RESUMO
BACKGROUND: The overstimulation of excitatory glutamatergic neurotransmission and the inhibition of Na(+),K(+)-ATPase enzymatic activity have both been implicated in neurotoxicity and are possibly related to the pathogenesis of epilepsy and neurodegenerative disorders. In the present study, we investigated whether glutamatergic stimulation by the glutamatergic agonists glutamate, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), kainate and N-methyl-d-aspartate (NMDA) modulates the Na(+),K(+)-ATPase and the K(+)-p-nitrophenylphosphatase activities in the crude synaptosomal fraction of the hippocampus and the frontal cortex of rats. RESULTS: Our results demonstrated that these glutamatergic agonists did not influence the activities of Na(+),K(+)-ATPase or K(+)-p-nitrophenylphosphatase in the brain structures analyzed. Assays with lower concentrations of ATP to analyze the preferential activity of the Na(+),K(+)-ATPase isoform with high affinity for ATP did not show any influence either. CONCLUSIONS: These findings suggest that under our experimental conditions, the stimulation of glutamatergic receptors does not influence the kinetics of the Na(+),K(+)-ATPase enzyme in the hippocampus and frontal cortex.
Assuntos
4-Nitrofenilfosfatase/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Lobo Frontal/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Lobo Frontal/enzimologia , Hipocampo/enzimologia , Ácido Caínico/farmacologia , Masculino , N-Metilaspartato/farmacologia , Ratos , Ratos Wistar , Transmissão Sináptica , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
In this work we demonstrated that promastigotes of Leishmania amazonensis exhibit an Mg-dependent ecto-ATPase activity, which is stimulated by heat shock. The Mg-dependent ATPase activity of cells grown at 22 and 28 degrees C was 41.0+/-5.2 nmol Pi/h x 10(7)cells and 184.2+/-21.0 nmol Pi/h x 10(7)cells, respectively. When both promastigotes were pre-incubated at 37 degrees C for 2h, the ATPase activity of cells grown at 22 degrees C was increased to 136.4+/-10.6 nmol Pi/h x 10(7) whereas that the ATPase activity of cells grown at 28 degrees C was not modified by the heat shock (189.8+/-10.3 nmol Pi/h x 10(7)cells). It was observed that Km of the enzyme from cells grown at 22 degrees C (Km=980.2+/-88.6 microM) was the same to the enzyme from cells grown at 28 degrees C (Km=901.4+/-91.9 microM). In addition, DIDS (4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid) and suramin, two inhibitors of ecto-ATPases, also inhibited similarly the ATPase activities from promastigotes grown at 22 and 28 degrees C. We also observed that cells grown at 22 degrees C exhibit the same ecto-phosphatase and ecto 3'- and 5'-nucleotidase activities than cells grown at 28 degrees C. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat-shock effect on ecto-ATPase activity of cells grown at 22 degrees C were exposed at 37 degrees C for 2h. A comparison between the stimulation of the Mg-dependent ecto-ATPase activity of virulent and avirulent promastigotes by the heat shock showed that avirulent promastigotes had a higher stimulation than virulent promastigotes after heat stress.
Assuntos
Adenosina Trifosfatases/metabolismo , Temperatura Alta , Leishmania mexicana/enzimologia , 4-Nitrofenilfosfatase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Brasil , Cricetinae , Humanos , Hidrólise , Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/parasitologia , Fatores de Tempo , VirulênciaRESUMO
The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg(2+) and K(+). Micromolar concentrations of Ca(2+) inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca(2+) concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca(2+). The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K(+) but the apparent affinity of the enzyme for Mg(2+) increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg(2+). Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca(2+) free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca(2+) and the activation improves the interaction of the enzyme with Mg(2+).
Assuntos
4-Nitrofenilfosfatase/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/fisiologia , Membrana Celular/enzimologia , Lipídeos/farmacologia , Magnésio/metabolismo , 4-Nitrofenilfosfatase/antagonistas & inibidores , 4-Nitrofenilfosfatase/sangue , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Química Encefálica , ATPases Transportadoras de Cálcio/sangue , Bovinos , Ativação Enzimática , Fosfatos/farmacologia , Fosfatidilcolinas/farmacologia , Potássio/farmacologia , Suínos , Vanadatos/farmacologiaRESUMO
Na(+)/K(+)-adenosinetriphosphatase (Na(+)/K(+)-ATPase) is of paramount importance for the proper functioning of the organism. The enzyme is involved in several aspects of brain function, such as the repolarization of the neuronal membranes and neurotransmitters uptake/release. Therefore, individual differences in the activity of brain Na(+)/K(+)-ATPase may result in differences in the functioning of the brain, which, in consequence, could lead to behavioral divergences. Individual differences in rearing, an exploratory behavior, have been shown to be genetically determined. In rats, the inhibition of the activity of Na(+)/K(+)-ATPase was reported to induce changes in the exploratory behavior. The aim of this work was to verify if subgroups of rats selected according to the number of rearings (high and low rearing subgroups) in the open field test differ in the activity of Na(+)/K(+)-ATPase in brain regions. Adult, male outbred Wistar rats were selected in the open field test according to the number of rearings in subgroups of high (HR) and low (LR) rearing responders. After a rest of about 20 days after the open field session, HR and LR rats were sacrificed. In the first experiment, frontal cortex, striatum, brainstem, hippocampus and the amygdala (including the overlying limbic cortex) were dissected. The reaction of dephosphorylation of Na(+)/K(+)-ATPase (K(+) stimulated p-nitrophenylphosphatase) was assayed in homogenates rich in synaptosomes. The results obtained showed a statistically significant higher activity of K(+)p-nitrophenylphosphatase only in the hippocampus of HR subgroup of rats. This result was replicated in two other subsequent experiments with different HR and LR subgroups of rats selected at different times of the year. Our data suggest that the difference in the activity of Na(+)/K(+)-ATPase in the hippocampus is innate and is involved in the expression of the rearing behavior.
Assuntos
4-Nitrofenilfosfatase/metabolismo , Comportamento Exploratório/fisiologia , Hipocampo/enzimologia , Neurônios/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , 4-Nitrofenilfosfatase/efeitos dos fármacos , Animais , Comportamento Exploratório/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Movimento/efeitos dos fármacos , Movimento/fisiologia , Neurônios/efeitos dos fármacos , Potássio/química , Potássio/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Ratos , Ratos WistarRESUMO
In the search of Na+,K(+)-ATPase modulators, we have reported the isolation by gel filtration and HPLC of a brain fraction, termed endobain E, which highly inhibits Na+,K(+)-ATPase activity. In the present study we compared some properties of endobain E with those of ascorbic acid. Kinetic experiments assaying synaptosomal membrane K(+)-p-nitrophenylphosphatase (K(+)-p-NPPase) activity in the presence of endobain E or ascorbic acid showed that in neither case did enzyme inhibition prove competitive in nature versus K+ or p-NPP concentration. At pH 5.0, endobain E and ascorbic acid maximal UV absorbance was 266 and 258 nm, respectively; alkalinization to pH 14.0 led to absorption drop and shift for endobain E but to absorbance disappearance for ascorbic acid. After cysteine treatment, endobain E absorbance decreased, whereas that of ascorbic acid remained unaltered; iodine treatment led to absorbance drop and shift for endobain E but to absorbance disappearance for ascorbic acid. HPLC analysis of endobain E disclosed the presence of two components: one eluting with retention time and UV spectrum indistinguishable from those of ascorbic acid and a second, as yet unidentified, both exerting Na+,K(+)-ATPase inhibition.
Assuntos
Ácido Ascórbico/farmacologia , Encéfalo/enzimologia , Ouabaína/análogos & derivados , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , 4-Nitrofenilfosfatase/metabolismo , Animais , Antioxidantes/farmacologia , Encéfalo/efeitos dos fármacos , Cisteína/farmacologia , Iodo/farmacologia , Cinética , Masculino , Ratos , Ratos Wistar , Espectrofotometria UltravioletaRESUMO
We have characterized phosphatase activity present on the external surface of Trichomonas vaginalis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at a rate of 134.3+/-14.8 nmol Pi/h per 10(7) cells. This phosphatase activity decreased by increasing the pH from 6.8 to 8.4, a pH range in which cell viability was maintained for at least 1 h. Experiments using classical inhibitors of acid phosphatases, such as ammonium molybdate and sodium fluoride, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate, [monoperoxo(picolinato)oxovanadate(V)] (mpV-PIC) and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), showed a decrease in this phosphatase activity, with different patterns of inhibition. Cytochemical analysis showed the localization of this enzyme on the parasite surface (cell body and flagellum) and in intracellular vacuoles. Phosphatase reaction products were also observed in exocytosed membrane-bound material.
Assuntos
Fosfatase Ácida/metabolismo , Membrana Celular/enzimologia , Trichomonas vaginalis/enzimologia , 4-Nitrofenilfosfatase/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Flagelos/enzimologia , Humanos , Especificidade por Substrato , Trichomonas vaginalis/crescimento & desenvolvimento , Vacúolos/enzimologiaRESUMO
Acid phosphatase activity, previously identified in Rhodnius prolixus oocytes, was studied during egg development. Fertilized eggs exhibited a five fold increase of total acid phosphatase activity during the first days of development. In contrast non-fertilized oviposited eggs showed no activation of this enzyme. An optimum pH of 4.0 for pNPP hydrolysis in a saturable linear reaction and a strong inhibition by lysosomal acid phosphatase inhibitors such as NaF (10 mM) and Na(+)/K(+) tartrate (0.5 mM) are the major biochemical properties of this enzyme. Fractionation of egg homogenates through gel filtration chromatography revealed a single peak of activity with a molecular mass of 94 kDa. The role of this enzyme in VT dephosphorylation was next evaluated. Western blots probed with anti-phosphoserine polyclonal antibody demonstrated that VT phosphoaminoacid content decreases during egg development. In vivo dephosphorylation during egg development was confirmed by following the removal of (32)P from (32)P-VT in metabolically labeled eggs. Vitellin was the only phosphorylated molecule able to inhibit pNPPase activity of partially purified acid phosphatase. These data indicate that acid phosphatase activation follows oocyte fertilization and this enzyme seems to be involved in VT dephosphorylation.
Assuntos
Fosfatase Ácida/metabolismo , Rhodnius/embriologia , Rhodnius/enzimologia , 4-Nitrofenilfosfatase/metabolismo , Animais , Feminino , Fertilização , Nitrofenóis/metabolismo , Oócitos/enzimologia , Oócitos/crescimento & desenvolvimento , Compostos Organofosforados/metabolismoRESUMO
In this study we report the effects of sulfated polysaccharides on the ecto-ATPase activity of intact cells of Leishmania tropica. Increasing concentrations of dextran sulfate stimulated progressively the ecto-ATPase activity, but did not modify other ecto-enzymes present on the surface of this parasite, such as 5'nucleotidase, 3'nucleotidase and a membrane-bound acid phosphatase activity. This stimulation was not observed when other sulfated polysaccharides such as chondroitin sulfates and heparin were tested. It depends on size and charge of the dextran sulfated molecule. When the cells were incubated in the presence of dextran sulfate Mr 8,000; 40,000 and 500,000 the stimulation of the ecto-ATPase activity was 11%; 23%; and 63%, respectively, and the stimulation was not observed when desulfated dextran (Mr 40,000) was used. The effects of dextran sulfate also depend on pH of the medium. At pH 7.5, the stimulation was over 60%, whereas at pH 8.5 only 25%. The effects of dextran sulfate 500,000 on the ecto-ATPase activity was totally abolished by spermidine and partially by putrescine, two polyamines synthesized and released by Leishmania.
Assuntos
Adenosina Trifosfatases/metabolismo , Sulfato de Dextrana/farmacologia , Leishmania tropica/enzimologia , 4-Nitrofenilfosfatase/metabolismo , Animais , Apirase/metabolismo , Sulfatos de Condroitina/farmacologia , Meios de Cultura , CinéticaRESUMO
Neurotensin (NT), a 13-amino acid peptide, is widely distributed in the brain and peripheral tissues of several mammalian species including man. In adult rat brain NT can bind to two distinct sites, one of high and the other of low affinity, corresponding to NT(1) and NT(2) receptor, respectively; structurally unrelated to these two, a third NT receptor (NT(3)) has been described. We have previously shown that Na(+), K(+)-ATPase is inhibited by NT when using ATP as substrate. In order to determine whether K(+)-stimulated dephosphorylation of this enzyme is involved, we tested NT effect by using p-nitrophenylphosphate, a non-natural substrate. K(+)-p-nitrophenylphosphatase activity was inhibited 42% by NT at 8.6 x 10(-6) M using an incubation medium containing 2 mM KCl but was unaffected in the presence of 5 or 20 mM KCl; however, with such KCl concentrations, NT was enabled to inhibit enzyme activity ( congruent with 35%) provided a suitable ATP:NaCl mixture (0.6:45.0 mM) was added. Mg(2+)-p-nitrophenylphosphatase activity remained unaltered at all conditions tested. Since SR 48692, a selective non-peptide NT(1) antagonist, abolished NT effect, involvement of NT(1) receptor in enzyme inhibition is suggested.
Assuntos
4-Nitrofenilfosfatase/antagonistas & inibidores , Neurotensina/farmacologia , Potássio/farmacologia , Receptores de Neurotensina/fisiologia , 4-Nitrofenilfosfatase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cinética , Magnésio/farmacologia , Masculino , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Fosforilação , Pirazóis/farmacologia , Quinolinas/farmacologia , Ratos , Ratos Wistar , Receptores de Neurotensina/antagonistas & inibidores , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/enzimologiaRESUMO
The plasma membrane (Ca(2+) + Mg(2+))ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca(2+) ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca(2+) strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca(2+)-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca(2+) is related to opposite long-term hydration effects on the substrate binding domain and the Ca(2+) binding domain.