scholarly journals On the distribution of Na+ pump sites in the frog skin

1977 ◽  
Vol 75 (3) ◽  
pp. 968-973 ◽  
Author(s):  
JW Mills ◽  
DR DiBona
Keyword(s):  
Binding Sites ◽  
Living Cell ◽  
Frog Skin ◽  
Cell Layer ◽  
Ouabain Binding ◽  
Ringer’S Solution ◽  
Na Pump ◽  
Stratum Spinosum ◽  
Sufficient Concentration ◽  
Sustained Increase

Exposure of the outside of the isolated frog skin to a Ringer's solution, made hypertonic by the addition of mannitol, causes a rapid and sustained increase in transepithelial permeability through a structural distortion-a focal blistering-of the "tight" junctions of the outermost living cell layer. [(3)H]ouabain, used as an autoradiographic marker for the Na+-pump (Na+-K+-adenosine triphosphatase), is usually unable to penetrate the frog skin from the outside solution, but when added to a hypertonic mannitol- Ringer's solution in the outside bath it readily penetrates the epithelium, presumably through the opened shunt pathway. Radioautographic analysis of [(3)H]ouabain binding sites revealed that most of ouabain enters from the outside solution binds to the sites on the cell membranes of the stratum spinosum, as was the case when it was applied from the inside bath in an earlier study. The outer living cell layer, the first to be exposed to ouabain, does not appear to be the major site for the Na+-pump, and therefore, is not likely to be responsible for most of the active pumping of Na+. This result demonstrates that previous failure to show a high density of Na+-pump sites on the cells of the outermost layer, when [(3)H]ouabain was applied from the inside solution, was not due to the inability of the marker to reach these cells at a sufficient concentration to reveal all pump sites. These results provide further support for a model of Na+-transport across the frog skin which distributes the active pump step on the inward facing membranes of all living cells.

scholarly journals Localization of Na+-pump sites in frog skin.

1977 ◽  
Vol 73 (1) ◽  
pp. 88-110 ◽  
Author(s):  
J W Mills ◽  
S A Ernst ◽  
D R DiBona
Keyword(s):  
Binding Sites ◽  
Living Cell ◽  
Frog Skin ◽  
Transepithelial Transport ◽  
Na Transport ◽  
Ouabain Binding ◽  
Ussing Chambers ◽  
Na Pump ◽  
Cell Layers ◽  
Grain Distribution

The localization of Na+-pump sites (Na+-K+-ATPase) in the frog skin epithelium was determined by a freeze-dry radioautographic method for identifying [3H]ouabain-binding sites. Ventral pelvic skins of Rana catesbeiana were mounted in Ussing chambers and exposed to 10(-6) M [3H]ouabain for 120 min, washed in ouabain-free Ringer's solution for 60 min, and then processed for radioautography. Ouabain-binding sites were localized on the inward facing (serosal) membranes of all the living cells. Quantitative analysis of grain distribution showed that the overwhelming majority of Na+-pump sites were localized deep to the outer living cell layer, i.e., in the stratum spinosum and stratum germinativum. Binding of ouabain was correlated with inhibition of Na+ transport. Specificity of ouabain binding to Na+-K+-ATPase was verified by demonstrating its sensitivity to the concentration of ligands (K+, ATP) that affect binding of ouabain to the enzyme. Additional studies supported the conclusion that the distribution of bound ouabain reflects the distribution of those pumps involved in the active transepithelial transport of Na+. After a 30-min exposure to [3H]ouabain, Na+ transport declined to a level that was significantly less than that in untreated paired controls, and analysis of grain distribution showed that over 90% of the ouabain-binding sites were localized to the inner cell layers. Furthermore, in skins where Na+ transport had been completely inhibited by exposure to 10(-5) M ouabain, the grain distribution was identical to that in skins exposed to 10(-6) M. The results support a model which depicts all the living cell layers functioning as a syncytium with regard to the active transepithelial transport of Na+.

Observations on ouabain binding and membrane phosphorylation by the sodium pump

1976 ◽  
Vol 193 (1112) ◽  
pp. 217-234 ◽  
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Kidney Cortex ◽  
Relative Molecular Mass ◽  
Type B ◽  
Ouabain Binding ◽  
Na Pump ◽  
Pump Mechanism ◽  
Morphological Differences ◽  
Equilibrium Dissociation

A study has been made of ouabain binding and the formation of phosphoprotein from ATP and inorganic phosphate (P i ) with plasma membranes from rabbit and guinea-pig kidney cortex. The aim of the work was first to see whether apparently conflicting results in the literature arise from membranes being prepared by different methods and, secondly, to evaluate the results in relation to the Na pump mechanism. Three different methods were used to prepare membranes, types A, Au and B. The preparations differed markedly when ouabain binding was supported by Mg alone both in the amount bound and in the affinity. Mgdependent binding was influenced by 1 mM P i but the extent of stimulation varied according to the preparations. The main effect of P i was to decrease the equilibrium dissociation constant marginally for type A membranes but eightfold for type B membranes. In contrast, the maximum number of binding sites was little affected. The membrane affinity for ouabain in relation to Mg and P i therefore depended on the method of preparation. In the reaction with Mg-ATP, type Au and B membranes were both phosphorylated to about the same extent. On the other hand, they reacted differently with P i , type B membranes being phosphorylated (in the presence of Mg and ouabain) to the same extent as with ATP, whereas under the same conditions, type Au membranes gave only 15 % of the phosphorylation found with ATP. The phosphoprotein, however formed, whether from ATP or P i , or type Au or type B membranes, migrated in the same way on gel electrophoresis to give a relative molecular mass of approximately 90000. With each preparation, over a tenfold range of ATPase activity, there was a constant value of 1.2 in the ratio of the maximum phosphorylation by ATP compared with the maximum number of ouabain-binding sites. These results show that membranes prepared in different ways exhibit some consistent properties of the Na pump but also striking anomalies. In view of likely morphological differences in the preparations, it is concluded that the inconsistent features, notably the responses to Mg and P i , are an unreliable guide to the pump mechanism.

Effect of methacholine on Na+ pump activity and ion content of dispersed avian salt gland cells

1981 ◽  
Vol 241 (1) ◽  
pp. R77-R86
Author(s):  
S. R. Hootman ◽  
S. A. Ernst
Keyword(s):  
Binding Sites ◽  
Gland Cell ◽  
Salt Gland ◽  
Ion Content ◽  
Ouabain Binding ◽  
Gland Cells ◽  
Na Pump ◽  
Pump Activity ◽  
Number Of Binding Sites ◽  
Dissociated Cells

The effects of the cholinergic agonist methacholine chloride (MCh) on cellular ion content and Na+ pump activity of dissociated duck salt gland cells were studied. Dispersed salt gland cells regulate intracellular ion levels in a ouabain-sensitive manner. MCh (0.5 mM) caused no detectable change in cell Na+ levels over the first 10 min of exposure of cells to the agonist but elicited decreases of 23 and 13%, respectively, in intracellular Cl- and K+ content. The rate of turnover of salt gland cell plasmalemmal Na+ pumps, as measured by [3H]ouabain binding to the dissociated cells, was markedly stimulated by 0.5 mM MCh, although the total number of binding sites at equilibrium remained unchanged. Replacement of medium Na+ with choline abolished the MCh-stimulated increase in ouabain binding but had no effect on the rate of glycoside binding in the absence of the agonist. Substitution of Cl- in the medium by NO3-, SO42-, or benzene sulfonate- reduced the stimulated component of Na+ pump turnover by 85-90%. Addition of 1 mM furosemide to the medium abolished the increase in ouabain binding and ouabain-sensitive oxygen consumption observed after exposure of salt gland cells to MCh. These data are consistent with the hypothesis that cholinergic stimulation of salt gland cells triggers a Cl--dependent uptake of Na+, which elicits a compensatory increase in Na+ pump turnover. In addition, the decrease in cellular Cl- content caused by MCh suggests that the agonist either directly or indirectly mediates an efflux of Cl- from the cells.

scholarly journals Up-regulation of sodium pump activity in Xenopus laevis oocytes by expression of heterologous β1 subunits of the sodium pump

1991 ◽  
Vol 279 (2) ◽  
pp. 329-336 ◽  
Author(s):  
G Schmalzing ◽  
S Gloor ◽  
H Omay ◽  
S Kröner ◽  
H Appelhans ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Binding Sites ◽  
Sodium Pump ◽  
Binding Capacity ◽  
Polyclonal Antiserum ◽  
Scatchard Analysis ◽  
Xenopus Laevis Oocytes ◽  
Ouabain Binding ◽  
Na Pump

Recent evidence suggests that the beta subunit of the Na+ pump is essential for the alpha subunit to express catalytic activity and for assembly of the holoenzyme in the plasma membrane. We report here that injection into Xenopus laevis oocytes of cRNAs specific for beta 1 subunit isoforms of the Na+ pump of four species (Torpedo californica, chicken, mouse and rat) causes a time-dependent increase in the number of ouabain-binding sites, both in the plasma membrane and in internal membranes. Expression of the beta 1 subunit of the Na+ pump of mouse and rat in the oocytes could be substantiated by immunoprecipitation using a polyclonal antiserum against the mouse beta 1 subunit. Scatchard analysis in permeabilized cells disclosed that the affinity for ouabain is unchanged after expression of each of the beta 1 subunits. A proportional increase in ouabain-sensitive 86Rb+ uptake indicates that the additionally expressed ouabain-binding sites on the cell surface represent functional Na+ pumps. The findings support the concept of Geering. Theulaz, Verrey, Häuptle & Rossier [(1989) Am. J. Physiol. 257, C851-C858] that beta 1 subunits expressed in oocytes associate with an excess of endogenous alpha subunits of the Na+ pump to form a hybrid enzyme. In addition, all of the beta 1 isoforms investigated in the present study were also capable of combining with the co-expressed alpha 1 subunit of the Torpedo Na+ pump to produce a functional enzyme. Injection of cRNA encoding for the Torpedo alpha 1 subunit alone had no effect on the ouabain-binding capacity of the surface and intracellular membranes of the oocyte.

Ouabain binding in tadpole ventral skin. II. Localization of Na pump sites

1987 ◽  
Vol 253 (3) ◽  
pp. R410-R417 ◽  
Author(s):  
D. H. Robinson ◽  
J. W. Mills
Keyword(s):  
Transport Properties ◽  
Binding Sites ◽  
Na Transport ◽  
Ouabain Binding ◽  
Na Pump ◽  
Homogenous Distribution ◽  
Ventral Skin

By use of [3H]ouabain autoradiography, the distribution of ouabain binding sites in the tadpole ventral skin and the change in the pattern of binding during metamorphosis were examined. In the tadpole the greatest grain density, and hence density of Na pumps, was found in the outer one-third of the epidermis. The pattern of binding changed at (Anat. Rec. 94: 7-23, 1946) stage 20 to a homogenous distribution. At stage 21 the highest grain density was in the middle third of the epidermis. The adultlike pattern of binding, with the highest density in the serosal two-thirds of the epidermis, was noted at stages 22 and 23. The average grain density through the entire tissue is approximately 2.4-fold higher in the stage 23 animal than in the tadpole with the significant increase in density occurring between stages 20 and 21. Since the adult Na transport characteristics are also not fully developed until stage 22 (Biochim. Biophys. Acta 692: 455-461, 1982), it is proposed that the development of the Na-pump characteristics is coincident with the development of other adult transport properties.

Food intake alters muscle protein gain with little effect on Na(+)-K(+)-ATPase and myosin isoforms in suckled rats

1997 ◽  
Vol 272 (5) ◽  
pp. R1461-R1471 ◽  
Author(s):  
M. L. Fiorotto ◽  
T. A. Davis
Keyword(s):  
Food Intake ◽  
Binding Sites ◽  
Nutrient Intake ◽  
Muscle Protein ◽  
Fiber Type ◽  
Muscle Size ◽  
Myosin Isoforms ◽  
Ouabain Binding ◽  
Rat Pups ◽  
Dna Contents

Biochemical maturation accompanies the rapid accretion of skeletal muscle in early life. We wished to determine whether changes in muscle protein accretion, induced by variations in food intake, altered the biochemical maturation of the soleus and the extensor digitorum longus (EDL) muscles. Rat pups were suckled in litters of 4, 10, or 16 to induce differences in food intake. At 21 days of age, muscle protein and DNA were quantitated and biochemical maturation was assessed from measurement of [3H]ouabain-binding site abundance and myosin isoform composition. Differences in food intake produced a twofold range in body and muscle weights and protein and DNA contents. Protein accretion was more sensitive to nutrient intake in the soleus than in the EDL. Serum 3-5,3'-triiodothyronine (T3) and insulin concentrations decreased with a reduction in food intake. Total ouabain-binding sites were not altered in either muscle and were independent of muscle size. Differences in myosin isoform composition were more pronounced for the soleus than the EDL, but were relatively small in magnitude. These results demonstrate that, whereas postnatal muscle protein accretion and circulating hormone concentrations are sensitive to food intake, the biochemical maturation is resilient. The immature muscle does not exhibit the fiber type-specific responses to malnutrition typical of mature muscle.

scholarly journals The Na,K-ATPase-Dependent Src Kinase Signaling Changes with Mesenteric Artery Diameter

2018 ◽  
Vol 19 (9) ◽  
pp. 2489 ◽  
Author(s):  
Lin Zhang ◽  
Christian Aalkjaer ◽  
Vladimir Matchkov
Keyword(s):  
Smooth Muscle ◽  
Binding Sites ◽  
Isometric Force ◽  
Src Kinase ◽  
Ouabain Binding ◽  
Arterial Diameter ◽  
Small Arteries ◽  
Functional Changes ◽  
High Affinity ◽  
Different Diameters

Inhibition of the Na,K-ATPase by ouabain potentiates vascular tone and agonist-induced contraction. These effects of ouabain varies between different reports. In this study, we assessed whether the pro-contractile effect of ouabain changes with arterial diameter and the molecular mechanism behind it. Rat mesenteric small arteries of different diameters (150–350 µm) were studied for noradrenaline-induced changes of isometric force and intracellular Ca2+ in smooth muscle cells. These functional changes were correlated to total Src kinase and Src phosphorylation assessed immunohistochemically. High-affinity ouabain-binding sites were semi-quantified with fluorescent ouabain. We found that potentiation of noradrenaline-sensitivity by ouabain correlates positively with an increase in arterial diameter. This was not due to differences in intracellular Ca2+ responses but due to sensitization of smooth muscle cell contractile machinery to Ca2+. This was associated with ouabain-induced Src activation, which increases with increasing arterial diameter. Total Src expression was similar in arteries of different diameters but the density of high-affinity ouabain binding sites increased with increasing arterial diameters. We suggested that ouabain binding induces more Src kinase activity in mesenteric small arteries with larger diameter leading to enhanced sensitization of the contractile machinery to Ca2+.

scholarly journals [3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

1992 ◽  
Vol 40 (6) ◽  
pp. 771-779 ◽  
Author(s):  
A A Maki ◽  
D G Baskin ◽  
W L Stahl
Keyword(s):  
Nervous System ◽  
Rat Brain ◽  
Cardiac Glycoside ◽  
Binding Sites ◽  
Quantitative Autoradiography ◽  
Affinity Binding ◽  
Ouabain Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Kd Values

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.

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