共查询到20条相似文献,搜索用时 46 毫秒
1.
A. V. Lopachev O. M. Lopacheva K. A. Nikiforova I. S. Filimonov T. N. Fedorova E. E. Akkuratov 《Biochemistry. Biokhimii?a》2018,83(2):140-151
Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein–protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase. 相似文献
2.
I. I. Krivoi T. M. Drabkina V. V. Kravtsova A. N. Vasiliev E. V. Vashchinkina A. V. Prokofiev I. V. Kubasov 《Biophysics》2006,51(5):799-804
It was found that ouabain and marinobufagenin, specific inhibitors of Na+,K+-ATPase, increased the contraction of the isolated rat diaphragm by ~15% (positive inotropic effect) at EC50 = 1.2 ± 0.3 nM and 0.3 ± 0.1 nM, respectively, which was indicative of the participation of the ouabain-sensitive Na+,K+-ATPase α2 isoform. Analysis of the dose-response curves for the effect of ouabain on the resting membrane potential of muscle fibers in the absence and in the presence of 100 nM acetylcholine (hyperpolarizing the membrane) showed the presence of two pools of Na+,K+-ATPase α2 that differed in affinity for ouabain. Only the high-affinity pool (IC50 ~ 9 nM) mediates the hyperpolarizing effect of nanomolar concentrations of acetylcholine. Most likely, it is this pool of that is involved in the positive inotropic effect of ouabain, which can be a mechanism of regulation of the muscle function by circulating endogenous inhibitors of Na+,K+-ATPase. 相似文献
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4.
Macarena Rojas Pablo Díaz Pablo León Alexis A. Gonzalez Magdalena González Víctor Barrientos 《Channels (Austin, Tex.)》2017,11(5):388-398
Renal sodium reabsorption depends on the activity of the Na+,K+-ATPase α/β heterodimer. Four α (α1–4) and 3 β (β1–3) subunit isoforms have been described. It is accepted that renal tubule cells express α1/β1 dimers. Aldosterone stimulates Na+,K+-ATPase activity and may modulate α1/β1 expression. However, some studies suggest the presence of β3 in the kidney. We hypothesized that the β3 isoform of the Na+,K+-ATPase is expressed in tubular cells of the distal nephron, and modulated by mineralocorticoids. We found that β3 is highly expressed in collecting duct of rodents, and that mineralocorticoids decreased the expression of β3. Thus, we describe a novel molecular mechanism of sodium pump modulation that may contribute to the effects of mineralocorticoids on sodium reabsorption. 相似文献
5.
Christopher?M. Stanley Dominique?G. Gagnon Adam Bernal Dylan?J. Meyer Joshua?J. Rosenthal Pablo Artigas 《Biophysical journal》2015,109(9):1852-1862
Cardiac cells express more than one isoform of the Na, K-ATPase (NKA), the heteromeric enzyme that creates the Na+ and K+ gradients across the plasmalemma. Cardiac isozymes contain one catalytic α-subunit isoform (α1, α2, or α3) associated with an auxiliary β-subunit isoform (β1 or β2). Past studies using biochemical approaches have revealed minor kinetic differences between isozymes formed by different α-β isoform combinations; these results make it difficult to understand the physiological requirement for multiple isoforms. In intact cells, however, NKA enzymes operate in a more complex environment, which includes a substantial transmembrane potential. We evaluated the voltage dependence of human cardiac NKA isozymes expressed in Xenopus oocytes, and of native NKA isozymes in rat ventricular myocytes, using normal mammalian physiological concentrations of Na+o and K+o. We demonstrate that although α1 and α3 pumps are functional at all physiologically relevant voltages, α2β1 pumps and α2β2 pumps are inhibited by ∼75% and ∼95%, respectively, at resting membrane potentials, and only activate appreciably upon depolarization. Furthermore, phospholemman (FXYD1) inhibits pump function without significantly altering the pump’s voltage dependence. Our observations provide a simple explanation for the physiological relevance of the α2 subunit (∼20% of total α subunits in rat ventricle): they act as a reserve and are recruited into action for extra pumping during the long-lasting cardiac action potential, where most of the Na+ entry occurs. This strong voltage dependence of α2 pumps also helps explain how cardiotonic steroids, which block NKA pumps, can be a beneficial treatment for heart failure: by only inhibiting the α2 pumps, they selectively reduce NKA activity during the cardiac action potential, leading to an increase in systolic Ca2+, due to reduced extrusion through the Na/Ca exchanger, without affecting resting Na+ and Ca2+ concentrations. 相似文献
6.
J. Lowe 《生物化学与生物物理学报:生物膜》2004,1661(1):40-46
We have previously demonstrated that Na+, K+-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the α subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of α isoforms (α1 and α2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na+, K+-ATPase activity 2.6-fold more sensitive to ouabain (I50=1.0±0.1 μM) than the activity of innervated membranes (I50=2.6±0.2 μM). As depicted in [3H]ouabain binding experiments, when the [3H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na+, K+-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of α1 and α2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na+, K+-ATPase α-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell. 相似文献
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9.
N. B. Bogdanov I. Yu. Petrushanko A. A. Boldyrev M. Gassmann A. Yu. Bogdanova 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2008,2(1):26-32
This study focuses on the oxygen-dependence of active and passive K+ fluxes across membranes of cerebellar granule cells of neonatal rats. Maximal Na+,K+-ATPase activity along with minimal passive K+ influx was observed within oxygen concentration range characteristic for neonatal rat cerebellum. Prolonged exposure to hypoxia as well as hyperoxia resulted in suppression of the Na+,K+-ATPase and activation of the passive K+ flux. Toxic effects of hypoxia could be partially prevented by inhibition of NO production with L-NAME. This was accomplished by suppression of Na+,K+-ATPase with subsequent reduction in ATP consumption concurrently with the reduction in passive K+ flux. Activation of the Na+,K+-ATPase by NO at physiological pO2 could be abolished by inhibition of NO synthase by L-NAME or soluble guanylyl cyclase with ODQ. However, treatment of cells with activator of PKG Rp-8-CTP did not mimic normoxic activation of the active K+ influx. Oxygen-induced responses under normoxic conditions were differentially mediated by α1 isoform of the Na+,K+-ATPase catalytic subunit, whereas α2/3 isoform was predominantly active under conditions of severe hypoxia. We conclude that both hypoxia and hyperoxia trigger a gradual dissipation of transmembrane K+ gradient and loss of excitability of cerebellar neurons. The latter may be partially reversed by suppression of NO production under hypoxic conditions 相似文献
10.
Takuto Fujii Takahiro Shimizu Hideki Sakai 《Biochemical and biophysical research communications》2010,399(4):683-687
K+-Cl− cotransporter-3 has two major amino terminal variants, KCC3a and KCC3b. In LLC-PK1 cells, exogenously expressed KCC3a co-immunoprecipitated with endogenous Na+,K+-ATPase α1-subunit (α1NaK), accompanying significant increases of the Na+,K+-ATPase activity. Exogenously expressed KCC3b did not co-immunoprecipitate with endogenous α1NaK inducing no change of the Na+,K+-ATPase activity. A KCC inhibitor attenuated the Na+,K+-ATPase activity in rat gastric mucosa in which KCC3a is predominantly expressed, while it had no effects on the Na+,K+-ATPase activity in rat kidney in which KCC3b is predominantly expressed. In these tissue samples, KCC3a co-immunoprecipitated with α1NaK, while KCC3b did not. Our results suggest that the NH2-terminus of KCC3a is a key region for association with α1NaK, and that KCC3a but not KCC3b can regulate the Na+,K+-ATPase activity. 相似文献
11.
Previous studies in expression systems have found different ion activation of the Na+/K+-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used
to quantify Na+,K+-ATPase activity, and the Na+ affinity of Na+,K+-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The
Na+ affinity was higher (K
m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with
glycolytic muscle. Na+,K+-ATPase isoform analysis implied that heterodimers containing the β1 isoform have a higher Na+ affinity than heterodimers containing the β2 isoform. Immunoprecipitation experiments demonstrated that dimers with α1 are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α2 isoform with ouabain suggested that heterodimers containing the α1 isoform have a higher Na+ affinity than heterodimers containing the α2 isoform. The estimated K
m values for Na+ are 4.0, 5.5, 7.5 and 13 mM for α1β1, α2β1, α1β2 and α2β2, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na+,K+-ATPase at physiological Na+ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different
isoform compositions. These differences may have consequences for ion balance across the muscle membrane. 相似文献
12.
《生物化学与生物物理学报:生物膜》2001,1510(1-2):118-124
Several Na+ transporters are functionally abnormal in the hypertensive rat. Here, we examined the effects of a high-salt load on renal Na+,K+-ATPase and the sodium-coupled glucose transporter (SGLT1) in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. The protein levels of Na+,K+-ATPase and SGLT1 in the DS rat were the same as those in the DR rat, and were not affected by the high-salt load. In the DS rat, a high-salt load decreased Na+,K+-ATPase activity, and this decrease coincided with a decrease in the apparent Mechaelis constant (Km) for ATP, but not with a change of maximum velocity (Vmax). On the contrary, a high-salt load increased SGLT1 activity in the DS rat, which coincided with an increase in the Vmax for α-methyl glucopyranoside. The protein level of phosphorylated tyrosine residues in Na+,K+-ATPase was decreased by the high-salt load in the DS rat. The amount of phosphorylated serine was not affected by the high-salt load in DR rats, and could not be detected in DS rats. On the other hand, the amount of phosphorylated serine residues in SGLT1 was increased by the high-salt load. However, the phosphorylated tyrosine was the same for all samples. Therefore, we concluded that the high-salt load changes the protein kinase levels in DS rats, and that the regulation of Na+,K+-ATPase and SGLT1 activity occurs via protein phosphorylation. 相似文献
13.
R Matsukawa N Terao M Hayakawa H Takiguchi 《Biochemical and biophysical research communications》1981,101(4):1305-1310
Prostagladin A2, which prevents intestinal ulcers produced by administration of nonsteroidal antiinflammatory compounds such as indomethacin, inhibited the Na+,K+-ATPase activity in basolateral plasma membrane of rat intestine significantly. Prostaglandin A2 inhibited mainly the Na+-dependent phosphorylation step in the overall reaction of Na+,K+-ATPase. This decrease of the Na+,K+-ATPase activity by prostaglandin A2 was due to the decrease of Vmax of the enzyme and of the affinity of the enzyme for Na+. It was also suggested that the presence of both Δ5,6 and Δ10,11 structure of prostaglandin A2 may be necessary for the inhibition of the Na+,K+-ATPase activity. 相似文献
14.
The N-terminus of the Na+,K+-ATPase α-subunit shows some homology to that of Shaker-B K+ channels; the latter has been shown to mediate the N-type channel inactivation in a ball-and-chain mechanism. When the Torpedo Na+,K+-ATPase is expressed in Xenopus oocytes and the pump is transformed into an ion channel with palytoxin (PTX), the channel exhibits a time-dependent inactivation gating at positive potentials. The inactivation gating is eliminated when the N-terminus is truncated by deleting the first 35 amino acids after the initial methionine. The inactivation gating is restored when a synthetic N-terminal peptide is applied to the truncated pumps at the intracellular surface. Truncated pumps generate no electrogenic current and exhibit an altered stoichiometry for active transport. Thus, the N-terminus of the α-subunit appears to act like an inactivation gate and performs a critical step in the Na+,K+-ATPase pumping function. 相似文献
15.
Mitzy Canessa José R. Romero Nelson Ruiz-Opazo Victoria L. M. Herrera 《The Journal of membrane biology》1993,134(2):107-122
The properties of the α1 Na+-K+ pump were compared in Dahl salt-sensitive (DS) and salt-resistant (DR) strains by measuring ouabain-sensitive luxes (mmol/liter cell x hr = FU, Mean ± se) in red blood cells (RBCs) and varying internal ( i ) and external ( o ) Na+ and K+ concentrations. Kinetic parameters of several modes of operation, i.e., Na+/ K+, K+/K+, Na+/Na+ exchanges, were characterized and analyzed for curve-fitting using the Enzfitter computer program. In unidirectional flux studies (n=12 rats of each strain) into fresh cells incubated in 140 mm Na+ + 5 mm K+, ouabain-sensitive K+ influx was substantially lower in the DS than in DR RBCs, while ouabain-sensitive Na+ efflux and Na i were similar in both strains. Thus, the coupling ratio between unidirectional Na+∶K+ fluxes was significantly higher in DS than in DR cells at similar RBC Na+ content. In the presence of 140 mm Na o , activation of ouabain-sensitive K+ influx by K o had a lower K m and V max in DS as estimated by the Garay equation (N=2.70 ± 0.33, K m 0.74 ± 0.09 mm; V max 2.87 ± 0.09 FU) than in DR rats (N=1.23 ± 0.36, K m 2.31 ± 0.16 mm; v max 5.70 ± 0.52 FU). However, the two kinetic parameters were similar following Na o removal. The activation of ouabain-sensitive K+ influx by Na i had significantly lower V max in DS (9.3 ± 0.4 FU) than in DR (14.5 ± 0.6 FU) RBCs but similar K m. These data suggest that the low K+ influx in DS cells is caused by a defect in modulation by Na o and Na i . Na+ efflux showed no differences in Na i activation or trans effects by Na o and K o , thus accounting for the different Na+∶K+ coupling ratio in the Dahl strains. Further evidence for the differences in the coupling of ouabain-sensitive fluxes was found in studies of net Na+ and K+ fluxes, where the net ouabain-sensitive Na+ losses showed similar magnitudes in the two Dahl strains while the net ouabainsensitive K+ gains were significantly greater in the DR than the DS RBCs. Ouabain-sensitive Na+ influx and K+ efflux were also measured in these rat RBCs. The inhibition of ouabain-sensitive Na+ influx by K o was fully competitive for the DS but not for the DR pumps. Thus, for DR pumps, K o could activate higher K+ influx in DR pumps without a complete inhibition of ouabain-sensitive Na+ influx. This behavior is consistent with K o interaction with distinct Na+ and K+ transport sites. In addition, the inhibition of K+ efflux by Na, was different between Dahl strains. Ouabain-sensitive K+ efflux at Na i level of 4.6 mmol/liter cell, was significantly higher in DS (3.86 ± 0.67 FU) than in DR (0.86 ± 0.14 FU) due to a threefold higher K50 for Na i -inhibition 9.66 ± 0.41 vs. 3.09 ± 0.11 mmol/liter cell. This finding indicates that Na+ modulation of K+ transport is altered at both sides of the membrane. The dissociation of Na+ modulatory sites of K+ transport from Na+ transport sites observed in RBCs of Dahl strains suggests that K+ transport by the Na+-K+ pump is controlled by Na+ allosteric sites different from the Na+ transport sites. The alterations in K+ transport may be related to the amino acid substitution (Leu/Gln276) reported for the cDNA of the α1 subunit of the Na+-K+ pump in the DS strain or to post-translational modifications during RBC maturation. These studies were supported by the following grants: NIH (HL-35664, HL-42120, HL-18318, HL-39267, HL-01967). J.R.R. is a Ford Foundation Predoctoral Fellow. A preliminary report of this work was presented at the International Conference on the Na+-K+ pump and 44th Annual Meeting of the Society of General Physiologists held at Woods Hole, MA, September 5–9, 1990, and published as an abstract in the J. Gen. Physiol. 96:70a, 1990. 相似文献
16.
Kuntal Dey Tapati Chakraborti Soumitra Roy Biswarup Ghosh Pulak Kar Sajal Chakraborti 《Life sciences》2010,86(13-14):473-481
AimsWe sought to identify, purify and partially characterize a protein inhibitor of Na+/K+-ATPase in cytosol of pulmonary artery smooth muscle.Main methods(i) By spectrophotometric assay, we identified an inhibitor of Na+/K+-ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na+/K+-ATPase α2β1 and α1β1 isozymes for determining some characteristics of the inhibitor.Key findingsWe identified a novel endogenous protein inhibitor of Na+/K+-ATPase having an apparent mol mass of ~ 70 kDa in the cytosolic fraction of the smooth muscle. The IC50 value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the α2β1 and α1β1 isozymes of the Na+/K+-ATPase; (ii) it interacted reversibly to the E1 site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na+/K+-ATPase exists as (αβ)2 diprotomer.SignificanceThe inhibitor binds to the Na+/K+-ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where α2 is more sensitive than α1. 相似文献
17.
Duane Gischewski Pereira Mariana Manzano Rendeiro Vanessa Faria Cortes Leandro Augusto Barbosa Luis Eduardo M. Quintas 《Journal of cellular biochemistry》2019,120(8):13107-13114
Despite the growing interest in the antitumor effect of cardiotonic steroids, combination treatments with well-established chemotherapy drugs like paclitaxel have been rarely investigated. Moreover, paclitaxel has been suggested as a Na+/K+-ATPase inhibitor. Here we investigated the effect of paclitaxel and digoxin alone or in combination on the viability of human lung (A549) and cervical cancer (HeLa) cell lines and the inhibitory effect of paclitaxel on several mammalian Na+/K+-ATPases. Although the viability of both tumor cell lines was concentration-dependently affected by digoxin treatment after 48 hours (A549 IC50 = 31 nM and HeLa IC50 = 151 nM), a partial effect was observed for paclitaxel, with a maximal inhibitory effect of 45% at 1000 nM with A549 and around 70% with HeLa cells (IC50 = 1 nM). Although the two drugs were cytotoxic, their combined effect in HeLa cells was revealed to be antagonistic, as estimated by the combination index. No direct inhibitory effect of paclitaxel was detected in human, pig, rat, and mouse Na+/K+-ATPase enzymes, but high concentrations of paclitaxel decreased the Na+/K+-ATPase activity in HeLa cells after 48 hours without affecting protein expression. Our findings demonstrate that, under our conditions, paclitaxel and digoxin cotreatment produce antagonistic cytotoxic effects in HeLa cells, and the mechanism of action of paclitaxel does not involve a direct inhibition of Na+/K+-ATPase. More studies shall be designed to evaluate the consequences of the interaction of cardiotonic steroids and chemotherapy drugs. 相似文献
18.
Juliana L. França Marcelo R. Pinto Malson N. Lucena Daniela P. Garçon Wagner C. Valenti John C. McNamara Francisco A. Leone 《The Journal of membrane biology》2013,246(7):529-543
The stimulation by Mg2+, Na+, K+, NH4 +, and ATP of (Na+, K+)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na+, K+)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg2+, Na+, and K+ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V M = 115.0 ± 2.3 U mg?1, K 0.5 = 0.10 ± 0.01 mmol L?1. Stimulation by Na+ (V M = 110.0 ± 3.3 U mg?1, K 0.5 = 1.30 ± 0.03 mmol L?1), Mg2+ (V M = 115.0 ± 4.6 U mg?1, K 0.5 = 0.96 ± 0.03 mmol L?1), NH4 + (V M = 141.0 ± 5.6 U mg?1, K 0.5 = 1.90 ± 0.04 mmol L?1), and K+ (V M = 120.0 ± 2.4 U mg?1, K M = 2.74 ± 0.08 mmol L?1) followed single saturation curves and, except for K+, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K I = 12.4 ± 1.3 mol L?1. Complementary inhibition studies suggest the presence of F0F1–, Na+-, or K+-ATPases, but not V(H+)- or Ca2+-ATPases, in the gill microsomal preparation. K+ and NH4 + synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4 +, and K+ of the gill enzyme. 相似文献
19.
The activity of human α-thrombin (EC 3.4.21.5) on small peptide substrates was enhanced by NaCl or KCl while tetramethylammonium chloride ((CH3)4NCl) or choline chloride (HO(CH2)2N(CH3)3Cl) which were used as ionic strength controls were without effect. The steady-state kinetic parameters of thrombin amidolysis of several peptidyl p-nitroanilide substrates were measured. Na+ enhanced thrombin activity by decreasing the Km,app (0.2 to 0.7-fold) of all substrates, as well as increasing thombin turnover (3.4 to 4.5-fold) of some substrates. The average KA for Na+for the four substrates examined was 3.5 × 10?2m. A comparison of the effects of Na+ vs K+ on thrombin hydrolysis of a single substrate indicated that both cations similarly decreased the Km,app (0.2 to 04.-fold) and increased thekcat,app (3.1 to 3.4-fold) except that higher K+ concentrations (KA = 2.8 × 10?1M) were required. The rate of inactivation of thrombin by the active site-directed inhibitor N-p-tosyl-lysine chloromethyl ketone under pseudo-first-order conditions was enhanced 3-fold by saturating NaCl. Also, the fibrinogen clotting activity of thrombin was enhanced by NaCl compared to the choline chloride control. Spectral studies demonstrated that thrombin titration by Na+ caused a positive ultraviolet difference spectrum with maxima at 281.5 and 288.5 nm (Δ?288.5 = +1067). The Km for Na+ was 2.3 × 10?2m which agrees with the kinetically determined KA for Na+. The results are consistent with Na+ binding to thrombin causing a conformational change in the active site. It is concluded that human α-thrombin is a monovalent cation-activated enzyme. 相似文献
20.
Torben Clausen 《The Journal of general physiology》2013,142(4):327-345
During excitation, muscle cells gain Na+ and lose K+, leading to a rise in extracellular K+ ([K+]o), depolarization, and loss of excitability. Recent studies support the idea that these events are important causes of muscle fatigue and that full use of the Na+,K+-ATPase (also known as the Na+,K+ pump) is often essential for adequate clearance of extracellular K+. As a result of their electrogenic action, Na+,K+ pumps also help reverse depolarization arising during excitation, hyperkalemia, and anoxia, or from cell damage resulting from exercise, rhabdomyolysis, or muscle diseases. The ability to evaluate Na+,K+-pump function and the capacity of the Na+,K+ pumps to fill these needs require quantification of the total content of Na+,K+ pumps in skeletal muscle. Inhibition of Na+,K+-pump activity, or a decrease in their content, reduces muscle contractility. Conversely, stimulation of the Na+,K+-pump transport rate or increasing the content of Na+,K+ pumps enhances muscle excitability and contractility. Measurements of [3H]ouabain binding to skeletal muscle in vivo or in vitro have enabled the reproducible quantification of the total content of Na+,K+ pumps in molar units in various animal species, and in both healthy people and individuals with various diseases. In contrast, measurements of 3-O-methylfluorescein phosphatase activity associated with the Na+,K+-ATPase may show inconsistent results. Measurements of Na+ and K+ fluxes in intact isolated muscles show that, after Na+ loading or intense excitation, all the Na+,K+ pumps are functional, allowing calculation of the maximum Na+,K+-pumping capacity, expressed in molar units/g muscle/min. The activity and content of Na+,K+ pumps are regulated by exercise, inactivity, K+ deficiency, fasting, age, and several hormones and pharmaceuticals. Studies on the α-subunit isoforms of the Na+,K+-ATPase have detected a relative increase in their number in response to exercise and the glucocorticoid dexamethasone but have not involved their quantification in molar units. Determination of ATPase activity in homogenates and plasma membranes obtained from muscle has shown ouabain-suppressible stimulatory effects of Na+ and K+.
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Open in a separate windowData were obtained from 10 different publications listed in the references of this paper. Each of them was selected for reporting the results of measurements of the content of [3H]ouabain-binding sites as well as α2-subunit abundance in the same muscle. Experimental details are given in the cited articles. The relative changes induced by the listed factors are given in percentages. Biopsies from human subjects were taken from the vastus lateralis muscle. The rat muscles were obtained from the hind limbs.
Introduction: Transport and content of Na+ and K+ in skeletal muscle
The Na+,K+-ATPase (also known as the Na+,K+ pump) is the major translator of metabolic energy in the form of ATP to electrical and chemical gradients for the two most common ions in the body. These gradients enable the generation of action potentials, which are essential for muscle cell function. Evaluation of the physiological and clinical significance of the Na+,K+ pumps requires measuring the transmembrane fluxes of Na+ and K+ in intact muscles or cultured muscle cells. The simplest approach involves incubating intact muscles isolated from small animals in temperature-controlled and oxygenated buffers with electrolyte and glucose concentration comparable to that normally present in blood plasma. Initial studies used cut hemi- or quarter-diaphragm muscles from rats, mice, or guinea pigs for incubation because these muscles were considered thin enough to allow adequate oxygenation under these conditions (Gemmill, 1940). However, such preparations have numerous cut muscle ends, allowing large passive movements of Na+ and K+ and free access of Ca2+ to the cell interior. This unavoidably boosts the energy required for active transport of Na+, K+, and Ca2+, and leads to impaired cell survival. Thus, in cut muscles, the components of O2 consumption and 42K uptake attributable to the Na+,K+ pump (i.e., the fraction suppressible by the cardiac glycoside ouabain, which binds to and inhibits the Na+,K+-ATPase) have been severely overestimated. (The ouabain-suppressible components of O2 consumption or 42K uptake are measured in isolated muscles incubated without or with ouabain and calculated as the difference.) Such overestimation led to the assumption that in skeletal muscle, the Na+,K+ pumps mediate a large fraction of total energy turnover, suggesting that a major part of the thermogenic action of thyroid hormone is caused by an increased rate of active Na+,K+ transport (Asano et al., 1976). In contrast, in intact resting muscle preparations, only 2–10% of the total energy turnover is used for active Na+,K+ transport (Creese, 1968; for details, see Clausen et al., 1991). Even during maximum contractile work in human muscles, only a small fraction (2%) (Medbø and Sejersted, 1990) of total energy release is used for the Na+,K+ pumps. Thus, in skeletal muscle, the thermogenic action of the Na+,K+ pumps is modest.For the analysis of Na+,K+ transport in skeletal muscle, isolated intact limb muscles are used, primarily mammalian soleus, extensor digitorum longus (EDL), extensor digitorum brevis, or epitrochlearis muscles. These preparations can survive during incubation for many hours and can undergo repeated excitation. More recently, the isolated rat sternohyoid muscle, which also offers thin dimensions, cellular integrity, and simple handling, has been introduced (Mu et al., 2011).Measurement of muscle Na+ and K+ content requires extraction. In the past, this was done by digesting the muscle preparation in nitric acid, a sometimes risky procedure. More recently, this approach has been superseded by homogenization of the tissue in 0.3 M trichloroacetic acid, followed by centrifugation to sediment the proteins (Kohn and Clausen, 1971). The clear supernatant may then be diluted for flame photometric determination of Na+ and K+ or counting of the isotopes 22Na or 42K. Because 42K has a short half-life (12.5 h) and is of limited availability, the K+ analogue 86Rb is often used as a tracer for K+. For the quantification of Na+,K+ pump–mediated (i.e., ouabain-suppressible) transport of K+, 86Rb gives the same results as 42K (Clausen et al., 1987; Dørup and Clausen, 1994). For other flux measurements, however, the results obtained with 86Rb differ appreciably from those obtained with 42K. For example, the fractional loss of 86Rb from intact resting rat soleus muscles is only 45% of that measured using 42K. Moreover, two agents shown to stimulate the Na+,K+ pumps in isolated rat soleus muscle, salbutamol (Clausen and Flatman, 1977) and rat calcitonin gene–related peptide (CGRP) (Andersen and Clausen, 1993), both induce a highly significant stimulation of 86Rb efflux from the same muscle (Dørup and Clausen, 1994). In contrast, these same agents induced a rapid but transient (20-min duration) inhibition of the fractional loss of 42K, indicating that a large fraction of the 42K lost from the cells is reaccumulated as a result of stimulation of the Na+,K+ pumps (Andersen and Clausen, 1993; Dørup and Clausen, 1994). Finally, in skeletal muscle, bumetanide, an inhibitor of the NaK2Cl cotransporter, produces no inhibition of 42K uptake but clear-cut inhibition of 86Rb uptake (see and22 in Dørup and Clausen, 1994). Thus, results obtained with 86Rb must be verified with 42K or flame photometric measurements of changes in intracellular Na+ and K+ content. The activity of the Na+,K+ pump depends on ATP supplied by glycolysis or oxidative phosphorylation. Excitability of isolated rat soleus or EDL muscles can be maintained in the presence of the electron transport inhibitor cyanide or during anoxia (Murphy and Clausen, 2007; Fredsted et al., 2012), whereas contractions are markedly suppressed by 2-deoxyglucose, which interferes with the production of glycolytic ATP. Thus, in keeping with the studies of Glitsch (2001), these data suggest that glycolytic ATP is a primary source of energy for the Na+,K+ pumps (see also Okamoto et al., 2001). More focused studies showed that Na+,K+-pump function is not adequately supported when cytoplasmic ATP is at the normal resting concentration of ∼8 mM, but that the addition of as little as 1 mM phosphoenol pyruvate produces a marked increase in Na+,K+-pump function that is supported by endogenous pyruvate kinase bound within the t-tubular triad (Dutka and Lamb, 2007). In anoxic rat EDL muscles, contractility could be restored by stimulating the Na+,K+ pumps with the β2 agonists salbutamol or terbutaline, effects that were abolished by ouabain or 2-deoxyglucose (Fredsted et al., 2012). Glycolytic ATP furnishes energy for contractile activity under the critical condition of anoxia. Because the Na+,K+ pumps are essential for the maintenance of excitability and contractions, it would not be surprising if they were also kept going on glycolytic ATP.Table 1.
Acute stimulation of the Na+,K+ pumps in skeletal muscleStimulating hormones, agents, and conditions | Mechanisms of action |
Epinephrine, norepinephrine | Stimulate generation of cAMP, which in turn activates the Na+,K+ pumps via PKA, increasing the affinity of the Na+K+ pumps for Na+ |
Isoproterenol, salbutamol, salmeterol | |
Other β2 agonists | |
β3 agonists | |
Calcitonins | Stimulate generation of cAMP |
CGRP | “ |
Amylin | “ |
cAMP, dibutyryl cAMP | Activates Na+,K+ pumps via PKA |
Theophylline | Inhibits phosphodiesterase A, which degrades cAMP. This leads to intracellular accumulation of cAMP. Theophylline is a degradation product of caffeine. |
Insulin, insulin-like growth factor I | Both act via the insulin receptors, increasing the affinity of Na+,K+ pumps for Na+ |
Monensin | A Na+ ionophore that increases [Na+]i, which directly stimulates the Na+,K+ pumps |
Veratridine | Augments the Na+ influx per action potential, thereby increasing [Na+]i |
Excitation | Augments [Na+] influx, thereby increasing [Na+]i |
Increasing temperature | When temperature increases 10°C, the rate of Na+,K+ pumping increases 2.3-fold |
ATP, ADP | Stimulate the Na+,K+ pumps via purinergic receptors |
Table 2.
Relative changes in [3H]ouabain binding and the α2 subunit of Na+,K+-ATPase in skeletal muscleReferences, muscle preparation, treatment | [3H]Ouabain binding | α2-subunit abundance |
Thompson et al., 2001, rats treated for 14 d with dexamethasone | +22–48% (P < 0.05) | +53% (P < 0.05) |
Juel et al, 2001, 1 h treadmill running 150-200 g rats, giant vesicles | +29% (P < 0.05) | +32% (P < 0.05) |
Green et al., 2004, 6 d submaximal cycling | +13% (P < 0.05) | +9% (P < 0.05) |
Sandiford et al., 2005, electrical stimulation of rat soleus | +16–21% (P < 0.05) | +38% (P < 0.05) |
Green et al., 2007, 16 h heavy intermittent cycling | +8% (P < 0.05) | +26% (P < 0.05) |
Green et al., 2008, 3 d submaximal cycling | +12% (P < 0.05) | +42% (P < 0.05) |
Green et al., 2009, chronic obstructive lung disease | −6% (NS) | +12% (P < 0.05) |
McKenna et al., 2012, young compared to aged subjects | −0.7% (NS) | −24% (P < 0.05) |
Boon et al., 2012, spinal cord injury in human subject | −47% (P < 0.05) | −52% (P < 0.05) |
Chibalin et al., 2012, nicotine pretreatment in 190 g rats | 0% (NS) | −25% (P < 0.05) |