RESUMO
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
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 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
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
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
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
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
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
L-2-Hydroxyglutaric (LGA) and D-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as L-2-hydroxyglutaric aciduria and D-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO(2) production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO(2) production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 1.9.3.1) in a dose-dependent manner (35-95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by D-2-hydroxyglutaric aciduria.
Assuntos
Córtex Cerebral/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Glutaratos/farmacologia , Músculo Esquelético/enzimologia , Animais , Dióxido de Carbono/metabolismo , Córtex Cerebral/efeitos dos fármacos , Relação Dose-Resposta a Droga , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Metabolismo Energético , Glucose/metabolismo , Humanos , Técnicas In Vitro , Cinética , Músculo Esquelético/efeitos dos fármacos , Ratos , EstereoisomerismoRESUMO
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
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.
Assuntos
Nucleotídeos de Adenina/sangue , Ilex paraguariensis , Animais , Hidrólise/efeitos dos fármacos , Masculino , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Ratos , Ratos Wistar , Água/farmacologiaRESUMO
In order to characterize the enzymes involved in the purine nucleotide catabolism as indicators of invasiveness and aggressiveness of malignant gliomas, the degradation of extracellular nucleotides by five different glioma cell lines was investigated and compared with primary astrocytes. Rapid hydrolysis of extracellular ATP and ADP by astrocytes was observed, whereas all glioma cell lines examined presented low rates of ATP hydrolysis. In contrast, ecto-5'-nucleotidase activity was increased in glioma cell lines when compared to astrocytes. Considering that ATP is recognized as a mitogenic factor that induces proliferation in human glioma cells, the substantial decrease in ATP and ADP hydrolysis observed in gliomas leads us to suggest that alterations in the ecto-nucleotidases pathway may represent an important mechanism associated with malignant transformation of glioma cell lines.
Assuntos
Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Astrócitos/metabolismo , 5'-Nucleotidase/metabolismo , Animais , Células Cultivadas , Espaço Extracelular/metabolismo , Glioma , Humanos , Cinética , L-Lactato Desidrogenase , Ratos , Células Tumorais CultivadasRESUMO
Primary astrocyte cultures from hippocampus, cortex and cerebellum presented different extracellular pattern of adenine nucleotide hydrolysis. The ATP/ADP hydrolysis ratio was 8:1 for hippocampal and cortical astrocytes and 5:1 for cerebellar astrocytes. The AMP hydrolysis in cerebellar astrocytes was seven-fold higher than in cortical or hippocampal cells. No accumulation of extracellular adenosine in all structures studied was observed. Dipyridamol increased significantly inosine levels in the extracellular medium of hippocampal and cortical, but not in cerebellar astrocytes medium. A higher expression of ecto-5'-nucleotidase was identified by RT-PCR in cerebellum. The differences observed may indicate functional heterogeneity of nucleotides in the brain.
Assuntos
Nucleotídeos de Adenina/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , 5'-Nucleotidase/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Dipiridamol/farmacologia , Espaço Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas de Transporte de Nucleosídeos/antagonistas & inibidores , Especificidade de Órgãos , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
An increase in ADP hydrolysis was observed in spinal cord synaptosomal fractions of 2-month-old Wistar male rats, when compared to other ages (1, 4 and 6 months of age), while no change in ATPase activity was observed. Conversely, in female rats, whilst no change in ADPase activity was observed in the spinal cord synaptosomal fraction, ATPase activity diminished with age, in 1-6-month-old animals. 5'-Nucleotidase activity was higher in the 4-month-old male and female rats in relation to 1 and 2-month-old animals. In the female rats, this activity continued to increase at least until 6 months of age. In conclusion, adenine nucleotides hydrolysis in synaptosomes from rat spinal cord is influenced by age and by gender. Since both ATP and adenosine may act as neuromodulators in the spinal cord, influencing several processes such as nociception, the regulation of ATP-metabolizing enzymes in spinal cord is probably important for the normal function of this tissue at different ages.
Assuntos
Adenosina Trifosfatases/metabolismo , Envelhecimento/fisiologia , Medula Espinal/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Análise de Variância , Animais , Feminino , Hidrólise , Masculino , Ratos , Ratos Wistar , Fatores Sexuais , Medula Espinal/citologia , Sinaptossomos/enzimologiaRESUMO
In the present work, we investigated the kinetics of the inhibition of Na(+), K(+)-ATPase activity caused by homocysteine (Hcy) in rat hippocampus. We also studied the interaction between Hcy and phenylalanine (Phe) and the kinetics of alanine (Ala) reversal of the inhibition of Na(+), K(+)-ATPase caused by Hcy. The apparent K(m) and V(max) of Na(+), K(+)-ATPase for ATP as substrate were 0.55mM and 2.0nmol Pi released per min per mg of protein, respectively. K(i) value was approximately 0.1mM, and the inhibition was of the non-competitive type. The results also showed a competition between Hcy and Phe. Ala per se did not alter this enzyme, but prevented the inhibitory effect caused by Hcy, suggesting a common binding site for these substances. It is proposed that the inhibition of Na(+), K(+)-ATPase by Hcy may be one of the mechanisms related to the neuronal dysfunction observed in human homocystinuria.
Assuntos
Hipocampo/metabolismo , Homocisteína/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Alanina/metabolismo , Animais , Cinética , Fenilalanina/metabolismo , Ratos , Ratos WistarRESUMO
Male Wistar rats were bilaterally implanted with indwelling cannulae in the caudal region of the posterior cingulate cortex. After recovery, animals were trained in a step-down inhibitory avoidance task (3.0-s, 0.4-mA foot shock) and received, immediately after training, a 0.5-microl infusion of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA; 1, 50 or 100 nM) or of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1, 25 or 50 nM). Animals were tested twice, 1.5 h and, again, 24 h after training, in order to examine the effects of these agents on short- and long-term memory, respectively. Only 50-nM DPCPX was effective in altering memory, promoting a facilitation. These results suggest that adenosine A1 receptors in the posterior cingulate cortex inhibit memory consolidation in a way that their blockade facilitates memory for inhibitory avoidance in rats.
Assuntos
Adenosina/análogos & derivados , Giro do Cíngulo/efeitos dos fármacos , Memória/efeitos dos fármacos , Antagonistas de Receptores Purinérgicos P1 , Xantinas/farmacologia , Adenosina/farmacologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Relação Dose-Resposta a Droga , Giro do Cíngulo/metabolismo , Masculino , Ratos , Receptores Purinérgicos P1/fisiologia , Retenção Psicológica/efeitos dos fármacosRESUMO
The low prevalence of coronary heart disease in premenopausal women and its increase after menopause are well established. Many studies have suggested that steroid hormones can inhibit platelet aggregation, reducing the cardiovascular risk. In addition, a number of studies have shown an effect of estrogen on vascular function. The process of haemostasis and thrombus formation can be also affected by adenine nucleotides and adenosine. Consequently, the 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. Thus, in this study, we examined the effect of ovariectomy (OVX), estradiol replacement therapy and the in vitro administration of 17beta-estradiol, dehydroisoandrosterone 3-sulfate (DHEAS) and pregnenolone (PREG) on the activity of the enzymes that degrade adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in the blood serum of female rats. OVX significantly increased the hydrolysis of ATP, ADP and AMP, whilst phosphodiesterase activity was unchanged. Estradiol replacement therapy significantly decreased the hydrolysis of the adenine nucleotides and of the substrate marker of phosphodiesterase. In vitro, the addition of steroid hormones did not have any effect on the nucleotide hydrolysis by rat serum. These results suggest the presence of a strong relation between these enzymes and the hormonal system. In addition, the alterations observed are important, because these enzymes control the nucleotides/nucleosides ratio in the circulation and thus the events related to haemostasis.
Assuntos
Nucleotídeos de Adenina/metabolismo , Terapia de Reposição Hormonal , Ovariectomia , 5'-Nucleotidase/metabolismo , Nucleotídeos de Adenina/sangue , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apirase/metabolismo , Sulfato de Desidroepiandrosterona/farmacologia , Estradiol/farmacologia , Feminino , Hormônios Esteroides Gonadais/farmacologia , Hidrólise , Pregnanolona/farmacologia , Ratos , Ratos WistarRESUMO
BACKGROUND AND METHODS: The in vitro effect of the nonsteroidal anti-inflammatory drug, acetylsalicylic acid (ASA), on the extracellular adenine nucleotide hydrolysis by intact rat blood platelets was studied. RESULTS: Our results demonstrate that aspirin, at final concentrations of 2.0 and 3.0 mM, inhibits ATP extracellular hydrolysis in vitro by approximately 17% and 21%, respectively. Aspirin, at a final concentration of 3.0 mM, also inhibited in vitro extracellular ADP hydrolysis by approximately 41%. The same concentrations of this drug, however, did not alter AMP hydrolysis by intact rat blood platelets under similar assay conditions. The kinetic analysis demonstrated that the inhibition of ADP and ATP hydrolysis by aspirin in rat platelets is of the uncompetitive type. CONCLUSION: In this study, we demonstrated an inhibitory effect of ASA upon E-NTPDase 3 activity of platelets from adult rats and discussed the significance of our findings.
Assuntos
Apirase/antagonistas & inibidores , Apirase/sangue , Aspirina/farmacologia , Plaquetas/enzimologia , Inibidores de Ciclo-Oxigenase/farmacologia , 5'-Nucleotidase/antagonistas & inibidores , Nucleotídeos de Adenina/metabolismo , Difosfato de Adenosina/sangue , Monofosfato de Adenosina/sangue , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/sangue , Animais , Plaquetas/efeitos dos fármacos , Técnicas In Vitro , Masculino , Ratos , Corantes de RosanilinaRESUMO
We have previously observed that, while acute stress induces analgesia, chronic stress causes a hyperalgesic response in male rats. No effect was observed in females. There is increasing evidence that both ATP and adenosine can modulate pain. Extracellular ATP and ADP are hydrolyzed by an apyrase in synaptosomes from the peripheral and central nervous systems. In the present study, we investigated the effect of chronic and acute stress on ATPase-ADPase and 5'-nucleotidase activities in spinal cord of male and female rats. Adult male and female Wistar rats were submitted to 1 h restraint stress/day for 1 day (acute) or 40 days (chronic) and were sacrificed 24 h later. ATPase-ADPase activities were assayed in the synaptosomal fraction obtained from the spinal cord of control and stressed animals. ADP hydrolysis was decreased 25% in chronically stressed males, while no change was observed on ATPase activity. There was an increase in the 5'-nucleotidase activity in the same group. No effect on ADPase, ATPase or on 5'-nucleotidase activity was observed in females with chronic stress, or after acute stress neither in males or females. Chronic stress reduced ADP hydrolysis and increased 5'-nucleotidase activity in the spinal cord in male rats.
Assuntos
Nucleotidases/metabolismo , Medula Espinal/enzimologia , Estresse Psicológico/enzimologia , 5'-Nucleotidase/metabolismo , Doença Aguda , Adenosina Trifosfatases/metabolismo , Animais , Apirase/metabolismo , Doença Crônica , Feminino , Masculino , Ratos , Ratos Wistar , Restrição Física , Caracteres Sexuais , Frações Subcelulares/enzimologia , Sinaptossomos/metabolismoRESUMO
The role of adenine nucleotides on vascular and platelet functions has long been established. Apyrase (CD39) takes part of a family of ecto-enzymes that hydrolyze adenosine diphosphate and adenosine triphosphate. The participation of apyrase in the thromboregulatory system is under study. An in vivo experimental model of acute arterial thrombosis was used to test the hypothesis that administering a soluble form of potato apyrase could prevent thrombus formation. Twenty-five white New Zealand male rabbits suffered balloon aortic endothelium denudation and, after 15 days, they were submitted to a thrombosis-triggering protocol with a procoagulant (Russel's viper venom) and epinephrine. After the thrombosis-triggering protocol, 12 animals received two soluble apyrase administrations intravenously (with 90 min intervals), while 13 control animals received no apyrase. Three hours after the triggering protocol, the animals were killed and the rate and area of arterial thrombosis were analyzed. The rate of thrombosis in the apyrase group was significantly lower than that of the control group (16.7 versus 69%, respectively; P = 0.015), as was the area of thrombosis (1.7 +/- 4.3 versus 21.7 +/- 37.4 mm2, respectively; P = 0.008). Our results confirm that apyrase participates in homeostasis through a potent anti-thrombotic effect.