The effect of EGTA and Ca++ in regulation of the brain Na/K-ATPase by noradrenaline.

BACKGROUND: The Na/K-ATPase activity of the brain synaptic plasma membranes (SPM) is regulated by noradrenaline (NA) and the synaptosomal factor SF (soluble protein obtained from the synaptosome cytosol). In the absence of SF, NA inhibits Na/K-ATPase, while, on addition of SF to the reaction medium, there is a NA-dependent activation of Na/K-ATPase. On the other hand, EGTA augments the Na/K-ATPase activity and attenuates the ability of NA to inhibit Na/K-ATPase. RESULTS: Considering that Ca2+ ion is a Na/K-ATPase modifier, it can be assumed that the effect of NA and SF is a Ca2+-dependent process. However, in the presence of 0.3 mM EGTA and 0.1 mM NA, the apparent inhibition constant for Ca2+ (at [Ca2+] > 0.3 mM) is not SF dependent, while the apparent activation constant for SF does not change at increasing Ca2+ concentration ([Ca2+] < 0.3 mM). At various Ca2+ concentrations (0.06, 0.35 and 0.6 mM), no significant changes occur in the mode of action of NA on the Na/K-ATPase activity in the presence of 5 microg/ml SF. EGTA also has no effect on the NA-independent activation of Na/K-ATPase evoked by high SF concentrations. CONCLUSIONS: Taking into account that in the absence of EGTA similar results have been obtained, it can be concluded that the effect of NA and SF on brain Na/K-ATPase is a Ca2+-independent process.

The Na/K-ATPase regulation by neurotransmitters in ontogeny.

Activity of the Na/K-ATPase from rat brain synaptic membranes is inhibited by NA (noradrenaline). However, during fractionation of cytozole from nerve endings, two non-homogeneous peaks are found (SF(a), 60-100 kD and SF( i ),;10 kD), which influence the Na/K-ATPase activity, both directly and SF(a) NA-dependently. Joint action of NA and synaptic factors (SF(a) and SF(i)) on the Na/K-ATPase, represents a sum of four different processes: 1) NA, without synaptic factors, inhibits the Na/K-ATPase; 2) At low SF(a) concentrations NA-dependent Na/K-ATPase activatory mechanism is evident; 3) At high SF(a) concentrations NA-independent Na/K-ATPase is activated; 4) The low-molecular SF(i) protein inhibits the Na/K-ATPase. Regulation of the Na/K-ATPase activity by NA, SF(a) and SF( i), obtained in similar conditions from two weeks old and one year old rats, is different. In older rats SF(i) is characterized with strong Na/K-ATPase inhibition; in younger rats SF(i) does not change the Na/K-ATPase activity. The NA- and SF(i) -dependent inhibition and activation ratio is different in young and elder rats. In two week olds NA/SF(i) activatory mechanism is stronger, while in one year olds NA-dependent inhibition of the Na/K-ATPase is prevailing. These experimental data indicate that regulation of the Na/K-ATPase activity has an important role in synaptic transmission and that this process has noteworthy, albeit presently unknown, functional importance in integrative activity of the brain.

[The regulation of the Na, K-ATPase system by neurotransmitters]. / Reguliatsiiia Na, K-ATFaznoi sistemy neirotransmitterami.

Factors regulating the activity of synaptosomal Na, K-ATPase have been found in the cytosol of nerve endings. The activatory effect of the factor increases in the presence of neurotransmitters regardless of their direct action on Na, K-ATPase. Synaptosomal Na, K-ATPase is not sensitive to the factor obtained from the cytosol of kidney tissue, or the cytosolic fraction obtained after sedimentation of microsomes. The effect of inhibiting low molecular ET(S) fraction on Na, K-ATPase activity is not mediated through noradrenaline, dopamine and serotonin as well by the system of secondary messengers. Factor stimulated by neurotransmitters activates the Na, K-ATPase system affecting the phosphorylating intermediates of the enzyme and putting the Na, K-ATPase system in the mode of simultaneous transport of Na and K ions.

[A kinetic analysis of the action of a synaptosomal factor on Na, K-ATPase]. / Kineticheskii analiz deistviia sinaptosomal'nogo faktora na Na, K-ATFazu.

A synaptosomal factor stimulated by neurotransmitters activates the Na, K-ATPase system effecting the phosphorylating intermediates moving the Na, K-ATPase system in the mode of simultaneous transport of Na+ and K+. This conclusion has been made during the analysis of kinetics of the effect of MgATP complex, free Mg2+ ions and ATP on Na, K-ATPase activity. Unlike the EGTA, the factor under study does not change the number of essential activators (ions of Na+ and K+) of the Na, K-ATPase system at the equimolar ATP and Mg2+ correlation.

[Kinetic research on the Na, K-ATPase system]. / Kineticheskoe issledovanie Na, K-ATFaznoi sistemy.

The velocity of Na, K-ATPase is studied as a function of MgATP, ATP and MG2+ concentrations. The kinetic analysis is used to substantiate the inclusion of certain intermediates and steps of their interconversion into the minimal model for Na, K-ATPase.

[Synaptosomal factors, regulating the activity of Na,K-ATPase and its sensitivity to neurotransmitters]. / Sinaptosomal'nye faktory, reguliruiushchie aktivnost'Na,K-ATPase i ee chuvstvitel'nost' k neirotransmitteram.

The factors regulating the activity of synaptosomal Na,K-ATPase have been found in nerve endings cytosol. One of these (Mr 10,000) essentially inhibits, whereas the other one (Mr 60,000) in the presence of norepinephrine, 5-hydroxytryptamine and dopamine activates the Na,K-ATPase of synaptosomes. Regardless of their direct action on Na,K-ATPase, the effect of the activating factor increases in the presence of neurotransmitters. In cell ksap obtained by microsome precipitation the activating factor is absent.

[Regulation of Na,K-ATPase by synaptosomal factors and neurotransmitters]. / Reguliatsiia Na, K-ATFazy sinaptosomal'nymi faktorami i neirotransmitterami.

The identical increase of Na, K-ATPase activity is caused by oxidated and reduced forms of noradrenaline, serotonin and dopamine through the synaptosomal activating factors. The synaptosomal inhibiting factor, orthovanadate and calcium ions independently inhibit Na, K-ATPase activity. The inhibition constant (Ki) for vanadate does not change in the presence of EDTA, whereas in the presence of synaptosomal factors regulating the Na, K-ATPase factors, noradrenaline causes drastic increase of Ki for vanadate. It has been concluded, that the data point to the existence of special regulating system of brain synaptosomal Na, K-ATPase.

[The molecular mechanism of the Na, K-ATPase system]. / Molekuliarnyi mekhanizm Na, K-ATFaznoi sistemy.

Some new properties of Na,K-ATPase system have been revealed using the kinetic analysis of the complex enzymic systems. The fundamental mechanism of Na,K-ATPase functioning has been interpreted and the minimum model including all known working modes of the enzyme under different conditions has been built. The existence of new unknown modes and properties of Na,K-ATPase is predicted and confirmed by different authors.

Functional significance of the effects of neurotransmitters on the Na+,K+-ATPase system.

The aim of the present experiments was to study the effects of the neurotransmitters acetylcholine, noradrenaline, 5-hydroxytryptamine, and dopamine on the Na+,K+-ATPase of rat brain synaptosomal fractions. It is shown that dopamine at low concentrations specifically inhibits the Na+,K+-ATPase of synaptic membranes from the brain regions rich in dopaminergic endings, but has no effect on the synaptosomal Na+,K+-ATPase from the other parts of brain. Acetylcholine and noradrenaline have similar specific effects on Na+,K+-ATPase from cholinergic and adrenergic synaptosomes. The Na+,K+-ATPase of synaptic membranes from the different brain regions, characterised by different distributions of cholinergic, adrenergic, and 5-hydroxytryptaminergic endings, show different reactions with neurotransmitters. These data indicate a functional significance of the effects of the neurotransmitters on the synaptosomal Na+,K+-ATPase.

[Regulation of the number of k-binding sites of Na,K-ATPase by adenosine triphosphate]. / Reguliatsiia kolichestva K-sviazyvaiushchikh tsentrov Na,K-ATPazy adenozintrifosfatom.

A possible existence of two functional states of Na,K-ATPase with different electrogenic coefficients has been experimentally proved. Regulation of electrogenicity is achieved by alteration in the number of K+ transport sites. A transition of Na,K-ATPase from one functional state to the other has been shown to occur during the binding of ATP free ions.

[New method for the study of the cation center of the Na,K-ATPase system]. / Novyi metod analiza kationnykh tsentrov Na,K-ATPaznoi sistemy.

An analysis of the shapes of the kinetic curves for the cationic sites of the Na,K-ATPase system revealed four completely activated Na-sites. Free ATP can change the so-called operation regime of Na,K-ATPase. At ATP excess two completely activated K-sites are detected, whereas at low ATP concentrations only one incompletely activated site is observed.

[Role of vanadium in regulation of oxidation-reduction reactions in membranes]. / Rol' vanadiia v reguliatsii okislitel'no-vosstanovitel'nykh reaktsii v membranakh.

The effect of vanadium on the microsomal membrane system of rat brain and heart was studied. The results obtained exclude the involvement of vanadium in redox processes occurring in brain microsomes. In heart microsomes vanadium, being a redox agent, can control important processes of heart metabolism, while NADH-vanadate-reductase may be a very effective regulator of local pH in heart intracellular membrane systems.

[Some properties of glial membrane Na, K-ATPase]. / Nekotorye svoistva Na,K-ATPazy glial'nykh membran.

Na,K-ATPase activity in glial membranes is rather low that in the nerve ending membranes, but is characterized by the same kind of Na+/K+-dependence. Glial Na,K-ATPase is insensitive to acetylcholine (ACh), 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA) while norepinephrine activates Na,K-ATPase at low concentrations and inhibits it at high concentrations. Participation of Na,K-ATPase in the regulatory mechanisms of the neuron-neuroglia relations is discussed.