Synaptosomal factors, regulating the activity of Na,K-ATPase and its sensitivity to neurotransmitters

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.     

A kinetic analysis of the action of a synaptosomal factor on Na, K-ATPase

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.     

Stimulation of Na,K-ATPase activity of frog skeletal muscle by insulin

Na,K-ATPase activity of a plasma membrane fraction obtained from frog skeletal muscles was increased approximately two-fold by exposing muscles to insulin, whereas the addition of insulin to a membrane preparation suspension has no effect on Na,K-ATPase activity. The effect of insulin on Na,K-ATPase activity of whole muscles was specific to insulin and insulin derivatives that had the ability of receptor-binding and was not inhibited by actinomycin D. Insulin also induced a development of Na,K-ATPase activity in muscles whose Na,K-ATPase activity had been blocked by ouabain-pretreating. Such a insulin action was inhibited by monensin. These observations suggest that insulin stimulates the monensin-sensitive intracellular transport of membrane proteins which should be responsible for the increase in Na/K pumping activity.     

Endogenous activators and inhibitors of Na, K-ATPase induced by acetylcholine

Preincubation of rat brain homogenates with acetylcholine (ACh) in concentrations of 10(-3)-10(-5) M for 60 minutes produces an essential increment (15-30%) in activity of microsomal Na, K-ATPase. Analogous effect was exerted by the acetylcholinesterase inhibitor eserine (10(-5)-10(-6) M). Acetylcholine has no effect in the presence of actinomycin D. Dialysis of microsomes isolated from the homogenate incubated with ACh leads to a decrease in the enzyme activity and release to the dialysate of low-molecular factor activating Na, K-ATPase of intact microsomes. The latter fact evidences the ACh-induced synthesis of activating factor and inhibition of Na, K-ATPase synthesis. After the animals are administered eserine (0.2-0.4 mg/kg), isolated microsomes show a reduced level of Na, K-ATPase (by 10-15%). Dialysis of microsomes leads to an appreciable elevation (by approximately 40%) of the enzyme activity and release into the dialysate of the inhibitory factor. The differences in the effects of eserine in vivo and in vitro suggest that during the impairment of brain integrity certain effects are excluded from the processes of the control over Na, K-ATPase activity. One of these may involve the impairment of intercellular interactions, for example, the disappearance of the effect on cholinoceptive cells of internuncial neurons that release inhibitory neurotransmitters (catecholamines).     

The Na,K-ATPase of nervous tissue

The Na,K-ATPase has been only partially purified from nervous tissue, yet it is clear that two forms (and +) of the catalytic subunit are present. is a component subunit of the glial Na,K-ATPase, which has a relatively low affinity for binding cardiac glycosides and + has been identified as a subunit of the Na,K-ATPase which has relatively high affinity for cardiac glycosides. The + form may also be sensitive to indirect modulation by neurotransmitters or hormones. The ratio of + / changes in the nervous system during development, and + appears to be the predominant species in adult neurones. Changes in Na,K-ATPase activity have been associated with several abnormalities in the nervous system, including epilepsy and altered nerve conduction velocity, but a causal relationship has not been definitively established. Although the Na,K-ATPase has a pivotal role in Na⁺ and K⁺ transport in the nervous system, a special role for the glial Na,K-ATPase in clearing extracellular K⁺ remains controversial.     

Some properties of thyroidal membrane adenosinetriphosphatase and iodide uptake: effects of basic polyamino acids.

Poly L-lysine, poly L-ornithine, and histone significantly inhibited the iodide uptake by the thyroid slices, as previously reported. These basic polymers diminshed Na, K-ATPase and concomitantly markedly elevated Mg-ATPase activity in the NaI-treated microsomal preparation and the plasma membrane fraction obtained from thyroid. Poly L-glutamic acid, which was noneffetive to the iodide uptake in vitro, did not show such phenomenon. K-dependent p-nitrophenylphosphatase activity which is considered to reflect the terminal step of the reaction sequence of Na, K-ATPase was also inhibited by poly L-lysine. The effects mentioned above of poly L-lysine and other basic polyamino acids on membrane ATPase system were only found in the preparations from thyroid. The inhibitory effect of these reagents on thyroidal iodide uptake was discussed in terms of the change in membrane ATPase activities.     

Postradiation changes in the activity of membrane-bound nerve tissue enzymes

A study was made of the effect of various radiation doses (1.75 to 12.25 Gy) on the enzyme activity of Na,K-ATPase system of the microsomal brain fraction of mongrel and Wistar rats. With a similar method of the fraction isolation different response of the activity of this enzyme was registered. Different radiosensitivity of M9-ATPase is responsible for the direction of changes in the Na,K-ATPase activity of the preparations.     

Regulation of Na,K-ATPase by synaptosomal factors and neurotransmitters

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.     

Stimulation of brain Na,K-ATPase by norepinephrine but not taurine

The effect of taurine on rat and hamster brain Na,K-ATPase was examined and compared to norepinephrine (NE) stimulation of the enzyme. Although NE stimulation of microsomal Na,K-ATPase was observed in the presence of the cell cytosolic fraction, taurine was without effect in the presence and absence of this fraction. Taurine also failed to modulate pubescent and mature hamster brain Na,K-ATPase. Presence or absence of ion chelators did not change taurine's effect. These results are discussed in relation to previous reports of taurine and catecholamine stimulation of Na,K-ATPase.     

Dopamine-induced exocytosis of Na,K-ATPase is dependent on activation of protein kinase C-epsilon and -delta

The purpose of this study was to define mechanisms by which dopamine (DA) regulates the Na,K-ATPase in alveolar epithelial type 2 (AT2) cells. The Na,K-ATPase activity increased by twofold in cells incubated with either 1 μM DA or a dopaminergic D₁ agonist, fenoldopam, but not with the dopaminergic D₂ agonist quinpirole. The increase in activity paralleled an increase in Na,K-ATPase α1 and β1 protein abundance in the basolateral membrane (BLM) of AT2 cells. This increase in protein abundance was mediated by the exocytosis of Na,K-pumps from late endosomal compartments into the BLM. Down-regulation of diacylglycerol-sensitive types of protein kinase C (PKC) by pretreatment with phorbol 12-myristate 13-acetate or inhibition with bisindolylmaleimide prevented the DA-mediated increase in Na,K-ATPase activity and exocytosis of Na,K-pumps to the BLM. Preincubation of AT2 cells with either 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983), a selective inhibitor of PKC-δ, or isozyme-specific inhibitor peptides for PKC-δ or PKC-ε inhibited the DA-mediated increase in Na,K-ATPase. PKC-δ and PKC-ε, but not PKC-α or -β, translocated from the cytosol to the membrane fraction after exposure to DA. PKC-δ– and PKC-ε–specific peptide agonists increased Na,K-ATPase protein abundance in the BLM. Accordingly, dopamine increased Na,K-ATPase activity in alveolar epithelial cells through the exocytosis of Na,K-pumps from late endosomes into the basolateral membrane in a mechanism-dependent activation of the novel protein kinase C isozymes PKC-δ and PKC-ε.     

Na, K-ATPase activity of the microsomal fraction of the rat brain exposed to insulin

The effect of insulin on the activity of Na, K-ATPase was studied in rat brain microsomes. Addition of insulin to the incubation medium in a dose of 0.18 U/ml coupled with strophanthine did not change the enzyme activity. The raising of the hormone dose to 0.36 U/ml was accompanied by inhibition of the enzyme activity. The incubation duration (10 and 30 min) did not influence the Na-pump. Preincubation of brain microsomes with insulin for 5 min significantly activated Na, K-ATPase. It has been thus demonstrated that insulin is capable of influencing the activity of Na, K-ATPase of rat brain microsomes in vitro. The effect obtained depends both on the dose of the hormone introduced into the incubation medium and the experimental conditions.     

Effects of anticonvulsive substances on Na,K-ATPase from the synaptic membranes of animal brain]

The distribution pattern of marker enzymes (Na, K-ATPase, acetylcholinesterase) in three fractions of synaptic membranes (SM) of rat brain were studied. The effects of three anticonvulsive agents on Na, K-ATPase from the total fraction of rat brain SM and purified membrane preparation from ox brain were estimated by different methods. Under optimal conditions (Na/K = 5) diphenylhydantoin (DPH) at a concentration of 0,1 mM activates Na, K-ATPase from the total SM fraction only in the absence of ouabain, whereas carbamazepine and pyrroxane taken at the same concentrations have no effect on Na, K-ATPase, irrespective of the type of the enzyme assay. DPH seems to compete with ouabain. Under non-optimal ionic conditions (Na/K = 250) all the anticonvulsive substances studied inhibit Na, K-ATPase of the total SM fraction. The mixture of hydrophobic agents (propylene glycol and ethanol) used to dissolve carbamazepine inhibits Na, K-ATPase from the total SM fraction only under non-optimal conditions. The inhibiting effect of the anticonvulsive substances under study on Na, K-ATPase from the purified membrane preparations is maximal at the concentration of 10(-6) M; at higher concentrations the effect is less pronounced.     

Characteristics of ATPase activity in the sarcolemma of longitudinal and circular muscles of the canine ileum

The subcellular fraction enriched in sarcolemmal vesicles was isolated from the longitudinal muscle (LM) and the circular muscle (CM) of the canine ileum by sucrose density gradient centrifugation. Treatment of the LM and CM membranes with sodium dodecylsulfate (0.2 mg/kg protein) led to a 3-fold increase in Na,K-ATPase activity (up to 24 and 39 mumol Pi/mg protein/h, respectively) and to a 90-95% inactivation of Mg-ATPase which was 2 and 8 times (for the CM and the LM, respectively) more active than Na,K-ATPase in the untreated sarcolemma. A specific inhibition of Na,K-ATPase activity by acetylcholine (Ach) and serotonin (ST) was observed which could de blocked in the presence of muscarinic and serotonin receptor antagonists. Sensitivity of the enzyme to ST was more than one order of magnitude higher than to Ach (IC50 = 10(-8) vs 1.2 x 10(-7) M). The inhibition of Na,K-ATPase activity by the neurotransmitters was more pronounced in the LM membranes (30-40%) than in the CM ones (10-20%). These data indicate that cell membranes of the LM and CM differ both in specific ATPase activities and the responsiveness of Na,K-ATPase to the receptor-mediated effects of Ach and ST.     

Membrane mechanisms of testosterone action]

The experiments on adult Wistar rats have shown that testosterone administration provoke hyperpolarization of hepatocytes and adrenocorticocytes plasmatic membranes. It was discovered that this hyperpolarization was caused by cell Na, K-ATPase activation. Inhibitors of protein biosynthesis prevent both testosterone-induced hyperpolarization and Na, K-ATPase activation. It is shown that some hyperpolarization factor appeared in the cytosol and blood serum under testosterone influence. It was suggested that this mechanism is mediated by cell genome.     

Enzymatic micromethod for determining Na, K-ATPase activity in rat cerebral cortex membranes]

A method for the assay of Na,K-ATPase activity of unpurified synaptosomal fraction obtained from the microquantities (2--3 mg of fresh tissue) of the rat cerebral cortex is described. This method is based on the fluorimetric determination of ADP formed in the course of ATPase reaction. The method is highly sensitive and may be used to determine the membrane preparations Na,K-ATPase activity with the protein content of 0.05--10.0 microgram per sample.     

Seasonal Changes in Microsomal Fraction Enriched with Na,K-ATPase from Kidneys of the Ground Squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis>

The Na,K-ATPase activity in microsomal fraction isolated from kidneys of winter hibernating ground squirrels was found to be 1.8–2.0-fold lower than that in active animals in summer. This is partially connected with a decrease in Na,K-ATPase protein content in these preparations (by 25%). Using antibodies to different isoforms of Na,K-ATPase α-subunit and analysis of enzyme inhibition by ouabain, it was found that the decrease in Na,K-ATPase activity during hibernation is not connected with change in isoenzyme composition. Seasonal changes of Na,K-ATPase a-subunit phosphory- lation level by endogenous protein kinases were not found. Proteins which could be potential regulators of Na,K-ATPase activity were not found among phosphorylated proteins of the microsomes. Analysis of the composition and properties of the lipid phase of microsomes showed that the total level of unsaturation of fatty acids and the lipid/protein ratio are not changed significantly during hibernation, whereas the cholesterol content in preparations from kidneys of hibernating ground squirrels is approximately twice higher than that in preparations from kidneys of active animals. However, using spin and fluorescent probes it was shown that this difference in cholesterol content does not affect the integral membrane micro-viscosity of microsomes. Using the cross-linking agent cupric phenanthroline, it was shown that Na,K-ATPase in mem- branes of microsomes from kidneys of hibernating ground squirrels is present in more aggregated state in comparison with membranes of microsomes from kidneys of active animals. We suggest that the decrease in Na,K-ATPase activity in kidneys of ground squirrels during hibernation is mainly connected with the aggregation of proteins in plasma membrane.     

Inhibition of Na,K-ATPase-suppressive activity of translationally controlled tumor protein by sorting nexin 6

Translationally controlled tumor protein (TCTP) has both extra- and intracellular functions. Our group recently reported that TCTP interacts with Na,K-ATPase and suppresses its activity. Our studies led to the identification of sorting nexin 6 (SNX6) which binds with TCTP as a potential negative regulator of TCTP. SNX6 does not interact directly with any cytoplasmic domains of Na,K-ATPase. However, when overexpressed, it restores the Na,K-ATPase activity suppressed by TCTP. This was confirmed by measurements of purified plasma membrane Na,K-ATPase activity after incubation with recombinant TCTP and SNX6. SNX6 alone has no effect on Na,K-ATPase activity, but activates Na,K-ATPase via inhibition of TCTP. Inhibition of endogenous TCTP by the overexpression of SNX6 or knockdown of TCTP expression by siTCTP increased Na,K-ATPase activity above the basal level. The interaction between SNX6 and TCTP thus appears to regulate Na,K-ATPase activity.     

Ethyl isopropylamiloride downregulates Na,K-ATPase gene expression which confers cytotoxicity in primary proximal tubule cell cultures

Our original attempt was to examine whether inhibition of Na/H exchange in proximal tubule would affect the expression of basolateral membrane protein Na,K-ATPase. Three amiloride analogues were tested within the range of 10(-6) M to 10(-4) M in primary cultures of proximal tubule cells. Only ethylisopropyl amiloride (EIPA) dose-dependently downregulated Na,K-ATPase activity in cultured proximal tubule cells. The time course study revealed that EIPA (10(-4) M) significantly decreased Na,K-ATPase alpha- and alpha-mRNA abundance within 4 hr and suppressed Na,K-ATPase alpha- and beta-mRNA levels by 76.3 +/- 4.5% and 85.5 +/- 5.8%, respectively, within 24 hr. The decrease in Na,K-ATPase mRNA was followed by a decrease in Na,K-ATPase activity by 22.5 +/- 10.8% and 48.8 +/- 5.9% within 12 and 24 hr, respectively, which could be reflected by a coordinate decrease in levels of both alpha- and mature beta-protein. The cell viability was not affected until 20 hr of EIPA treatment, when an increase in LDH release and cell detachment was observed. Because EIPA rapidly decreased intracellular pH (pHi) to 6.7 within 2 hr and raising pHi to 6.6 by metabolic acidosis could not elicit changes in Na,K-ATPase activity, EIPA-induced downregulation of Na,K-ATPase should not be mediated through H+. In view of the time course of EIPA effects on Na,K-ATPase subunit mRNA, protein, activity and cell toxicity, the cytotoxic effect is likely resulted from a decrease in Na,K-ATPase activity. Take together, we conclude that EIPA induces downregulation of Na,K-ATPase expression via both pre- and post-translational mechanisms, which confers cytotoxic effects on proximal tubule cells.     

Changes in the Na, K-ATPase of neuronal membranes in penicillin-induced epileptic foci in the cerebral cortex of the rat]

The relationship between electrophysiological changes and Na, K-ATPase activity of neuronal membranes in sodium penicillin-induced epileptic foci was studied. Na,K-ATPase activity is inhibited both in the primary focus and in homotopic contralateral area during latent period and in the stage of forming epileptic activity. In the stage of marked convulsive activity Na, K-ATPase is inhibited only in the primary focus. It is shown that penicillin at a concentration range of 2 x 10(-6)--2 x 10(-3) M does not influence Na,K-ATPase activity of crude synaptosomes of the rat brain cortex. It is suggested that Na,K-ATPase inactivation may serve as a pathogenetic factor in the development of convulsive process.