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1.
A kinetic comparison of cardiac glycoside interactions with Na+,K+-ATPases from skeletal and cardiac muscle and from kidney 总被引:3,自引:0,他引:3
E T Wallick B J Pitts L K Lane A Schwartz 《Archives of biochemistry and biophysics》1980,202(2):442-449
The rates of association of [3H]ouabain to Na+,K+-ATPase and the rates of dissociation of the enzyme-ouabain complexes were determined for enzymes isolated from dog skeletal muscle, beef heart muscle, and lamb kidney medulla. The rates of association were strongly influenced by the presence of ligands such as magnesium, sodium, potassium, ATP, and inorganic phosphate. For a particular set of binding ligands, the rates of association did not vary much amongst the three enzymes studied, although enzyme from skeletal muscle was the fastest. In contrast, the rates of dissociation were relatively independent of the ligand conditions. The rates of dissociation also varied greatly amongst the enzyme sources, with skeletal muscle Na+,K+-ATPase being the fastest. Although the major determinant of the affinity of the Na+,K+-ATPase for ouabain is the rate of dissociation, the rate of association also plays a role. Since the binding of ouabain to the Na+,K+-ATPase in the presence of magnesium, ATP, sodium, and potassium is very slow, it is difficult to obtain an I50 (equilibrium) value for the inhibition of hydrolytic activity by ouabain. If measurements of activity are made after a long period of time (3 h), the affinity of the enzyme for ouabain, estimated from inhibition of Na+,K+-ATPase activity, approached the value calculated from [3H]ouabain binding. The ratio of the I50 value for ouabagenin to that for ouabain for the skeletal muscle enzyme was the same as that for cardiac muscle enzyme, indicating that the sugar moiety of ouabain was interacting with the receptor of both enzymes. It is apparent, therefore, that the absence of a sugar binding site in skeletal Na+,K+-ATPase is not the reason for the faster dissociation rate of this enzyme. 相似文献
2.
[3H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K+. By utilising high K+ incubations to estimate non-specific [3H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 · 10?7 M and the total maximum binding to be 2.33 · 105 sites/cell. Ouabain inhibition of (Na+, K+)-pump function was monitored by the cellular uptake of B6Rb over 5 min. The larger fraction of B6Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 · 10?7 M. The cellular distribution of the ( was investigated using [3H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [3H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes. 相似文献
3.
M H Lin D R Romsos T Akera G A Leveille 《Biochemical and biophysical research communications》1978,80(2):398-404
[3H]-Ouabain binding to muscle preparations was utilized to estimate the number of Na+,K+-ATPase enzyme units in hindlimbs from 8 week old lean and obese mice. Specific [3H]-ouabain binding per mg particulate protein was 36% lower in obese mice; whereas, the affinity of the binding sites for ouabain was similar in obese and lean mice. Since obese mice had less muscle than lean mice, the number of Na+,K+-ATPase enzyme units in hindlimbs from obese mice was less than half the number observed in lean mice. 相似文献
4.
C Inagaki M Martinez-Maldonado A Schwartz 《Archives of biochemistry and biophysics》1973,158(1):421-434
Binding of [14C]ethaerynic acid [EA]at concentrations of EA from 10?4m to 10?2m to a membrane preparation containing Na+,K+-ATPase activity in vitro occurred in a nonsaturable manner; binding was stimulated by Na+ or K+, but was not affected by Mg2+ and/or ATP. [14C]EA significantly bound to a microsomal preparation with low Na+,K+-ATPase activity as well as to a heat-denatured enzyme; this binding reaction was not stimulated by Na+. These observations suggest that EA binds non-specifically or to nonspecific sites on membrane preparations. Nonselective binding of [14C]EA to subcellular particles after fractionation of slices also suggested the presence of nonspecific EA binding sites in vivo. In vitro [3H]ouabain binding to medullary and cortical Na+,K+-ATPase preparations was partially reduced by pretreatment with EA. On the other hand, [14C]EA binding to Na+,K+-ATPase was not affected by pretreatment of the preparation with ouabain (10?6m to 5 × 10?4m). EA reduced the sensitivity of [3H]ouabain binding to the enzyme preparation to Na4 and K+.EA was infused (0.1, 1.0, and 10 mg/min) into one renal artery of hydropenic dogs. A prompt natriuresis in the infused kidney occurred. Similar changes were observed in the contralateral kidney 20 min after starting the infusion. Both kidneys were removed 30 min after the beginning of the infusion, and Na+,K+-ATPase was isolated from the cortex and the medulla. Enzyme activity from cortex and medulla of either kidney was not significantly different from enzyme activity from cortex and medulla of control, uninfused dogs, regardless of dose of EA or method of enzyme isolation. Furthermore, in vitro binding of [3H]ouabain to Na+,K+-ATPase membrane preparations from cortex and medulla was the same for experimental and control kidneys. In vitro incubation of 2 × 10?3m EA with a membrane preparation caused the same inhibition of ATPase activity when the enzyme was isolated either from control or EA-infused dogs. The inhibition could not be reversed by recentrifugation or rehomogenization of the enzyme. Our results do not support the concept that Na+,K+-ATPase is a pharmacological receptor for ethacrynic acid. 相似文献
5.
E.J.J. Van Zoelen L.G.J. Tertoolen J. Boonstra P.T. Van Der Saag S.W. De Laat 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,720(3):223-234
1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na+ relative to K+, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K+ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na+. Under these conditions the pumping activity of the Neuro-2A (Na++K+)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na++K+)-ATPase for ouabain, as measured by direct [3H]ouabain-binding studies and by inhibition studies of active K+ uptake. In the presence of 3.5 mM ATP and the absence of external K+ both techniques indicate an apparent dissociation constant for ouabain of 2·10?6 M. Neuro-2A cells contain (3.5±0.7)·105 ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7±0.4)·10?20 mol K+/min per copy of (Na++K+)-ATPase at room temperature. 相似文献
6.
Antonelli Marta C. Lieste Marcela Costa Mercado Rosalio Hernández-R Jorge 《Neurochemical research》1998,23(7):939-944
Previous results showed that Na+/K+-ATPase may have a functional relationship with the neurotransmitter serotonin which activates the glial sodium pump in the rat brain. Both the reaction rate (V) of Na+/K+-ATPase activity and [3H]ouabain binding were significantly increased in the presence of serotonin. It is not known, however, which isoform is involved in the Na+/K+-ATPase response to serotonin and its regional distribution. Quantitative autoradiography of [3H]ouabain binding to rat brain slices was employed at different [3H]ouabain concentrations in order to gain information on both the distribution and the possible isoform involved. The results showed that 1500 nM [3H]ouabain binding was sensitive to serotonin 10–3 M and significantly increased in the following brain regions: frontal cortex, areas CA1, CA2, and CA3 of the hippocampus, presubiculum, zona incerta, caudate putamen and the amygdaloid area, confirming and extending previous results. An effect of serotonin on brain but not kidney tissue at high, 1500 nM, and the lack of effect at low, 50 nM [3H]ouabain concentrations, strongly suggests the participation of the 2 isoform in the response of the pump to the neurotransmitter. Glial cells showed stimulation of ouabain binding by serotonin at ouabain concentrations above 350 nM. The present results open interesting questions related to the brain regions involved and the K+ handling by the glial 2 isoform of the pump. 相似文献
7.
《Biochemical and biophysical research communications》1980,96(4):1518-1523
The presence of an endogenous ‘ouabain like’ compound in rat brain is demonstrated based on the ability of acid acetone extracts of brain to inhibit [3H] ouabain binding and Na+,K+-ATPase activity. Partial purification of the inhibitory activity was achieved by methanol and trichloroacetic acid fractionations followed by Sephadex G-25 chromatography. The results are discussed with relation to the possible role of the endogenous ‘ouabain like’ compound in the regulation of the Na+,K+ pump activity. 相似文献
8.
9.
Laurence Bianchini Barthélémy Fossat Jacqueline Porthé-Nibelle Brahim Lahlou 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1990,160(1):93-98
Summary Na+, K+ exchanges were studied in isolated hepatocytes of the rainbow trout, Salmo gairdneri. Ouabain at 10–4
M produced maximal inhibition (95%) of K+ uptake and enhanced intracellular Na+ accumulation, showing that active fluxes account for a very large proportion of Na+ and K+ exchanges. Inhibition of the Na–K pump by ouabain was significant at low concentrations (10–8
M). When external K+ concentration was reduced from 7 mM to 0.5 mM, half maximum inhibition (IC50) of K+ uptake was obtained at a 22-fold lower concentration of ouabain confirming that ouabain and potassium compete at the same pump site. Time-course analysis of [3H]ouabain binding indicated a two-component kinetics: one component saturable and dependent on K+ concentration in the medium, the other linear and independent of external K+. The ouabain binding site number, determined by Scatchard plots, remained constant (ca. 2.5·105 per cell) and independent of the external K+ concentration (7, 0.5 or 0 mM), while the dissociation constant (KD) decreased from 4.2 M to 7.3 nM when K+ was removed from the Hank's medium. These ouabain binding sites are characterized by an exceptionally low turnover rate (400 min–1), as estimated from ouabain-sensitive K+ flux, in comparison to those described in other cell types of higher vertebrates. At each external K+ concentration studied, the inhibition of K+ uptake and ouabain binding measured as a function of ouabain concentration indicated a strict correlation between the degree of K pump inhibition and the amount of bound glycoside. 相似文献
10.
[3H]Ouabain binding in frog and toad urinary bladder was investigated by short-circuit current (SCC), scintillation counting and authoradiographic techniques. SCC data and analysis of tissue digests following serosal exposure to ouabain showed that ouabain binding and inhibition of Na+ transport was completely reversible in toad bladder whereas, in frog bladder, [3H] ouabain was tightly bound and Na+ transport remained suppressed even after a 60-min washout. Mucosal exposure of frog bladder to [3H]ouabain or serosal exposure after preincubation with unlabeled ouabain led to a marked reduction in binding. Specificity of binding was assessed further by adjusting the concentration of cecrtain (Na+?K+)-ATPase ligands (K+, ATP) to levels known to reduce ouabain binding. High K+ concentrations and depletion of endogenous ATP by incubation under anoxic conditions resulted in a significant drop in [3H]ouabain binding. Autoradiographic analysis showed that grains are localized primarily to the basolateral plasma membranes of the granular cells, providing direct morphological evidence for the location of Na+ pumps at these sites. Although autoradiographs did not provide sufficient resolution to rule out unequivocally ouabain binding to the mitochondria-rich cell, morphological evidence suggests that grain densities are significatly higher between adjacent granular cells than between granular cell-mitochondria-rich cell interfaces. 相似文献
11.
《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1999,122(1):101-108
Effects of two triterpene glycosides, isolated from the holothurian Psolus fabricii, on rat brain Na+,K+-ATPase (Na,K-pump; EC 3.6.1.3) were investigated. Psolusosides A and B (PsA and PsB) inhibited rat brain Na+,K+-ATPase with I50 values of 1×10−4 M and 3×10−4 M, respectively. PsA significantly stimulated [3H]ATP binding to Na+,K+-ATPase, weakly increased [3H]ouabain binding to the enzyme, and inhibited K+-phosphatase activity to a smaller degree than the total reaction of ATP hydrolysis. In contrast, PsB decreased [3H]ATP binding to Na+,K+-ATPase, and had no effect on [3H]ouabain binding to the enzyme. K+-Phosphatase activity was inhibited by PsB in parallel with Na+,K+-ATPase activity. The fluorescence intensity of tryptophanyl residues of Na+,K+-ATPase was increased by PsA and decreased by PsB in a dose-dependent manner. The excimer formation of pyrene, a hydrophobic fluorescent probe, was decreased by PsA only. The different characteristics of inhibition mode for these substances were explained by peculiarities of their chemical structures and distinctive affinity to membrane cholesterol. 相似文献
12.
Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na+/K+ pump. 86Rb+-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40–60 min, and was used to monitor the activity of the Na+/K+ pump. Ouabain (10?3 mol/I) inhibited the steady-state uptake of 86Rb+ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive 86Rb+-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. 86Rb+-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na+/K+ pump activity by ouabain and a concomitant shift in the intracellular Na+:K+ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na+/K+ pump and the glucose transport system. 相似文献
13.
14.
Anush Deghoyan Anna Nikoghosyan Armenuhi Heqimyan 《Electromagnetic biology and medicine》2014,33(1):58-67
Age-dependent effect of Static Magnetic Field (SMF) on rats in a condition of active and inactive Na+/K+ pump was studied for comparison of brain tissues hydration state changes and magnetic sensitivity. Influence of 15?min 0, 2 Tesla (T) SMF on brain tissue hydration of three aged groups of male albino rats was studied. Tyrode’s physiological solution and 10?4?M ouabain was used for intraperitoneal injections. For animal immobilization, the liquid nitrogen was used and the definition of tissue water content was performed by tissue drying method. Initial water content in brain tissues of young animals is significantly higher than in those of adult and aged ones. SMF exposure leads to decrease of water content in brain tissues of young animals and increase in brain tissues of adult and aged ones. In case of ouabain-poisoned animals, SMF gives reversal effects on brain tissue’s hydration both in young and aged animals, while no significant effect on adults is observed. It is suggested that initial state of tissue hydration could play a crucial role in animal age-dependent magnetic sensitivity and the main reason for this could be age-dependent dysfunction of Na+/K+ pump. 相似文献
15.
Harmaline inhibits K+ influx into primary cell cultures of ground squirrel kidneys to a greater extent than either ouabain or furosemide. A concentration of 200 μM harmaline was required to inhibit half of the total K+ influx; this effect was also seen at low temperature (5°C), and in another species (hamster). Although kinetic analysis of K+ influx indicates that harmaline does not compete with extracellular K+, harmaline did reduce the binding of [3H]ouabain to the cells. K+ efflux was also reduced. Therefore, harmaline may inhibit the furosemide-sensitive Na+/K+ cotransport system as well as the ouabain-sensitive Na+/K+ pump. 相似文献
16.
The effects of alloxan-diabetes on the partial reaction of (Na++K+)-ATPase, K+-activated para-nitrophenylphosphatase, and on ouabain binding were studied in isolated adult dog heart myocytes. The Km of K+-activated para-nitrophenylphosphatase for K+ activation was increased from 2.5 to 7.7 mM with no change in Vmax. The Scatchard plots for ouabain binding between control and diabetic animals were indistinguishable. These results indicate that in acute diabetes induced by alloxan, the number of Na+-K+ pumping sites in the heart is not altered but the affinity of the system for K+ is decreased. It is suggested that the decrease in K+ affinity of the (Na++K+)-ATPase enzyme system is at least in part responsible for the altered K+ homeostasis in the diabetic state. 相似文献
17.
The effect of the Na/K-ATPase inhibitor ouabain on phosphoinositide (Ptdlns) hydrolysis was studied in rat brain cortical slices. Ouabain induced a dose-dependent accumulation of inositol phosphates (InsPs) which was much higher in neonatal rats (1570±40% of basal) than in adult animals (287±18% of basal). For this reason, all experiments were conducted with 7 day-old rats. Strophantidin caused a similar stimulation of Ptdlns hydrolysis, although it was less potent than ouabain. The order of potency for ouabain-stimulated InsPs accumulation in brain areas was hippocampus>cortex>brainstem>cerebellum. The effect of ouabain was not blocked by antagonists for the muscarinic, alpha1-adrenergic and glutamate receptors. Also ineffective were the K+ channel blockers 4-aminopyridine and tetraethylammonium, the sodium channel blocker tetrodotoxin, and the calcium channel blocker verapamil, whereas the Na/Ca exchanger blocker amiloride partially antagonized the effect of ouabain. The accumulation of InsPs induced by ouabain was additive to that of carbachol and norepinephrine, as well as to that induced by high K+ and veratrine, but not to that of glutamate. Removal of Na+ ions from the incubation buffer completely prevented the accumulation of InsPs induced by ouabain. The effect of ouabain was also dependent upon extracellular calcium and was under negative feedback control of protein kinase C. Despite the higher effect of ouabain on Ptdlns hydrolysis of immature rats, the density of [3H]ouabain binding sites, as well as the activity of Na/K-ATPase were higher in adult animals. Furthermore, a poor correlation was found between ouabain-stimulated Ptdlns hydrolysis and [3H]ouabain binding in brain regions. These results suggest an involvement of Na+ pump in the hydrolysis of Ptdlns, possibly related to an effect on Na+ and Ca2+ homeostasis. The immature rat appear to be an useful model for studying the relationship between Na/K-ATPase and inositol metabolism. 相似文献
18.
The effect of adrenaline on the K+-activated hyperpolarization of frog skeletal muscle fibres was studied. The amplitude of K+-activated hyperpolarization, which was produced when the external K+ concentration was changed from 0 to 2 mM, was markedly increased in the presence of adrenaline. In the presence of ouabain (1 × 10?5 M), which completely and reversibly eliminated the K+-activated hyperpolarization, adrenaline caused no significant changes in both the membrane potential and conductance under the condition where the K+-activated hyperpolarization was supposed to be produced. These results suggested that adrenaline accelerated the electrogenic Na+ pump which produced the K+-activated hyperpolarization. 相似文献
19.
Neuronal tissues from Manduca sexta, the tobacco hornworm, Hyalophora cecropia, the silkmoth and Danaus plexippus, the Monarch Butterfly, contain Na+K+-ATPase which is sensitive to cardiac glycoside (ouabain). The Km for K+ stimulation of Na+K+-ATPase in M. sexta and D. plexippus is 2.2 mM and for Na+ stimulation in D. plexippus, 6.0 mM. In vitro ouabain concentrations of 1.0 × 10?5 M and 5.0 × 10?5 M in the presence of 7.5 mM K+ inhibited Na+K+-ATPase activity in H. cecropia and M. sexta by 50% respectively. Na+K+-ATPase from D. plexippus was approximately 300 times less sensitive. High concentrations (10?3 M in haemolymph) of ouabain had no effect on M. sexta in vivo. This is largely explained by haemolymph K+ (>; 30 mM) antagonizing the binding of ouabain to Na+K+-ATPase. As demonstrated in vitro, 30 mM K+ totally protects Na+K+-ATPase from inhibition by 7.5 × 10?3 M ouabain in D. plexippus and protects the enzyme by 65% in M. sexta. At least part of the physiological burden incurred in utilization of cardiac glycoside ingestion and storage for protection from predation, however, is probably related to the toxic effects of cardiac glycosides on neuronal Na+K+-ATPase. 相似文献
20.
The effect of various mitogens was studied on sodium (Na+) potassium (K+) transport, 3H-ouabain binding, and adenosine triphosphatase (ATPase) activity in human and sheep peripheral lymphocytes. Concanavalin A (ConA), phytohemagglutinin (PHA), horse anti-lymphocyte serum (ALS), and anti-IgG antisera, in order of decreasing potency, stimulated in particular the ouabain-sensitive K+ pump influx, while the cardiac glycoside-insensitive K+ leak flux was only slightly affected. Sheep lymphocytes primed in vivo with human IgG as antigen also responded with K+ pump flux activation when exposed to the antigen in vitro. Both PHA and ConA also stimulated active Na+ efflux in human lymphocytes. Apparently these mitogens activate the Na+K+ pump system in the lymphocyte membrane—an assumption supported by the finding of a significant activation of the ouabain-sensitive Na+K+-ATPase. From rate studies of 3H-ouabain binding carried out at 37 °C in presence and absence of sodium azide, and at 0 °C, it is concluded that PHA alters the rate of ouabain uptake to these cells. Thus PHA may alter the affinity of the pump for ouabain, equivalent to an increased cation turnover per pump site. However, our findings do not completely discount the possibility that PHA also increases the total number of ouabain molecules bound and therefore of Na+K+ pumps. 相似文献