The work is a study of the influence of Ca2+ (0.01 - 1 mM) on neuronal CI-, HCO3-, -ATPase complex: an enzyme that is a CI--pump which is functionally and structurally coupled to GABAA-receptors. It is found that infl...The work is a study of the influence of Ca2+ (0.01 - 1 mM) on neuronal CI-, HCO3-, -ATPase complex: an enzyme that is a CI--pump which is functionally and structurally coupled to GABAA-receptors. It is found that influence of Ca2+ on the multifunctional complex starts at concentration of 50·M and at concentration of 0.1 mM, it reduces the “basal” one and increases the CI-, HCO3-, -stimulated Mg2+-ATPase activities. GABA (0.1 - 100μM) activates the “basal” Mg2+-ATPase activity in the ab-sence of calcium. The effect of GABA on the enzyme in the presence of 0.01 ·M Ca2+ does not change. At the same time, 1 mM Ca2+eliminates the GABA effect on the “basal” Mg2+-ATPase activity. Competitive blocker of GABAA-receptors bicuculline (5 - 20 μM) in the absence of Ca2+ ions elimi-nates the stimulation of the “basal” Mg2+-ATPase by anions. When 0.25 mM Ca2+ is added to the in-cubation medium the inhibitory bicuculline effect on the enzyme does not appear. We found that 0.1 mM o-vanadate (protein tyrosine phosphatase blocker) reduces the GABA-activated ATPase activity. At the same time, 0.1 mM genistein (a protein tyrosine kinase blocker) has no effect on enzyme activity. In the presence of Ca2+ (0.25 mM), the effect of o-vanadate on the “basal” and CI-, HCO3-, -ATPase activities does not appear. It is shown for the first time that high concentrations of Ca2+prevent the action of GABAA-ergic ligands on the study ATPase. It is assumed that there is the involvement of protein kinases and protein phosphatases in the modulation of the enzyme activity by calcium. The observed effect of calcium on the ATPase may play an important role in the study of the mechanisms of epileptogenesis and seizure activity.展开更多
This work examines the influence of Cl- (2.5 - 125 mM) and HCO3- (2 - 30 mM) on the Cl-/HCO3- - ATPase complex of the neuronal membrane and this enzyme is a Cl--pump that is coupled to GABAA receptors. The greatest (4...This work examines the influence of Cl- (2.5 - 125 mM) and HCO3- (2 - 30 mM) on the Cl-/HCO3- - ATPase complex of the neuronal membrane and this enzyme is a Cl--pump that is coupled to GABAA receptors. The greatest (44%) activating effect on the enzyme is found with HCO3- (20 - 30 mM), while the maximum activity occurs in the presence of a ratio of ~25 mM HCO3- /~5mM Cl-. Blockers of the GABAA receptor, namely bicuculline (10 - 50 μM) and picrotoxin (50 - 100 μM), inhibit this anion activation, whereas the HCO3- -ATPase activity is not sensitive to these ligands. Autoradiographic analysis of the spectrum of the partially purified enzyme phosphorylated with [γ-32P]ATP allowed us to distinguish three major 32P-labeled protein whose molecular weight are about 57, 53, and 48 kDa. In the presence of 5 mM Cl-/25mM HCO3- and 100 μM picrotoxin, the intensity of the phosphorylation of bands significantly decreased, thereby confirming the assumption about coupled of binding sites for anions and GABAA-ergic ligands. It was suggested scheme of Cl--transport through the plasma membrane by utilizing neuronal Cl-/ -HCO3- ATPase in the low (5 mM) Cl- and high (25 mM) HCO3- concentrations. The data demonstrated for the first time that the GABAA-coupled Cl-/ HCO3- -ATPase from rat brain neuronal membranes is maximally activated at a Cl-/HCO3- ratio of 1:5 and it remains stable at high concentrations of substrate and buffer.展开更多
文摘The work is a study of the influence of Ca2+ (0.01 - 1 mM) on neuronal CI-, HCO3-, -ATPase complex: an enzyme that is a CI--pump which is functionally and structurally coupled to GABAA-receptors. It is found that influence of Ca2+ on the multifunctional complex starts at concentration of 50·M and at concentration of 0.1 mM, it reduces the “basal” one and increases the CI-, HCO3-, -stimulated Mg2+-ATPase activities. GABA (0.1 - 100μM) activates the “basal” Mg2+-ATPase activity in the ab-sence of calcium. The effect of GABA on the enzyme in the presence of 0.01 ·M Ca2+ does not change. At the same time, 1 mM Ca2+eliminates the GABA effect on the “basal” Mg2+-ATPase activity. Competitive blocker of GABAA-receptors bicuculline (5 - 20 μM) in the absence of Ca2+ ions elimi-nates the stimulation of the “basal” Mg2+-ATPase by anions. When 0.25 mM Ca2+ is added to the in-cubation medium the inhibitory bicuculline effect on the enzyme does not appear. We found that 0.1 mM o-vanadate (protein tyrosine phosphatase blocker) reduces the GABA-activated ATPase activity. At the same time, 0.1 mM genistein (a protein tyrosine kinase blocker) has no effect on enzyme activity. In the presence of Ca2+ (0.25 mM), the effect of o-vanadate on the “basal” and CI-, HCO3-, -ATPase activities does not appear. It is shown for the first time that high concentrations of Ca2+prevent the action of GABAA-ergic ligands on the study ATPase. It is assumed that there is the involvement of protein kinases and protein phosphatases in the modulation of the enzyme activity by calcium. The observed effect of calcium on the ATPase may play an important role in the study of the mechanisms of epileptogenesis and seizure activity.
文摘This work examines the influence of Cl- (2.5 - 125 mM) and HCO3- (2 - 30 mM) on the Cl-/HCO3- - ATPase complex of the neuronal membrane and this enzyme is a Cl--pump that is coupled to GABAA receptors. The greatest (44%) activating effect on the enzyme is found with HCO3- (20 - 30 mM), while the maximum activity occurs in the presence of a ratio of ~25 mM HCO3- /~5mM Cl-. Blockers of the GABAA receptor, namely bicuculline (10 - 50 μM) and picrotoxin (50 - 100 μM), inhibit this anion activation, whereas the HCO3- -ATPase activity is not sensitive to these ligands. Autoradiographic analysis of the spectrum of the partially purified enzyme phosphorylated with [γ-32P]ATP allowed us to distinguish three major 32P-labeled protein whose molecular weight are about 57, 53, and 48 kDa. In the presence of 5 mM Cl-/25mM HCO3- and 100 μM picrotoxin, the intensity of the phosphorylation of bands significantly decreased, thereby confirming the assumption about coupled of binding sites for anions and GABAA-ergic ligands. It was suggested scheme of Cl--transport through the plasma membrane by utilizing neuronal Cl-/ -HCO3- ATPase in the low (5 mM) Cl- and high (25 mM) HCO3- concentrations. The data demonstrated for the first time that the GABAA-coupled Cl-/ HCO3- -ATPase from rat brain neuronal membranes is maximally activated at a Cl-/HCO3- ratio of 1:5 and it remains stable at high concentrations of substrate and buffer.
