Effects of prostaglandin A2 on Na+, K+-ATPase activity in basolateral plasma membrane of rat intestine in vitro

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 Δ^5,6 and Δ^10,11 structure of prostaglandin A₂ may be necessary for the inhibition of the Na⁺,K⁺-ATPase activity.     

Ca2+ mediates the effect of inhibition of Na+-K+-ATPase on the basolateral K+ channels in the rat CCD

We investigated the effect ofinhibiting Na⁺-K⁺-ATPase on the basolateral18-pS K⁺ channel in the cortical collecting duct (CCD) ofthe rat kidney. Inhibiting Na⁺-K⁺-ATPase withstrophanthidin decreased the activity of the 18-pS K⁺channel and increased the intracellular Ca²⁺ to 420 nM.Removal of extracellular Ca²⁺ abolished the effect ofstrophanthidin. When intracellular Ca²⁺ was raised with 5 µM ionomycin or A-23187 to 300, 400, and 500 nM, the activity of the18-pS K⁺ channel in cell-attached patches fell by 40, 85, and 96%, respectively. To explore the mechanism ofCa²⁺-induced inhibition, the effect of 400 nMCa²⁺ on channel activity was studied in the presence ofcalphostin C, an inhibitor of protein kinase C, or KN-93 and KN-62,inhibitors of calmodulin-dependent kinase II. Addition of calphostin Cor KN-93 or KN-62 failed to block the inhibitory effect of highconcentrations of Ca²⁺. This suggested that the inhibitoryeffect of high concentrations of Ca²⁺ was not mediated byprotein kinase C or calmodulin-dependent kinase II pathways. To examinethe possibility that the inhibitory effect of high concentrations ofCa²⁺ was mediated by the interaction of nitric oxide withsuperoxide, we investigated the effect of 400 nM Ca²⁺ onchannel activity in the presence of 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) orN-nitro-L-arginine methyl ester.Pretreatment of the tubules with 4,5-dihydroxy-1,3-benzenedisulfonicacid or N-nitro-L-arginine methylester completely abolished the inhibitory effect of 400 nMCa²⁺ on channel activity. Moreover, application of4,5-dihydroxy-1,3-benzenedisulfonic acid reversed the inhibitory effectof strophanthidin. We conclude that the effect of inhibitingNa⁺-K⁺-ATPase is mediated by intracellularCa²⁺ and the inhibitory effect of high concentrations ofCa²⁺ is the result of interaction of nitric oxide with superoxide.

     

Kinetic studies on Na+/K+-ATPase and inhibition of Na+/K+-ATPase by ATP

Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na+/K+-ATPase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (deltarHm) but also the kinetic data (Km and Vmax). At 310.15K and pH 7.4, the molar reaction enthalpy (deltarHm) was measured as -40.514 +/- 0.9kJmol(-1). The Michaelis constant (Km) was determined to be 0.479 +/- 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na+/K+-ATPase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a Km value of about 0.5mM and free ATP was a competitive inhibitor with a Ki value of 0.253 mM.     

Stimulation of p-nitrophenylphosphatase activity of Na+/K+-ATPase by NaCl with oligomycin or ATP

It is known that the addition of NaCl with oligomycin or ATP stimulates ouabain-sensitive and K+-dependent p-nitrophenylphosphatase (pNPPase) activity of Na+/K+-ATPase. We investigated the mechanism of the stimulation. The combination of oligomycin and NaCl increased the affinity of pNPPase activity for K+. When the ratio of Na+ to Rb+ was 10 in the presence of oligomycin, Rb+-binding and pNPPase activity reached a maximal level and Na+ was occluded. Phosphorylation of Na+/K+-ATPase by p-nitrophenylphosphate (pNPP) was not affected by oligomycin. Because oligomycin stabilizes the Na+-occluded E1 state of Na+/K+-ATPase, it seemed that the Na+-occluded E1 state increased the affinity of the phosphoenzyme formed from pNPP for K+. On the other hand, the combination of ATP and NaCl also increased the affinity of pNPPase for K+ and activated ATPase activity. Both activities were affected by the ligand conditions. Oligomycin noncompetitively affected the activation of pNPPase by NaCl and ATP. Nonhydrolyzable ATP analogues could not substitute for ATP. As NaE1P, which is the high-energy phosphoenzyme formed from ATP with Na+, is also the Na+-occluded E1 state, it is suggested that the Na+-occluded E1 state increases the affinity of the phosphoenzyme from pNPP for K+ through the interaction between alpha subunits. Therefore, membrane-bound Na+/K+-ATPase would function as at least an (alphabeta)2-diprotomer with interacting alpha subunits at the phosphorylation step.     

Effect of prostaglandin F2 alpha on Na+-K+-ATPase activity in luteal membranes

Slices of rat corpora lutea (CL) incubated with prostaglandin F2 alpha (PGF2 alpha) in Krebs-Hensenleit (K-H) Ringer solution showed a decrease in Na+-K+-ATPase activity after 60 min of incubation. However, PGF2 alpha in vitro did not alter Na+-K+-ATPase activity of isolated luteal membrane fractions. Following PGF2 alpha-induced in vivo luteal regression, reduction of Vmax and elevation of the activation energy above transition temperature of the lipid phase of the membrane occurred without changes in Km, optimum pH and transition temperature. These results suggest that reduction of Na+-K+-ATPase activity after PGF2 alpha treatment may be due to reduction in the number of enzyme molecules or to masking of the active site of the enzyme without any change in enzyme characteristics. In addition, a change in membrane-bound enzyme activity may be an early step in PGF2 alpha-induced luteolysis.     

Na+-ATPase is a different entity from the (Na+ + K+)-ATPase in rat kidney basolateral plasma membranes

In this work, we present evidence in agreement with the hypothesis that there exist two Na+-stimulated ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells: (1) (Na+ + K+)-ATPase activity, which is inhibited by ouabain and by treating the membranes with trypsin, is insensitive to furosemide and reaches maximal activity upon treatment with SDS at an SDS/protein ratio of 1.6; (2) the Na+-ATPase activity, which is insensitive to ouabain and to trypsin treatment, is inhibited by furosemide and reaches maximal activity upon treatment with SDS at an SDS/protein ratio of 0.4.     

Development of (Na+-K+)-ATPase in rat cerebrum: correlation with Na+-dependent phosphorylation and K+-paranitrophenylphosphatase.

Abstract— The activities of (Na⁺ K⁺)-ATPase and its proposed partial reactions, K ⁺-pNPPase and Na ⁺-dependent phosphorylation, all increase tenfold relative to microsomal protein between 5 days prior to birth and 60 days postnatally in NaI-treated rat cerebral microsomes, and all reach half of their adult values between the fifth and tenth postnatal day. These increases are concurrent with the most rapid changes in cerebral wet weight. Increases in the amount of the related phosphorylatable polypeptide during development. as estimated by densitometry of Coomassie-stained polyacrylamide gels after electrophoresis of constant amounts of microsomal protein dissolved in sodium dodecylsulfate, parallel the increments in levels of Na ⁺-dependent phosphorylation. The fraction of total phosphorylation that is Na ⁺-dependent increases steadily during development. suggesting a precursor role for some of the Na ⁺-independent fraction. The results are consistent with a single biosynthetic control for the enzymatic sites critical to the partial reactions of (Na ⁺-K ⁺)-ATPase. No changes in turnover number or affinity for substrate or ligands were found during development. Little similarity was noted among the age-related changes of Mg ²⁺ -ATPase activity. Mg ²⁺ -paranitrophenylphosphatase activity, and Na⁺-independent phosphorylation levels. The most rapid changes in (Na⁺-K⁺)-ATPase take place during the period corresponding to glial proliferation and neuronal arborization.     

番茄碱对人红细胞膜Na+-K+-ATPase活性影响的研究

以低渗法从新鲜健康人红细胞中制得膜Na^ -K^ -ATPase,研究了番茄碱(tomatine)X~人红细胞膜Na^ -K^ -ATPase活性的影响。实验结果表明,反应体系中的tomatine浓度低于1mmol／L时,对不依赖钙调蛋白(CAM)激活的Na^ -K^ -ATPase(称之为基本酶活)影响不大,浓度达1mmol／L时,该酶活性仍保持在95％左右;而在此浓度范围内,tomatine对依赖CaM的Na^ -K^ -ATPase有明显的抑制作用,其IC50值为0．16mmol／L．说明tomatine对膜酶Na^ -K^ -ATPase活性的影响可能是通过阻断CaM激活的途径而起作用,从而为进一步研究番茄碱的作用机制奠定了基础。     

Effects of batrachotoxin on electroplax Na + -K + -ATPase and levels of ATP in rat muscle

—Batrachotoxin (BTX) in low concentrations (20 nm ) depolarizes electrically excitable membranes (Albuquerque , Daly and Witkop , 1971). At these levels, BTX does not inhibit Na⁺-K⁺-ATPase. At much higher concentrations (60 μm ) BTX partially inhibits Na⁺-K⁺-ATPase from electroplax of Electrophorus electricus. In contrast to inhibition by cardiac glycosides, the inhibition of Na⁺-K⁺-ATPase by batrachotoxin is not antagonized by KCl. BTX had no effect on ATP levels in stimulated nerve muscle preparations at the time when sustained contracture was initiated by the drug. Phosphocreatine levels were decreased and levels of glucose-6-phosphate and 6-phosphogluconate were increased, while levels of fructose-1,6-diphosphate and α-ketoglutarate were unchanged. It is concluded that the inhibition of Na⁺-K⁺-ATPase or lowering of ATP levels by BTX is not significantly involved in the membrane depolarization produced by the toxin.     

Mechanism of apical K+ channel modulation in principal renal tubule cells. Effect of inhibition of basolateral Na(+)-K(+)-ATPase

The effects of inhibition of the basolateral Na(+)-K(+)-ATPase (pump) on the apical low-conductance K+ channel of principal cells in rat cortical collecting duct (CCD) were studied with patch-clamp techniques. Inhibition of pump activity by removal of K+ from the bath solution or addition of strophanthidin reversibly reduced K+ channel activity in cell-attached patches to 36% of the control value. The effect of pump inhibition on K+ channel activity was dependent on the presence of extracellular Ca2+, since removal of Ca2+ in the bath solution abolished the inhibitory effect of 0 mM K+ bath. The intracellular [Ca2+] (measured with fura-2) was significantly increased, from 125 nM (control) to 335 nM (0 mM K+ bath) or 408 nM (0.2 mM strophanthidin), during inhibition of pump activity. In contrast, cell pH decreased only moderately, from 7.45 to 7.35. Raising intracellular Ca2+ by addition of 2 microM ionomycin mimicked the effect of pump inhibition on K+ channel activity. 0.1 mM amiloride also significantly reduced the inhibitory effect of the K+ removal. Because the apical low-conductance K channel in inside-out patches is not sensitive to Ca2+ (Wang, W., A. Schwab, and G. Giebisch, 1990. American Journal of Physiology. 259:F494-F502), it is suggested that the inhibitory effect of Ca2+ is mediated by a Ca(2+)-dependent signal transduction pathway. This view was supported in experiments in which application of 200 nM staurosporine, a potent inhibitor of Ca(2+)- dependent protein kinase C (PKC), markedly diminished the effect of the pump inhibition on channel activity. We conclude that a Ca(2+)- dependent protein kinase such as PKC plays a key role in the downregulation of apical low-conductance K+ channel activity during inhibition of the basolateral Na(+)-K(+)-ATPase.     

Development of (Na+ -K+)-ATPase in rat hindbrain: increments in parallel with Na+-dependent phosphorylation and K+-pnitrophenylphosphatase.

Rat hindbrain NaI-enriched microsomal (Na⁺-K⁺)-ATPase activity, K⁺-pNPPase activity, and Na⁺-dependent steady-state phosphorylation levels all increase approx 10-fold relative to microsomal protein between 5 days prenatally and 60 days postnatally. These activities, as well as the mean wet weight of the hindbrain, are at half of their 60 day values shortly after the 10th postnatal day. For all ages, these hindbrain activities average over twice those found in the forebrain in a companion paper (Bertoni & Siegel , 1978). Increases during development in the amount of the related phosphorylatable polypeptide, estimated by densitometry of stained polyacrylamide gels containing fixed amounts of microsomal protein dissolved in SDS, are in agreement with increases in steady state levels of Na⁺-dependent phosphorylation. The fraction of total phosphorylation that is Na⁺-dependent rises steadily during development consistent with, but not obligatorily due to, a conversion of some of the previously Na⁺-independent portion. Mg2⁺-ATPase and Mg²⁺ -pNPPase activities and steady-state Na⁺,-independent phosphorylation levels do not increase in parallel during development. These observations add further support to the proposed partial reaction scheme for (Na⁺- K⁺)-ATPase. The major increments in (Na ⁺-K⁺)-ATPase occur simultaneously with the deposition of specialized plasma membranes, particularly in the molecular layer of the cerebellum, as described in previous studies of rat hind brain.     

Uptake of aminoglycoside antibiotics into brush-border membrane vesicles and inhibition of (Na+ + K+)-ATPase activity of basolateral membrane

The effects of aminoglycoside antibiotics on plasma membranes were studied using rat renal basolateral and brush-border membrane vesicles. 3',4'-Dideoxykanamycin was bound to the basolateral membrane and brush-border membrane vesicles. They had a single class of binding sites with nearly the same constant, and the basolateral membrane vesicles had more binding sites than those of the brush-border membrane. Dideoxykanamycin B was transported into the intravesicular space of brush-border membrane vesicles, but not into that of basolateral membrane vesicles. The (Na+ + K+)-ATPase activity of the plasma membrane fraction prepared from the kidney of rat administered with dideoxykanamycin B intravenously decreased significantly. Aminoglycoside antibiotics entrapped in the basolateral membrane vesicles inhibited (Na+ + K+)-ATPase activity, but those added to the basolateral membrane vesicles externally failed to do so. The activity of (Na+ + K+)-ATPase was non-competitively inhibited by gentamicin. It is thus concluded that aminoglycoside antibiotics are taken up into the renal proximal tubular cells across the brush-border membrane and inhibit the (Na+ + K+)-ATPase activity of basolateral membrane. This inhibition may possibly disrupt the balance of cellular electrolytes, leading to a cellular dysfunction, and consequently to the development of aminoglycoside antibiotics' nephrotoxicity.     

Effect of external ATP on the plasma membrane permeability and (Na+ +K+)-ATPase activity of mouse neuroblastoma cells

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 X 10(-6)M. Neuro-2A cells contain (3.5 +/- 0.7) X 10(5) ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7 +/- 0.4) X 10(-20) mol K+/min per copy of (Na+ +K+)-ATPase at room temperature.     

S-adenosyl-L-methionine modulates Na+ + K+-ATPase activity in rat colonic basolateral membranes.

Rat colonic basolateral membranes were incubated with S-adenosyl-L-[methyl-3H]methionine (0.3 mM) at 37 degrees C for 2 h at pH 9.0. This resulted in an increase in the specific activity of Na+ + K+-ATPase by 60%. Kinetic parameter analysis revealed a 2-fold increase in the Vmax. of this enzymatic activity, whereas the Km for ATP was unchanged. The methylation inhibitor S-adenosyl-L-homocysteine (2 mM) significantly reduced these S-adenosyl-L-methionine-stimulated increases in specific activity and the Vmax. of Na+ + K+-ATPase. S-Adenosyl-L-methionine treatment of basolateral membranes was also found to significantly increase the fluidity of these preparations, as assessed by steady-state fluorescence polarization techniques using the fluorophore 1,6-diphenyl-1,3,5-hexatriene; S-adenosyl-L-homocysteine (2 mM) again markedly reduced this S-adenosyl-L-methionine-induced increase in fluidity. While transmethylation reactions involving phospholipids, non-polar lipids and proteins were all found to exist in rat colonic basolateral membranes, based on a number of observations, the results of the present studies suggest that transmethylation of membrane phospholipids, but not membrane non-polar lipids or proteins, influenced the fluidity of basolateral membranes which, in turn, modified Na+ + K+-ATPase activity in these membranes.     

Specific inhibition of ouabain sensitive and K+-dependent p-nitrophenylphosphatase by polyamines.

The ouabain sensitive and K⁺-dependent p-nitrophenyl-phosphatase was inhibited by polyamines. The order of effectiveness was spermine spermidine putrescine = cadaverine. The half maximum inhibition concentration of spermine was approximately 0.03 mM and 0.8 mM in the presence of 0.5 mM and 3.0 mM KCl in the reaction mixtures, respectively. Basic amino acids and hydroxylamine inhibited slightly. Other amines such as glycine and histamine were without effect. Spermine did not inhibit other membrane bound phosphatases, such as glucose-6-phosphatase, 5′-nucleotidase, alkaline phosphatase and ouabain insensitive p-nitrophenylphosphatase activity at pH 7.5