR) when compared to Wistar normotensive rats. We, herein, present data to demonstrate interactions between rat platelet angiotensinergic and adenosinergic systems that could contribute to the understanding and treatment of cardiovascular diseases such as hypertension, thrombosis and arteriosclerosis.
Assuntos
Trifosfato de Adenosina/metabolismo , Angiotensina II/farmacologia , Plaquetas/enzimologia , Hipertensão/genética , Vasoconstritores/farmacologia , 5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Hidrólise , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Masculino , Fenótipo , Diester Fosfórico Hidrolases/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Receptor Tipo 1 de Angiotensina/metabolismo , Timidina Monofosfato/análogos & derivados , Timidina Monofosfato/metabolismoRESUMO
The effect of methylprednisolone on the hydrolysis of adenine nucleotides by rat blood serum enzymes was studied. Adult male Wistar rats were submitted to three different treatments with synthetic steroid methylprednisolone: one dose of 50 mg/kg, i.p. (acute); or oral doses of 6 mg/kg dissolved in drinking water for 15 (sub-chronic) or 30 (chronic) days. Decreased ADP hydrolysis was observed after acute and sub-chronic treatments. Furthermore, ATP, ADP and AMP hydrolysis decreased after chronic treatment. These alterations may constitute one of the mechanisms that mediate the development of some of the side effects associated with corticosteroid use.
Assuntos
Nucleotídeos de Adenina/sangue , Metilprednisolona/farmacologia , Envelhecimento/metabolismo , Animais , Hidrólise , Masculino , Ratos , Ratos WistarRESUMO
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.
Assuntos
Carcinoma 256 de Walker/enzimologia , Nucleotídeos/metabolismo , 5'-Nucleotidase/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cátions Bivalentes/metabolismo , Concentração de Íons de Hidrogênio , Cinética , L-Lactato Desidrogenase/metabolismo , Masculino , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The main objective of the present study was to evaluate the in vivo (acute and chronic) and in vitro effects of proline on serum nucleotide hydrolysis. For acute administration, 29-day-old rats received one subcutaneous injection of proline (18.2 (micromol/g body weight) or an equivalent volume of 0.9% saline solution (control) and were sacrificed 1 h, 3 h or 12 h later. Results showed that acute proline administration provoked a decrease in ATP (42%) and ADP (49%) hydrolysis when rats were sacrificed 1 h after the injection. Furthermore, in rats killed 3 h and 12 h after acute injection, no change in nucleotide hydrolysis were observed. For chronic treatment, buffered proline was injected subcutaneously twice a day at 10 h intervals from the 6(th) to the 28(th) day of age. Rats were sacrificed 3 h or 12 h after the last injection. Chronic administration of proline did not alter the nucleotide hydrolysis when the rats were killed 12 h after the last injection, but decreased ATP (15%) and ADP (32%) hydrolysis when rats were sacrificed 3 h after the last injection. The in vitro effect of proline (3.0 microM - 1.0 mM) on serum nucleotide hydrolysis was also investigated; results showed that 1.0 mM of proline significantly increased ATP (45%), ADP (55%) and AMP (49%) hydrolysis. The data indicate that proline in vivo and in vitro alters nucleotide hydrolysis, which may be involved in the pathogeny of hyperprolinemic patients.
Assuntos
Nucleotídeos/sangue , Nucleotídeos/metabolismo , Prolina/farmacologia , Animais , Hidrólise/efeitos dos fármacos , Masculino , Prolina/administração & dosagem , Ratos , Ratos WistarRESUMO
Despite the extensive research on the pharmacology of L-arginine, there are only few data on its antithrombotic properties. We studied the effect of oral L-arginine administration in a model of arterial thrombosis in rabbits divided into three groups: group 1, group without intervention; group 2, control group, treated with normal diet and submitted to the thrombosis-triggering protocol; group 3, treated for 2 weeks with L-arginine (2.25%) prior the protocol. L-Arginine did not alter platelet aggregation nor coagulation parameters but reduced vascular activities of both ADPase (49.1 +/- 8.5 versus 28.9 +/- 8.3 versus 18.8 +/- 10.3 nmoles inorganic phosphate/min per mg protein; mean +/- SD; group 1 versus group 2 versus group 3, respectively; ANOVA F = 19.21; P < 0.0001) and ATPase (97.8 +/- 15.8 versus 52.1 +/- 11.6 versus 31.9 +/- 16.3 nmoles inorganic phosphate/min per mg protein; mean +/- SD; group 1 versus group 2 versus group 3, respectively; ANOVA, F = 34.65; P < 0.0001). L-Arginine did not reduce the thrombi area (17.1 mm, 9.02 and 48.07, versus 27.04 mm, 25.4 and 70.39, median, percentile 25 and 75 respectively, P = 0.079; group 2 versus group 3, respectively). In conclusion, oral L-arginine administration did not inhibit thrombosis, and, conversely, it significantly reduced the arterial wall ADPase and ATPase activities. This effect may limit its antithrombotic properties.
Assuntos
Adenosina Trifosfatases/efeitos dos fármacos , Apirase/efeitos dos fármacos , Arginina/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Trombose/prevenção & controle , Adenosina Trifosfatases/metabolismo , Administração Oral , Análise de Variância , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Apirase/metabolismo , Arginina/administração & dosagem , Coagulação Sanguínea/efeitos dos fármacos , Distribuição de Qui-Quadrado , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/enzimologia , Masculino , Modelos Animais , Coelhos , Estatísticas não Paramétricas , Trombose/etiologia , Trombose/patologiaRESUMO
Extracellular nucleotides ATP, ADP, AMP and adenosine are well known signaling molecules of the cardiovascular system that are involved in several physiological processes: cell proliferation, platelet aggregation, inflammatory processes and vascular tonus. The levels of these molecules are controlled by ecto-NTPDases and ecto-5'-nucleotidase/CD73 (ecto-5'-NT/CD73) actions, which are responsible for the complete ATP degradation to adenosine. The thyroid hormones, thyroxine (T(4)) and triiodothyronine (T(3)), play important roles in the vascular system promoting vasodilatation. Here we investigated the influence of thyroid hormones on the enzyme cascade that catalyzes the interconversion of purine nucleotides in vascular smooth muscle cells (VSMC). Exposure of VSMCs to 50nM T(3) or T(4) did not change ATP and ADP hydrolysis significantly. However, the same treatment caused an increase of 75% in AMP hydrolysis, which was time-dependent but dose-independent. Moreover, T(3) treatment significantly increased ecto-5'-NT/CD73 mRNA expression, which suggests a genomic effect of this hormone upon ecto-5'-NT/CD73. In addition to the importance of the ecto-5'-NT in cell proliferation and differentiation, its overexpression could result in higher extracellular levels of adenosine, an important local vasodilatator molecule.
Assuntos
5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Nucleotídeos de Adenina/metabolismo , Animais , Aorta/citologia , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Hidrólise/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de TempoRESUMO
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.
Assuntos
Nucleotídeos de Adenina/sangue , Inibidores Enzimáticos/farmacologia , Homocisteína/farmacologia , 5'-Nucleotidase/metabolismo , Difosfato de Adenosina/sangue , Monofosfato de Adenosina/sangue , Trifosfato de Adenosina/sangue , Animais , Relação Dose-Resposta a Droga , Hidrólise , Concentração Inibidora 50 , Masculino , Pirofosfatases/metabolismo , Ratos , Ratos WistarRESUMO
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 (tumour-promoting molecule) in the circulation.
Assuntos
Nucleotídeos de Adenina/metabolismo , Plaquetas/metabolismo , Carcinoma 256 de Walker/sangue , Soro/metabolismo , Nucleotídeos de Adenina/antagonistas & inibidores , Animais , Plaquetas/enzimologia , Carcinoma 256 de Walker/enzimologia , Hidrólise , Pirofosfatases/sangue , Ratos , Soro/enzimologia , Células Tumorais CultivadasRESUMO
There is evidence that estrogen is associated with a reduction on cardiovascular disease risk through inhibition of platelet aggregation and action on vascular function. The process of haemostasis can also be affected by adenine nucleotides and adenosine. Consequently, regulation of enzymes that hydrolyze these nucleotides in the bloodstream is essential in the modulation of the processes of platelet aggregation, vasodilatation and coronary flow. Ecto-ATP diphosphohydrolase and ecto-5'-nucleotidase from platelets are enzymes related to nucleotide hydrolysis. In the present study, we examined the effect of ovariectomy (OVX) and estradiol replacement therapy (ER) on the activity of the enzymes that degrade adenine nucleotides in platelets of female rats. The OVX group significantly decrease the hydrolysis of ATP, ADP and AMP by 42, 52 and 29.3%, respectively, when compared to a control group. ER did not reverse the inhibition of nucleotide hydrolysis observed in OVX rats. Our findings indicate that hormonal deprivation affects ATP, ADP and AMP hydrolysis by platelets and consequently the level of these nucleotides and adenosine in the circulation. Since, ADP is the most important platelet agonist and recruiting agent present in the microenvironment of the thrombus, our findings was contribute to a better comprehension of the cardiovascular complications described in alterations of sexual hormonal status.
Assuntos
Nucleotídeos de Adenina/metabolismo , Plaquetas/metabolismo , Terapia de Reposição de Estrogênios/efeitos adversos , Ovariectomia/efeitos adversos , 5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Plaquetas/enzimologia , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Ativação Enzimática , Feminino , Hidrólise , Ratos , Ratos WistarRESUMO
It has been reported that animals submitted to repeated restraint stress present various adaptation responses which are dependent on the sex. These adaptations include changes in nociception and adenine nucleotide hydrolysis. In this study, we report the effect of chronic administration of a gonadal steroid (17beta-estradiol) on ATP, ADP and AMP hydrolysis in spinal cord synaptosomes of adult ovariectomized (OVX) Wistar rats submitted to repeated restraint stress over 40 days. We also measured nociceptive threshold in these animals using the tail-flick test. The results show that tail-flick latencies were decreased in both stressed groups, OVX and OVX rats receiving estradiol replacement therapy, indicating reduced nociceptive threshold after exposure to repeated stress. Repeated restraint stress caused no effect on ATPase or ADPase activities. On the other hand, AMP hydrolysis in spinal cord synaptosomes from repeatedly stressed rats was decreased in OVX rats compared to non-stressed OVX ones, indicating reduced extracellular adenosine production; this effect was reversed by hormonal replacement. These observations suggest that nociceptive sensitivity to noxious stimuli is affected by repeated stress and that modulation of neurotransmission by adenine nucleotides in spinal cord may be altered by the interaction of sexual hormones and psychological factors, such as exposure to stress.
Assuntos
Adenosina Trifosfatases/metabolismo , Estradiol/administração & dosagem , Nociceptores/fisiopatologia , Medula Espinal/citologia , Estresse Fisiológico/enzimologia , Sinaptossomos/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Análise de Variância , Animais , Feminino , Hidrólise/efeitos dos fármacos , Ovariectomia/métodos , Medição da Dor/métodos , Ratos , Ratos Wistar , Tempo de Reação/efeitos dos fármacos , Restrição Física/métodos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Sinaptossomos/enzimologiaRESUMO
Glioblastomas are the most common form of primary tumors of the central nervous system (CNS) and despite treatment, patients with these tumors have a very poor prognosis. ATP and other nucleotides and nucleosides are very important signaling molecule in physiological and pathological conditions in the CNS. ATP is degraded very slowly by gliomas when compared to astrocytes, potentially resulting in the accumulation of extracellular ATP around gliomas. Cell lysis caused by excitotoxic death or by tumor resection may liberate intracellular ATP, a known mitotic factor for glioma cells. The aim of this study is to examine the effects on cytotoxicity induced by extracellular ATP in U138-MG human glioma cell line and C6 rat glioma cell line compared to hippocampal organotypic cell cultures. The cytotoxicity of ATP (0.1, 0.5, 5 mM) was measured using propidium iodide and LDH assays. Caspases assay was performed to identify apoptotic cell death. Results showed that the glioma cells present resistance to death induced by ATP when compared with a normal tissue. High ATP concentrations (5 mM) induced cell death after 24 h in organotypic cell cultures but not in glioma cell lines. Our data indicate that ATP released in these situations can induce cell death of the normal tissue surrounding the tumor, potentially opening space to the fast growth and invasion of the tumor.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Líquido Extracelular , Glioma/fisiopatologia , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Glioma/patologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Humanos , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Concentração Osmolar , Agonistas do Receptor Purinérgico P2 , Ratos , Ratos Wistar , Receptores Purinérgicos P2X7RESUMO
Mate is a xanthine-containing beverage, which is prepared as an infusion of the dried and ground leaves of Ilex paraguariensis St. Hil. (Aquifoliacea). Previous reports have shown that Ilex paraguariensis has the highest levels of caffeine and theobromine when compared to other Ilex species. Furthermore, mate is able to interfere in the circulatory system, acting as a diuretic and hypotensive agent. Many processes of vascular injury result in the release of adenine nucleotides, which exert a variety of effects. Nucleoside 5' tri- and diphosphates may be hydrolyzed by members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. The synchronic action of a NTPDase and a 5'-nucleotidase promotes the catabolism of ATP to adenosine, which is able to control the extracellular nucleotides/nucleosides ratio. The chronic ingestion of aqueous extract of Ilex paraguariensis by rats during 15 days significantly decreased ATP (55%), ADP (50%) and AMP (40%) hydrolysis in blood serum. These results suggest changes in the balance of purine levels induced by Ilex paraguariensis ingestion. Considering the potential effects of Ilex paraguariensis in the circulatory system, these results may be relevant since NTPDases are a novel drug target for the treatment of cardiovascular diseases.