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+.

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.

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.

pHi determines rate of sodium transport in frog skin: results of a new method to determine pHi

1994 ◽  
Vol 266 (3) ◽  
pp. F367-F374 ◽  
Author(s):  
R. Rick
Keyword(s):  
Frog Skin ◽  
Nuclear Potential ◽  
Na Channel ◽  
Cytoplasmic Ph ◽  
Na Transport ◽  
Principal Cells ◽  
Weak Organic Acid ◽  
Transepithelial Na Transport ◽  
Cell Layers ◽  
Important Regulator

The pH of the isolated frog skin epithelium was determined on a cellular and subcellular level based on the distribution of a weak organic acid, 4-bromobenzoic acid. The indicator is detectable by X-ray microanalysis due to the presence of an element label. The results show that the pH of principal cells, but not the Na concentration, is closely correlated with the rate of transepithelial Na transport. Acidification leads to an inhibition of Na transport, regardless of whether the change was spontaneous or experimentally induced. Under the conditions of this study, the pH of principal cells was not well regulated. At a bath pH of 7.0, large pH differences between the cell layers were detectable. In mitochondria-rich cells, the pH was a function of the intracellular Cl concentration but not the Na transport rate. The cytoplasmic pH consistently exceeded the nuclear pH. The nuclear-cytoplasmic pH differential in principal cells amounted to 0.3 pH units, which is equivalent to a nuclear potential of -17 mV. The results support the view that the intracellular pH (pHi) is an important regulator of transepithelial Na transport. Regulation is primarily achieved at the level of the apical Na channel, making the Na influx the rate-limiting step in Na reabsorption.

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.

Transepithelial transport kinetics and Na entry in frog skin: effects of novobiocin

1976 ◽  
Vol 231 (6) ◽  
pp. 1866-1874 ◽  
Author(s):  
LJ Cruz ◽  
TU Biber
Keyword(s):  
Kinetic Parameters ◽  
Frog Skin ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Close Correlation ◽  
Transport Kinetics ◽  
Transepithelial Transport ◽  
Linear Component ◽  
Na Transport ◽  
Transepithelial Na Transport

Na+ entry across the outer surface of frog skin and transepithelial Na transport were studied simultaneously at different [Na] in either the presence or absence of novobiocin by direct measurements of J12 (unidirectional uptake) and Io (short-circuit current). J12 consisted of two components: one linear, the other saturable. The kinetic parameters of the saturating components in controls were close to the kinetic parameters of overall transepithelial transport (Jm12 = 1.68+/-0.13 mleq cm-2h-1; Io =1.80+/-0.14 mueq cm-2h-1. K12 = 6.02+/-1.27 mM;Kio=6.12+/-1.33 mM). Novobiocin significantly augmented net transepithelial Na transport by increasing J13. J31 remained unaffected. A 1:1 relationship between the saturating component of J12 and Io was observed in both treated and untreated skins at all [Na] tested. (Jm12Iom, k12, and Kio were significantly larger in treated skins, but despite very drastic changes in transport rates, a close correlation between kinetic parameters of entry step and transepithelial transport was maintained. This suggests that the kinetics of transepithelial transport may simply reflect those of the rate-limiting step: the Na entry across the outer barrier of the skin. The results indicate that the linear component of J12 is not involved in transepithelial transport kinetics.

Feeding induces lipid accumulation and increased Na+ transport in in vitro Necturus antrum

1990 ◽  
Vol 259 (6) ◽  
pp. G998-G1009
Author(s):  
M. J. Rutten ◽  
C. D. Moore ◽  
R. Delcore ◽  
L. Y. Cheung
Keyword(s):  
Electron Microscopy ◽  
Lipid Accumulation ◽  
Corn Oil ◽  
Short Circuit ◽  
Nile Red ◽  
Transepithelial Transport ◽  
Na Transport ◽  
Ussing Chambers ◽  
Lipid Granules

We investigated the effects of feeding on lipid accumulation and transepithelial transport using in vitro Necturus gastric antral mucosae. Antra from fed Necturi were examined for lipid accumulation using light, fluorescence, histochemical, and electron microscopy. Ussing chambers were used for measurement of potential difference (PD), transepithelial resistance (Rt), short-circuit current (Isc), and unidirectional fluxes of 22Na+ and [3H]mannitol. Light microscopy of antra from 2-day postfed animals showed many intracellular lipid granules in surface mucous epithelial cells. These granules could be distinguished from other intracellular organelles by their high affinity for osmium and the lipid fluorescent probe Nile red. Glycoprotein cytochemical staining showed these granules to be distinct from the epithelial cell mucous granules. Electron microscopy showed the lipid granules to be part of a membranous reticular network. Two-day postfed animals also had a approximately 3.5-fold increase in amiloride-sensitive Isc and PD, a decrease in Rt, and an increased luminal-to-serosal Na+ fluxes. Transepithelial [3H]mannitol fluxes were low and remained unchanged in both fasted and 2-day postfed animals. After 2 days of feeding, the PD and Isc began to decrease followed by a secondary increase in Rt. Feeding Necturi a corn oil diet did not induce the appearance of either cellular lipid or alterations in Isc but produced a transient increase in Rt. Our data show that feeding (goldfish) to Necturi causes an increase in both lipid accumulation and amiloride-sensitive Na+ transport in gastric antral cells.

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 Autoradiographic localization of tritiated ouabain-sensitive sodium pump sites in ion transporting epithelia.

1980 ◽  
Vol 28 (1) ◽  
pp. 72-77 ◽  
Author(s):  
S A Ernst ◽  
J W Mills
Keyword(s):  
Sodium Pump ◽  
Close Correlation ◽  
Electrolyte Transport ◽  
Na Transport ◽  
Ouabain Binding ◽  
Intact Tissue ◽  
Na Pump ◽  
Saturation Kinetics ◽  
A Cell ◽  
Heterogeneous Tissues

The freeze-dry autoradiographic method devised originally by Stirling (J Cell Biol 53:704, 1972) to localize Na+ pump sites with (3H)ouabain is reviewed. Biochemical, physiological, and autoradiographic data are discussed which establish that ouabain binding to intact tissue conforms to rigid criteria for high Na+ pump specificity. Among these are that glycoside binding exhibits saturation kinetics, ligand dependence, and close correlation with degrees of inhibition of Na+-K+-ATPase and Na+ transport. Moreover, localization of Na+ pump sites by this technique shows a cell and membrane specificity which mirrors that obtained by cytochemical and immunocytochemical methods. In addition to resolving cell-specific patterns of localization in heterogeneous tissues, the demonstration of Na+-K+-ATPase by these techniques indicates that Na+ pumps are distributed uniformly along plasmalemmal surfaces and are restricted to the basolateral interface in reabsorptive and secretory epithelia despite the opposing polarity of net transepithelial electrolyte transport.

scholarly journals Interaction of ouabain with the Na(+) pump in intact epithelial cells

1981 ◽  
Vol 88 (3) ◽  
pp. 637-643 ◽  
Author(s):  
JW Mills ◽  
ADC MacKnight ◽  
JA Jarrell ◽  
JM Dayer ◽  
DA Ausiello
Keyword(s):  
Epithelial Cells ◽  
Quantitative Measure ◽  
Free Medium ◽  
Na Transport ◽  
Pump Mode ◽  
Ouabain Binding ◽  
Na Pump ◽  
Phosphorylated Form ◽  
Binding Rate ◽  
Volume Recovery

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.

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.

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