Activation by lithium ions of the inside sodium sites in (Na+ + K+)-ATPase.

1. Purified pig kidney ATPase was incubated in 30--160 mM Tris-HCl with various monovalent cations. 130 mM LiCl stimulated a ouabain-sensitive ATP hydrolysis (about 5% of the maximal (Na+ + K) activity), whereas 160 mM Tris-HCl did not stimulate hydrolysis. Similar results were obtained with human red blood cell broken membranes. 2. In the absence of Na+ and with 130 mM LiCl, the ATPase activity as a function of KCl concentration showed an initial slight inhibition (50 micrometer KCl) followed by an activation (maximal at 0.2 mM KCl) and a further inhibition, which was total at mM KCl. In the absence of LiCl, the rate of hydrolysis was not affected by any of the KCl concentrations investigated. 3. The lithium-activation curve for ATPase activity in the absence of both Na+ and K+ had sigmoid characteristics. It also showed a marked dependence on the total LiCl + Tris-HCl concentration, being inhibited at high concentrations. This inhibition was more noticeable at low LiCl concentrations. 4. In the absence of Na+, 130 mM Li+ showed promoted phosphorylation of ATPase from 1 to 3 mM ATP in the presence of Mg2+. In enzyme treated with N-ethylmaleimide, the levels of phosphorylation in Li+-containing solutions, amounted to 40% of those in Na+- and up to 7 times of those in K+-containing solutions. 5. The total (Na+ + K+)-ATPase activity was markedly inhibited at high buffer concentrations (Tris-HCl, Imidazole-HCl and tetramethylammonium-HEPES gave similar results) in cases when either the concentration of Na+ or K+ (or both) was below saturation. On the other hand, the maximal (Na+ + K+)-ATPase activity was not affected (or very slightly) by the buffer concentration. 6. Under standard conditions (Tris-HCl + NaCl = 160 mM) the Na+-activation curve of Na+-ATPase had a steep rise between 0 and 2.5 mM, a fall between 2.5 and 20 mM and a further increase between 20 and 130 mM. With 30 mM Tris-HCl, the curve rose more steeply, inhibition was noticeable at 2.5 mM Na+ and was completed at 5 mM Na+. With Tris-HCl + NaCl = 280 mM, the amount of activation decreased and inhibition at intermediate Na+ concentrations was not detected.     

Inhibition of membrane Na(+)-K+ Atpase of the brain, liver and RBC in rats administered di(2-ethyl hexyl) phthalate (DEHP) a plasticizer used in polyvinyl chloride (PVC) blood storage bags

Significant amounts of di(2-ethylhexyl) phthalate (DEHP) leach out into blood stored in DEHP plasticized polyvinyl chloride (PVC) bags resulting in the exposure of recipients of blood transfusion to this compound. The aim of this study was to find out whether DEHP at these low levels has any effect on the activity of membrane Na(+)-K+ ATPase, since a decrease in this enzyme activity has been reported to take place in a number of disorders like neurodegenerative and psychiatric disorders, coronary artery disease and stroke, syndrome-X, tumours etc. DEHP was administered (ip) at a low dose of 750 microg/100 g body weight to rats and the activity of membrane Na(+)-K+ ATPase in liver, brain and RBC was estimated. Histopathology of brain, activity of HMG CoA reductase (a major rate limiting enzyme in the isoprenoid pathway of which digoxin, the physiological inhibitor of Na(+)-K+ ATPase is a product), intracellular concentration of Ca2+ and Mg2+ in RBC (which is altered as a result of inhibition of Na(+)-K+ ATPase) were also studied. (In the light of the observation of increase of intracellular Ca2+ load and intracellular depletion of Mg2+ when Na(+)-K+ ATPase is inhibited). Histopathology of brain revealed areas of degeneration in the rats administered DEHP. There was significant inhibition of membrane Na(+)-K+ ATPase in brain, liver and RBC. Intracellular Ca2+ increased in the RBC while intracellular Mg2+ decreased. However activity of hepatic HMG CoA reductase decreased. Activity of Na(+)-K+ ATPase and HMG CoA reductase, however returned to normal levels within 7 days of stopping administration of DEHP. The inhibition of membrane Na(+)-K+ ATPase activity by DEHP may indicate the possibility of predisposing recipients of transfusion of blood or hemodialysis to the various disorders mentioned above. However since this effect is reversed when DEHP administration is stopped, it may not be a serious problem in the case of a few transfusion; but in patients receiving repeated blood transfusion as in thalassemia patients or patients undergoing hemodialysis, possibility of this risk has to be considered. This inhibition is a direct effect of DEHP or its metabolites, since activity of HMG CoA reductase, (an enzyme which catalyses a major rate limiting step in the isoprenoid pathway by which digoxin, the physiological inhibitor of Na(+)-K+ ATPase is synthesized) showed a decrease.     

Effects of rubratoxin B on the kinetics of cationic and substrate activation of (Na+-K+)-ATPase and p-nitrophenyl phosphatase.

Rubratoxin B, a lactone-containing bisanhydride metabolite of certain toxigenic molds, inhibited (Na+-K+)-stimulated ATPase activity of mouse brain microsomes in a dose-dependent manner with an estimated IC50 of 6 x 10(-6) M. Hydrolysis of ATP was linear with time and enzyme concentration, with or without rubratoxin in reaction mixtures. Altered pH and activity curves for (Na+-K+)-ATPase demonstrated comparable inhibition by rubratoxin in buffered acidic, neutral, and alkaline pH ranges. Kinetic studies of cationic-substrate activation of (Na+-K+)-ATPase indicated classical competitive inhibition for Na+ and K+. Results also showed competitive inhibition for K+ activated p-nitrophenyl phosphatase as demonstrated by altered binding site parameters without change in the catalytic velocity of dephosphorylation of the enzyme . phosphoryl complex. Noncompetitive inhibition with regards to activation by ATP and p-nitrophenyl phosphate was indicated by altered Vmax values with no change in Km values. Inhibition was partially restored by repeated washings. Preincubation with sulfhydryl agents protected the enzyme from inhibition. Cumulative inhibition studies with rubratoxin and ouabain indicated possible interaction between the two inhibitors of (Na+-K+)-ATPase. Rubratoxin appeared to exert its effects on (Na+-K+)-ATPase by interacting at Na+ and K+ sites.     

Inhibition of Mg2+ and Na(+)-K+ ATPases in selected tissues of fish, Cyprinus carpio under fenvalerate toxicity.

The changes in the magnesium adenosine triphosphatase (Mg2+ ATPase) and sodium-potassium adenosine triphosphatase (Na(+)-K+ ATPase) in gill, brain, liver and muscle tissues of freshwater fish, Cyprinus carpio at 6, 12, 24 and 48 hr exposure periods were studied after subjecting to sublethal concentration (10 micrograms/lit) of fenvalerate. Mg2+ ATPase and Na(+)-K+ ATPase activities were inhibited in all the tissues of fenvalerate exposed fish. The per cent inhibition increased with increase in the period of exposure and the possible reasons for the inhibition patterns are discussed.     

Alpha 1-adrenergic receptor involvement in norepinephrine-ethanol inhibition of rat brain Na+ -K+ ATPase and in ethanol tolerance

Norepinephrine (NE) sensitization of rat brain Na+ -K+ ATPase to ethanol (EtOH) inhibition appears to be mediated by alpha 1-adrenoreceptors, since it was reversed by prazosin and WB-4101 (alpha 1-receptor antagonists) in a concentration-dependent manner, but not by yohimbine and piperoxan (alpha 2-receptor antagonists). In addition, clonidine (alpha 2-agonist) and methoxamine (central receptor type uncertain) produced very little sensitization. Chronic EtOH administration to rats for 3 weeks produced tolerance to the hypothermic effect of test doses of EtOH (3 g/kg, i.p.) and a decreased inhibitory effect of NE + EtOH on the enzyme in vitro. This inhibition was still prevented by prazosin and WB-4101. However, the binding of tritiated WB-4101 and prazosin to brain membrane preparations from control and EtOH-tolerant rats revealed that the maximum number of binding sites (Bmax) and the dissociation constant (KD) of alpha 1-adrenoreceptors were decreased after tolerance development. These changes in numbers and binding properties of alpha 1-adrenoreceptors probably account for the decreased NE sensitization of the ATPase to EtOH inhibition in preparations from EtOH-tolerant rats.     

Modulation of benthiocarb in vitro inhibited neonate rat (Wistar strain) brain Na+K+-ATPase by norepinephrine

Effect in vitro of benthiocarb, an organocarbamate herbicide on neonate rat (3 day old) brain was studied to understand the interaction of benthiocarb with Na+K+-ATPase. Na+K+-ATPase of the developing rat brain was selected as an index enzyme since alterations in the Na+K+-ATPase activity leads to neuronal dysfunction. The assay of Na+K+-ATPase in the presence of 1-8 mu moles of benthiocarb showed decreased activity and a concentration dependent inhibition of Na+K+-ATPase was noticed upto 7 mu moles of benthiocarb. Based on IC50 values (median concentration), 50% inhibition of the enzyme was observed with 5 mu moles of benthiocarb. Norepinephrine (NE) was selected to study the modulation of benthiocarb inhibited enzyme. Maximum increase (76.7%) of Na+K+-ATPase was noticed with 35 mu moles of NE and effective concentration (EC50) of NE which produced 50% activation of the enzyme was found to be 20 mu moles. This study suggests that NE acts as a protective agent in reversing the benthiocarb in vitro inhibited neonate rat brain Na+K+-ATPase.     

Effects of steroid hormones on total brain Na(+)-k+ ATPase activity in Oreochromis mossambicus

The effects of administration of cortisol, corticosterone, testosterone, progesterone and a synthetic estrogen. diethylstilbestrol (DES) on total brain Na(+)-K+- ATPase were investigated in tilapia, O. mossambicus. Exogenous administration of 0.125 and 0.25 microg/g body weight of glucocorticoids and 0.125, 0.25 and 0.5 microg/g body weight of DES for 5 days significantly stimulated Na+(-) K+ ATPase activity by 14-41% in the brain, while 0.5 microg/g body weight of glucocorticoids did not evoke any response on the activity of the enzyme. Progesterone (0.125 and 0.25 microg/g body weight) administration significantly decreased the enzyme activity by 21-36% and high dose (0.5 microg/g body weight) was ineffective. Testosterone exhibited a biphasic effect on Na(+)-K+ ATPase activity--a low dose stimulated by 14% while middle and high doses inhibited it by 19-24%. The results seem to be the first report on the effect of steroids on brain ATPase activity in a teleost. When 0.25microg/g body weight of actinomycin D or puromycin was administered prior to the treatment of similar doses of hormones, the inhibitors significantly inhibited the effect of the hormones by 24-52%. This clearly shows that the effect of the hormones was sensitive to the action of inhibitors suggesting a possible genomic mode of action under long-term treatment. The results suggest that cortisol, corticosterone and DES may possibly stimulate the co-transport of glucose and excitation of membrane potential while progesterone and testosterone inhibit them in the brain of O. mossambicus by regulating the activity of Na(+)-K+ ATPase.     

Effects of cadmium and mercury on Na(+)-K+, ATPase and uptake of 3H-dopamine in rat brain synaptosomes.

Effects in vivo of cadmium (Cd), mercury (Hg) and methylmercury (CH3Hg) on Na(+)-K+ ATPase and uptake of 3H-dopamine (DA) in rat brain synaptosomes were studied. These heavy metals significantly inhibited the Na(+)-K+ ATPase activity in a dose-dependent manner. Similarly, inhibition of DA uptake by synaptosomes was also observed in rats treated with these metals. Intraperitoneal route of metal administration was found to be more effective than per os treatment. Mercuric compounds compared to Cd elicited a higher inhibition of Na(+)-K+ ATPase and DA uptake in rat brain synaptosomes.     

Interleukin-1 beta inhibits Na+-K+ ATPase activity and protein expression in cardiac myocytes

Recent studies have shown that heart diseases are always accompanied with high levels of IL-1beta and a decrease in Na+-K+ ATPase concentrations. This work studies the involvement of the cytokine in the observed changes in the pump. Rats were injected intraperitoneally with 400 mg of IL-1beta and 4 h later, the heart was isolated and a crude homogenate of the right and left ventricles was prepared and tested for Na+-K+ ATPase activity and protein expression. IL-1beta inhibited by around 70% the activity of the ATPase in the left and right ventricles. This inhibition of the pump was ascribed to a decrease in its protein expression as demonstrated by western blot analysis. A dose and time response study conducted on isolated cardiac myocytes confirmed the inhibitory role of the cytokine on the ATPase and showed that IL-1beta exerts its maximal down-regulatory effect at 2 h and at a dose of 20 ng/ml. The cytokine caused also an up-regulation of the NaKCl2 cotransporter. Both MEK and p38MAPK were shown to be involved in the signaling pathway activated by the cytokine. It can be concluded that the decrease in the Na+-K+ ATPase concentration observed in heart diseases is a consequence of the accompanying high levels of IL-1beta, and may be responsible for the different symptoms that accompany cardiac ischemia.     

Protection against cadmium toxicity and enzyme inhibition by dithiothreitol

In the present in vivo studies the alterations in cation transporting enzymes of the brain, kidney and liver tissues were assessed at intervals between 0 to 48 h after a single, acute (10 mg kg-1, i.p.) dose of cadmium (Cd). The inhibition of Na+-K+-ATPase during the first 24 h does not parallel the changes in K+-PNPPase suggesting differential effects on phosphorylation and dephosphorylation steps of the overall ATPase reaction. Between 30 min to 2 h the inhibition in enzyme activity was steep (27 per cent in brain, 54 per cent in liver) followed by a rapid reversal between 2-6 h. This critical period may correspond to the time of induction of metallothionein. This enzyme reversal was followed by a significant decrease in Na+-K+ ATPase (40-68 per cent) and K+-PNPPase (44-60 per cent) between 24 to 48 h. A similar pattern was observed in Ca2+-ATPase in all the three tissues. A 33 per cent mortality was observed in rats after 48 h of cadmium challenge. Administration of dithiothreitol (DTT, 20 mg kg-1, i.p.) to CdCl2 pretreated rats at 24 h resulted in mortality reduced from 33 per cent to 0 and reversal in the inhibition of Na+-K+-ATPase in brain and kidney and Ca2+-ATPase in brain. Since protection of brain and kidney enzymes by DTT paralleled its protection against Cd toxicity, their inhibition by Cd may, in part, constitute the biochemical basis of Cd toxicity.     

Captopril inhibits ouabain-sensitive Na+/K+-ATPase

Captopril has been reported to inhibit ouabain-sensitive Na+/K+-ATPase activity in erythrocyte membrane fragments. We investigated the effect of captopril on two physiological measures of Na+/K+ pump activity: 22Na+ efflux from human erythrocytes and K+-induced relaxation of rat tail artery segments. Captopril inhibited 22Na+ efflux from erythrocytes in a concentration-dependent fashion, with 50% inhibition of total 22Na+ efflux at a concentration of 4.8 X 10(-3) M. The inhibition produced by captopril (5 X 10(-3) M) and ouabain (10(-4) M) was not greater than that produced by ouabain alone (65.3 vs. 66.9%, respectively), and captopril inhibited 50% of ouabain-sensitive 22Na+ efflux at a concentration of 2.0 X 10(-3) M. Inhibition by captopril of ouabain-sensitive 22Na efflux was not explained by changes in intracellular sodium concentration, inhibition of angiotensin-converting enzyme or a sulfhydryl effect. Utilizing rat tail arteries pre-contracted with norepinephrine (NE) or serotonin (5HT) in K+-free solutions, we demonstrated dose-related inhibition of K+-induced relaxation by captopril (10(-6) to 10(-4) M). Concentrations above 10(-4) M did not significantly inhibit K+-induced relaxation but did decrease contractile responses to NE, although not to 5HT. Inhibition of K+-induced relaxation by captopril was not affected by saralasin, teprotide or indomethacin. We conclude that captopril can inhibit membrane Na+/K+-ATPase in intact red blood cells and vascular smooth muscle cells. The mechanism of pump suppression is uncertain, but inhibition of ATPase should be considered when high concentrations of captopril are employed in physiological studies.     

Na+ channel and Na+-K+ ATPase involvement in norepinephrine- and veratridine-stimulated metabolism in perfused rat hind limb.

In the constant flow perfused rat hind limb, norepinephrine (NE) evoked increases in oxygen uptake (VO2) and lactate efflux (LE) were inhibited by the cardiac glycoside ouabain (1 mM), without interrupting the NE-mediated vasoconstriction. The membrane labilizer veratridine, previously shown to increase VO2 and LE, without increasing perfusion pressure, was also shown to be inhibited by the cardiac glycoside ouabain, as well as by the ouabain analogues digitoxin and digoxin. The stimulatory actions of veratridine on VO2 were inhibitable by low doses of the specific sodium channel blocker tetrodotoxin (TTX), while NE effects were unaffected, suggesting that NE may be acting via a TTX-insensitive sodium channel. It is concluded that agents such as NE (a vasoconstrictor) or veratridine (a membrane labilizer), which stimulate VO2 in the perfused rat hind limb, do so by increasing Na+ influx. The observed increases in oxygen consumption and LE are due to Na+-K+ ATPase activity to pump Na+ out of the cell at the expense of ATP turnover. Energy dissipation due to Na+ cycling may be a form of facultative thermogenesis attributable to NE that can be stimulated by membrane labilizers such as veratridine in the constant flow perfused rat hind limb.     

Salinity dependent Na+-K+ATPase activity in gills of the euryhaline crab Chasmagnathus granulata

The occurrence and response of Na+-K+ATPase specific activity to environmental salinity changes were studied in gill extracts of all of the gills of the euryhaline crab Chasmagnathus granulata from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). All of the gills exhibited a salinity dependent Na+-K+ATPase activity, although the pattern of response to environmental salinity was different among gills. As described in other euryhaline crabs highest Na+-K+ATPase specific activity was found in posterior gills (6 to 8), which, with exception of gill 6, increased upon acclimation to reduced salinity. However, a high increase of activity also occurred in anterior gills (1 to 5) in diluted media. Furthermore, both short and long term differential changes of Na+-K+ATPase activity occurred among the gills after the transfer of crabs to reduced salinity. The fact that variations of Na+-K+ATPase activity in the gills were concomitant with the transition from osmoconformity to ionoregulation suggests that this enzyme is a component of the branchial ionoregulatory mechanisms at the biochemical level in this crab.     

The effect of cytoplasmic K+ on the activity of the Na+/K(+)-ATPase.

Experiments with the reconstituted (Na+ + K+)-ATPase show that besides the ATP-dependent cytoplasmic Na(+)-K+ competition for Na+ activation there is a high affinity inhibitory effect of cytoplasmic K+. In contrast to the high affinity K+ inhibition seen with the unsided preparation at a low ATP especially at a low temperature, the high affinity inhibition by cytoplasmic K+ does not disappear when the ATP concentration an-or the temperature is increased. The high affinity inhibition by cytoplasmic K+ is also observed with Cs+, Li+ or K+ as the extracellular cation, but the fractional inhibition is much less pronounced than with Na+ as the extracellular cation. The results suggest that either there are two populations of enzyme, one with the normal ATP dependent cytoplasmic Na(+)-K+ competition, and another which due to the preparative procedure has lost this ATP sensitivity. Or that the normal enzyme has two pathways for the transition from E2-P to E1ATP. One on which the enzyme with the translocated ion binds cytoplasmic K+ with a high affinity but not ATP, and another on which ATP is bound but not K+. A kinetic model which can accommodate this is suggested.     

Enhancement of [3H]DAGO1 binding to rat brain by low concentrations of monovalent cations

The effects of mono- and di-valent cations and the nonhydrolyzable guanyl nucleotide derivative 5'-guanylimidodiphosphate (Gpp(NH)p) on the binding of the selective, high affinity mu-opiate receptor agonist, [3H]DAGO ([3H]Tyr-D-Ala-Gly-Mephe-Gly-ol), to rat brain membranes were studied in a low ionic strength 5 mM Tris-HCl buffer. Na+ and Li+ (50 mM) maximally increased [3H]DAGO binding (EC50 values for Na+, 2.9 mM and Li+, 6.2 mM) by revealing a population of low affinity binding sites. The density of high affinity [3H]DAGO binding sites was unaffected by Na+ and Li+, but was maximally increased by 50 mM K+ and Rb+ (EC50 values for K+, 8.5 mM and Rb+, 12.9 mM). Divalent cations (Ca2+, Mg2+; 50 mM) inhibited [3H]DAGO binding. Gpp(NH)p decreased the affinity of [3H]DAGO binding, an effect that was enhanced by Na+ but not by K+. The binding of the mu-agonist [3H]dihydromorphine was unaffected by 50 mM Na+ in 5 mM Tris-HCl. In 50 mM Tris-HCl, Na+ (50 mM) inhibited [3H]DAGO binding by decreasing the density of high affinity binding sites and promoting low affinity binding. The effects of Na+ in 5 mM and 50 mM Tris-HCl were also investigated on the binding of other opiate receptor agonists and antagonists. [3H]D-Ala-D-Leu-enkephalin binding was increased and inhibited. [3H]etorphine binding increased and was unchanged, and both [3H]bremazocine and [3H]naloxone binding increased by 50 mM Na+ in 5 mM and 50 mM Tris-HCl, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the expression of cardiac Na+-K+ ATPase subunits in the UM-X7.1 cardiomyopathic hamster

Previous studies have shown that cardiac Na+ -K+ ATPase activity in the UM-X7.1 hamster strain is decreased at an early stage of genetic cardiomyopathy and remains depressed; however, the mechanism for this decrease is unknown. The objective of the present study was to assess whether changes in the expression of cardiac Na+-K+ ATPase subunits in control and UM-X7.1 cardiomyopathic hamsters are associated with alterations in the enzyme activity. Accordingly, we examined sarcolemmal Na+-K+ ATPase activity as well as protein content and mRNA levels for the alpha1, alpha2, alpha3 and beta1-subunit of the Na+-K+ ATPase in 250-day-old UM-X7.1 and age-matched, control Syrian hamsters; this age corresponds to the severe stage of heart failure in the UM-X7.1 hamster. Na+-K+ ATPase activity in UM-X7.1 hearts was decreased compared to controls (9.0 +/- 0.8 versus 5.6 +/- 0.8 micromol Pi/mg protein/hr). Western blot analysis revealed that the protein content of Na+-K+ ATPase alpha1- and beta1-subunits were increased to 164 +/- 27% and 146 +/- 22% in UM-X7.1 hearts respectively, whereas that of the alpha2- and alpha3-subunits were decreased to 82 +/- 5% and 69 +/- 11% of control values. The results of Northern blot analysis for mRNA levels were consistent with the protein levels; mRNA levels for the alpha1- and beta1-subunits in UM-X7.1 hearts were elevated to 165 +/- 14% and 151 +/- 10%, but the alpha2-subunit was decreased to 60 +/- 8% of the control value. We were unable to detect mRNA for the alpha3-subunit in either UM-X7. 1 or control hearts. These data suggest that the marked depression of Na+-K+ ATPase activity in UM-X7.1 cardiomyopathic hearts may be due to changes in the expression of subunits for this enzyme.     

Epinephrine stimulates the Na+-K+ ATPase in isolated rat jejunal crypt cells

The effect of epinephrine on the activity of the Na+-K+ ATPase was studied in isolated rat jejunal cells. The activity of the pump was assessed by measuring the ouabain inhibitable K+ accumulation by the enterocytes using 86Rb as a tracer. Epinephrine stimulated significantly the Na+-K+ ATPase in crypt cells but not in villus cells. This effect was still apparent in presence of propranolol and prazocin but disappeared in presence of yohimbine. Amiloride did not affect the epinephrine-induced stimulation. Calcium channel blockers and dibutyryl cAMP enhanced the activity of the pump, and exerted respectively overlapping and additive effects with epinephrine, when added simultaneously. The calcium ionophore A23187 inhibited the basal activity of the ATPase and the stimulatory effect of epinephrine disappeared in its presence. These results suggest that epinephrine stimulates the Na+-K+ ATPase in jejunal crypt cells by activating alpha2 receptors and decreasing intracellular calcium, and not by altering cAMP levels.     

Effect of ABA on the Activity of Mitochondrial Membrane Bound Na+-K+ ATPase

Effect of ABA on the activity of mitochondrial membrane bound Na+-K+ATPase during isolation of mitochondria from soybean cotyledons, there was an increased activity of the mitochondrial membrane bound Na+-K+ATPase if abscisic acid (ABA) was added to the medium when soybean seedling were grown at 27 ℃ or 16℃, 40 mol/L ABA could change the turning point temperature of Arrhenius the activation energies (Ea) of Na+-K+ATPase from 36.6℃ or 22.7℃ decreased to 30. 3℃ or 17.8℃ respectively. The Km value and S0.5 value for this enzyme with ABA was higher than that without ABA. Hill coefficient (n) of this enzyme with ABA was 1.01 and without ABA was 1.89. The o2 uptake of mitochondria also increased. These results showed that the temperature of phase transition of mitochondrial membrane were decreased by ABA treatment.     

Mechanisms by which Li+ stimulates the (Na+ and K+)-dependent ATPase.

The addition of LiCl stimulated the (Na+ + K+)-dependent ATPase activity of a rat brain enzyme preparation. Stimulation was greatest in high Na+/low K+ media and at low Mg-ATP concentrations. Apparent affinities for Li+ were estimated at the alpha-sites (moderate-affinity sites for K+ demonstrable in terms of activation of the associated K+-dependent phosphatase reaction), at the beta-sites (high-affinity sites for K+ demonstrable in terms of activation of the overall ATPase reaction), and at the Na+ sites for activation. The relative efficacy of Li+ was estimated in terms of the apparent maximal velocity of the phosphatase and ATPase reactions when Li+ was substituted for K+, and also in terms of the relative effect of Li+ on the apparent Km for Mg-ATP. With these data, and previously determined values for the apparent affinities of K+ and Na+ at these same sites, quantitative kinetic models for the stimulation were examined. A composite model is required in which Li+ stimulates by relieving inhibition due to K+ and Na+ (i) by competing with K+ for the alpha-sites on the enzyme through which K+ decreases the apparent affinity for Mg-ATP and (ii) by competing with Na+ at low-affinity inhibitory sites, which may represent the external sites at which Na+ is discharged by the membrane Na+/K pump that this enzyme represents. Both these sites of action for Li+ would thus lie, in vivo, on the cell exterior.     

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.