Interaction of catecholamines and ethanol on the kinetics of rat brain (Na+ + K+)-ATPase

The effects of catecholamines (CA) and ethanol (EtOH), singly and in combination, on the kinetics of rat brain (Na+ + K+)-ATPase were studied. Addition of 0.05 M EtOH alone did not change Vmax or Km for K+, Na+, Mg2+ and ATP. Addition of 0.1 mM dopamine (DA) or noradrenaline (NA) alone stimulated the enzyme activity in presence of vanadium-containing ATP as substrate, but not with vanadium-free ATP except in the presence of high Mg2+ : ATP ratios. CA alone decreased the Km slightly for K+ and by about 50% for ATP, increased it for Mg2+ and did not change it for Na+. However, the combination of DA or NA + EtOH produced a marked inhibition which was competitive for K+, and uncompetitive or mixed for Mg2+, Na+ and ATP. The inhibitory effect of NA + EtOH was abolished in 20 mM K+. These findings suggest that NA sensitizes the enzyme to EtOH inhibition at physiological K+ concentrations, by conformational change away from the outwardly facing K+-binding E2P for to the inwardly facing Na+-binding E1P form.     

Effects of ethanol withdrawal, stress and amphetamine on rat brain (Na+ + K+)-ATPase.

Rats were fed liquid diets containing ethanol (EtOH) or sucrose for 4 weeks, and killed at various times after removal of EtOH. In those having access to the diet up to the time of sacrifice, EtOH caused no increase in (Na⁺ + K⁺)-ATPase activity of whole brain homogenates. However, activity was increased during the period 12–48 hr after withdrawal of EtOH, and was greatest at 24 hr. Fractionation of the homogenate showed that the increase was confined to the lysed synaptosomal fraction. Activity was also increased at 16 hr after one acute dose of EtOH (5 g/kg). The increased ATPase activity during withdrawal could be blocked by administration of another dose of EtOH (3 g/kg) 1 hr before sacrifice, in both acutely and chronically EtOH-treated rats. This is consistent with enhanced sensitivity of noradrenaline-stimulated enzyme to inhibition by EtOH in vitro. ATPase activity was also increased by amphetamine in a dose-dependent manner, both in vivo and in vitro, and by forced swimming. The rise in (Na⁺ + K⁺)-ATPase activity during EtOH withdrawal is interpreted as an activation by conformational change, secondary to catecholamine release due to stress, rather than an adaptive response to chronic EtOH exposure.     

Enzyme modifications that alter interactions of K+ and cardioactive steroids with (Na+ + K+)-dependent ATPase

Treatment of a purified (Na ⁺ + K ⁺-dependent ATPase preparation from dog kidney medulla with acetic anhydride or trinitrobenzene sulfonate (TNBS) produced a dose-dependent irreversible inactivation of the (Na ⁺ + K ⁺)-ATPase and K ⁺-phosphatase reactions catalyzed by the enzyme. Both K ⁺ and the cardioactive steroid strophanthidin protected against inactivation. Prior treatment with concentrations of acetic anhydride or TNBS that produced only partial inactivation also modified the residual activity of the enzyme, as manifested by an increase in the K_0.5 for K ⁺ as activator of the phosphatase reaction (mediated through the moderate-affinity α-sites for K ⁺) but not in the K_0.5, for K ⁺ as activator of the ATPase reaction (mediated through the high-affinity β-sites for K ⁺): correspondingly. the I ₅₀ values (concentrations required to produce 50 per cent inhibition) for ouabain and strophanthidin as inhibitors of the ATPase reaction were increased, and the binding of [³H]ouabain was decreased, by such treatment. Ouabain activated the phosphatase reaction in the absence of K ⁺ and. alter similar treatment, the apparent affinity for ouabain as activator was also decreased. These experiments demonstrate interactions between cardioactive steroids at their sites on the extracellular face of this transmembrane enzyme and K ⁺ at its moderate-affinity α-sites on the intracellular face. and further indicate that K ⁺ can modulate enzyme-drug interactions at such sites, as well as through K ⁺ -sites on the extracellular face of the enzyme.     

Effects of ethanol on ouabain inhibition of mouse brain (Na+,K+)ATPase activity

Plots of ouabain inhibition of mouse cerebral cortical (Na+,K+)ATPase activity fitted a two-site model significantly better than a one-site model, consistent with the presence of two forms of the enzyme with different affinities for ouabain. The fraction of enzyme activity with high affinity for ouabain (HAO: Ki = 500 nM), suggested to be localized neuronally, constituted the major portion (60-70%) of activity. Ouabain inhibition of both components of enzyme activity was reduced as KCl concentrations were increased. In vitro, only high concentrations of ethanol affected (Na+,K+)ATPase activity and ouabain inhibition of activity. Ethanol (500 mM) selectively reduced the activity, and increased the sensitivity to ouabain inhibition, of the HAO component, with no significant effect on the low-affinity (LAO) component. On the other hand, following chronic treatment of mice with ethanol in vivo, in a paradigm that produced tolerance and physical dependence, the sensitivity to ouabain of the HAO form of the enzyme was selectively increased. The relative proportions, and the activities of the HAO and LAO components, were not altered. The effects of ethanol, added in vitro, on the HAO component were decreased in ethanol-tolerant animals. The selective effect of chronic ethanol ingestion on (Na+,K+)ATPase activity indicates the specificity of action of ethanol in the CNS.     

Effects of ethanol on rat brain (Na + K)ATPase from native and delipidized synaptic membranes

The role of lipids in the effect of ethanol on synaptosomal (Na + K)ATPase was studied using native and partially delipidized synaptosomal membranes from control and alcoholic rats. A biphasic effect of alcohol was observed with the (N + K)ATPase from control membranes. Ethanol at low concentrations (less than 100 mM) appears to enhance the enzyme activity, but at higher concentrations (greater than 300 mM) was inhibitory. The biphasic response to ethanol was also observed with the (Na + K)ATPase isolated from alcoholic animals; however, in this case the enzyme showed a resistance to the inhibitory effect of ethanol. Delipidization of synaptic membranes with Lubrol WX or phospholipase A practically abolishes the effects of alcohol on (Na + K)ATPase from both control and alcoholic animals. It thus seems that the effects of ethanol are due mainly to their interaction with the lipids surrounding the enzyme. Furthermore, addition of ethanol to native membranes did not change the Vmax and Km for K+. However, when ethanol at the same concentration was added to delipidized membranes, a decrease in Km with no change in Vmax was observed. Ethanol under these conditions apparently interacts also with the enzyme protein. On the other hand, chronic ethanol intake produces an increase of both Vmax and Km for K+. However, when alcohol was added in vitro, there were no changes in the kinetic parameters of either native or delipidized membranes. These data indicate that although the effects of ethanol on synaptosomal (Na + K)ATPase are mainly due to its interaction with the lipid microenvironment of the enzyme, a direct ethanol action on the enzyme protein also occurs. Our data further suggest that chronic ethanol treatment alters enzyme sensitivity to the effect of ethanol which may be related to the membrane-lipid composition and/or to changes in the conformation of the enzyme protein.     

A possible role of catecholamines and (Na+ + K+)ATPase in the ethanol withdrawl syndrome

Chronic ethanol intoxication leads to an increase in the intracellular Na+/K+ ratio. It is suggested that this derangement is counteracted by catecholamines via an activation of (Na+ + K+)ATPase. This hypothesis is discussed in relation to the symptomatology of ethanol withdrawal.     

(Na+ + K+)ATPase inhibition by Palythoa extracts--chemical nature of the inhibitor and kinetics of inhibition.

Crude toxic extracts obtained by ethanol extraction from the coelenterate Palythoa caribaeorum were shown to possess strong (Na⁺ + K⁺)ATPase inhibitory activity on enzyme preparations from the electroplax of Etectrophorus electricus. The toxic and inhibitory effects were foun to be separable. Chromatographie, spectrophotofluorimetric, electrophoretic and biological data demonstrate that the inhibitor is serotonin. It is a non-competitive inhibitor for Na⁺ and ATP but is a competitive inhibitor for K⁺. In enzyme preparations of a specific activity of 1.5 μM P_i/min, I₅₀ is of the order of 1 mM.     

Effect of 6-aminonicotinamide on monoamine oxidase and Na+K+ATPase activity in different regions of rat brain

The monoamine oxidase activity in the cerebral hemispheres decreased significantly after 2, 4, 8 and 16 hr of 6-amino-nicotinamide (35 mg/kg body weight, i.p.) administration. In the cerebellum, the MAO activity was not affected significantly. In the brain stem, however, a significant increase was observed after 2 hr of drug administration followed by a gradual decrease at later time intervals. The Na+K+ATPase activity in the cerebral hemispheres was increased significantly at 2 and 4 hr of 6-aminonicotinamide administration. This was followed by a gradual decrease at later time intervals. In the cerebellum, like monoamine oxidase, the Na+K+ATPase did not change significantly. The brain stem showed a decrease at 2 hr of drug administration, followed by a significant increase at 4 hr and then a gradual decrease to near control values.     

Effects of monovalent cations on (Na+ + K+)-ATPase in rat brain slices.

The influence of monovalent cations on membrane (Na + K+)-ATPase was estimated in vitro in intact cells from the oxygen consumption of rat brain cortical slices. High concentrations of K+, Rb+ or Cs+ stimulated the respiration in the presence of Na+. This stimulation was antagonized by ouabain in a concentration- and time-dependent manner. Additionally, only combinations of monovalent cations, that stimulate (Na+ + K+)-ATPase, increased oxygen consumption, indicating that the stimulated portion of respiration is realted to the (Na+ + K+)-ATPase activity. Low concentrations of Rb+ and Cs+, however, failed to affect oxygen consumption. Li+ slightly and transiently stimulated oxygen uptake at low concentrations and inhibited it at higher concentrations. Low concentrations of Tl+ also stimulated respiration in a K+-free medium. However, the inhibitory effects of Tl+ were predominant at higher concentrations or in the presence of K+. Thus, monovalent cations can alter (Na+ + K+)-ATPase activity. While Rb+ and Li+ produce opposite effects on this enzyme system under certain conditions, these actions do not seem to be related to the antidepressant action of Rb+ and the antimanic action of Li+.     

Action of methanethiol on membrane (Na+ , K+)-ATPase of rat brain.

The action of methanethiol (CH₃SH) on rat brain (Na⁺, K⁺)-ATPase was examined. The results show that CH₃SH acts at several sites on the enzyme system. The effects are characterized by an inhibition of the ATPase activity, but a concurrent stimulation of the associated K⁺-dependent phosphatase. The inhibitory effect of CH₃SH was of an apparently mixed type with respect to the activation of the ATPase by Na⁺ or by ATP suggesting that CH₃SH may inhibit the formation of phosphoenzyme intermediate in the ATPase reaction, and the inhibition may not be fully reversed by increasing Na⁺. Methanethiol inhibited the activation of the ATPase by K⁺ in an apparently uncompetitive manner, whereas it produced a competitive stimulation of the K⁺ activation of the K⁺-dependent phosphatase activity by increasing the affinity of K⁺ for the enzyme. There was no significant change in the apparent K_m for the substrate p-nitrophenyl phosphate for the phosphatase activity. These effects of CH₃SH may be relevant to its toxicity, and offer a possible molecular site of its action with implications for the encephalopathy of hepatic failure.     

Physiological functions of (Na+ + K+)-adenosinetriphosphatase (ATPase) isozymes

Since Sweadner demonstrated that there are two isozymes (alpha and alpha(+) isoforms) of (Na+ + K+)-adenosine triphosphatase (ATPase) in the brain about 10 years ago, the isozymes have been extensively studied at biochemical and molecular levels. We started the studies on the isozymes of (Na+ + K+)-ATPase soon after finding that pyrithiamin, an antimetabolite of thiamin is a selective inhibitor of the alpha(+)isoform. This review summarizes the previous studies on the isozymes of (Na+ + K+)-ATPase, which are now classified into alpha 1, alpha 2, and alpha 3.     

Effect of chronic ethanol treatment on specific [3H]ouabain binding to (Na+ + K+)-ATPase in different areas of cat brain.

Chronic ethanol administration to cats increased specific [3H]ouabain binding by 63% in cerebral cortex, 47% in cerebellum, 84% in amygdala, and 100% in hippocampus when the binding assays were performed in the presence of 160 nM [3H]ouabain. There was no significant change in specific [3H]ouabain binding in hypothalamus, thalamus, corpus striatum, and brain stem following chronic ethanol ingestion. Scatchard analysis revealed that enhancement of specific [3H]ouabain binding following chronic ethanol treatment in some areas of cat brain is primarily due to changes in densities of ouabain binding sites. Since ouabain is a specific inhibitor of (Na+ + K+)-ATPase the present observations suggest that the molecular mechanism for the enhancement of (Na+ + K+)-ATPase activity after chronic ethanol ingestion may be due to increased net rate of synthesis of (Na+ + K+)-ATPase molecules or exposure of non-functional enzyme system following conformational change of plasma membrane.     

Effect of ethanol and acetaldehyde on the (Na+ +K+)-activated adenosine triphosphatase activity of cardiac plasma membranes.

Ethanol and acetaldehyde inhibited the (Na⁺ + K⁺)-activated ATPase activity of plasma membranes prepared from the guinea-pig heart. The degree of inhibition was dose-dependent and antagonized by the K⁺ concentration in the reaction mixture. The inhibition is not attributable to increase in osmolality. The presence of ethanol or acetaldehyde in the reaction mixture was necessary for the inhibitory effect. Plasma membranes treated with ethanol or acetaldehyde and subsequently washed showed no impairment of (Na⁺ + K⁺)-activated ATPase activity. Prolonged exposure of the plasma membranes to a low concentration of ethanol was ineffective in increasing the inhibition of ATPase activity.     

Interaction of ouabain with (Na+ + K+)ATPase from human heart and from guinea-pig heart.

(Na⁺ + K⁺)ATPase (ATP phosphohydrolase, EC 3.6.1.3.) has been prepared from human heart and guinea-pig heart, with respective specific activities of 10–15 μmol P_i · mg^?1 · h^?1 and of 25–30 μmol P_i · mg^?1 · h^?1. Residual Mg²⁺—ATPase activities were about 5 per cent. The parameters of (Na⁺ + K⁺)ATPase activity and of ouabain-interaction have been compared: (1) Half-maximal activity concentrations and Hill coefficients of Na⁺, K⁺, Mg²⁺ and ATP were similar for the two species. The apparent activation energies calculated from Arrhenius plots were also similar. A transition was observed at about 23°C. (2) Human heart (Na⁺ + K⁺)ATPase was 10 times more sensitive to ouabain-inhibition than that of guinea-pig. Hunter-Downs plots showed a competitive inhibition for K⁺ at low K⁺ concentration and noncompetitive inhibition at high concentration. (3) The Scatchard plot for [³H]ouabain binding was upward-concave with human heart and linear with guinea-pig heart. (4) The dissociation kinetics of [³H]ouabain from human preparations studied by an isotopic dilution technique indicated two classes of binding sites with k_d of 0.058 min^?1 and 0.0092 min^?1. The dissociation kinetics with guinea-pig heart indicated one single class of binding sites with a k_d of 0.43 min^?1. (5) The time-course of 0.2 μM [³H]ouabain binding showed pseudo-first order association kinetics in man and in guinea-pig. k_a for the two classes of binding sites in man were therefore similar, respectively equal to 3.4 × 10⁶ min^?1 · M^?1 and to 3.7 × 10⁶ min^?1 · M^?1 · k_a for guinea-pig heart was equal to 2.3.10⁶ min^?1 · M^?1. (6) In guinea-pig heart, K_D c from Scatchard plot and from k_d/k_a ratio were equal to the inhibition constant K_i calculated from Hunter-Downs plot indicating that the binding sites were closely related to (Na⁺ + K⁺)ATPase inhibition. (7) In human heart, K_D of the low affinity binding sites was close to K_i, whereas K_D of the high affinity binding sites was several times lower. This suggests that only low affinity binding sites might be involved in (Na⁺ + K⁺)ATPase inhibition by ouabain.     

An inborn alcohol tolerance in alcohol-preferring rats. The lack of relationship between tolerance to ethanol and the brain microsomal (Na+K+) ATPase activity

Alcohol tolerance, measured as performance on the tilting plane after ethanol injection (2.5 g/kg body weight i.p.), was examined in the AA strain of rats which voluntarily drink large amounts of alcohol, in the ANA strain which drink very little alcohol, and in ordinary albino rats which consume intermediary amounts of alcohol. The AAs showed the highest innate ethanol tolerance; the ANAs had the lowest innate tolerance. After chronic alcohol administration, both AA and ANA strains increased their tolerance, but the AAs remained superior to the ANAs. The chronic ethanol treatment did not significantly change the (Na⁺K⁺) ATPase activity of the crude microsomal fraction of the brains of the strains.