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.

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.

Homeoviscous adaptation and thermal compensation of sodium pump of trout erythrocytes

1991 ◽  
Vol 260 (5) ◽  
pp. R916-R924 ◽  
Author(s):  
R. S. Raynard ◽  
A. R. Cossins
Keyword(s):  
Cold Acclimation ◽  
Scatchard Analysis ◽  
Transport Activity ◽  
Turnover Number ◽  
Ouabain Binding ◽  
Homeoviscous Adaptation ◽  
Membrane Order ◽  
Na Pump ◽  
Pump Activity ◽  
Cholesterol Supplementation

The effects of thermal acclimation of trout on the transport activity and turnover number of the erythrocyte Na+ pump have been determined. Na+ pump activity was estimated by measuring the ouabain-sensitive K+ influx in Na(+)-loaded cells and the number of active pumps determined by Scatchard analysis of [3H]ouabain binding and by correlation of ouabain binding with pump inhibition. Cold acclimation was associated with an increase in pump activity of up to 60%, although the furosemide-sensitive and residual fluxes were unaffected. The number of ouabain binding sites was similar in both acclimation groups at approximately 21,000-23,000 sites/cell. This means that cold acclimation induced an increase in the transport turnover number of pump molecules from approximately 6 to 9 s-1. Cold acclimation was also associated with a decrease in membrane order as indicated by steady-state fluorescence polarization of the membrane probe, 1,3-diphenyl-1,3,5-hexatriene, with a homeoviscous efficacy of 25-41%. That membrane order may influence pump transport activity is supported by experiments on cholesterol supplementation, which caused both an increase in membrane order and a decrease in pump turnover number. The degree of pump compensation was dependent on the season, with greatest responses in the late spring and declining responses through to winter. By contrast, changes in membrane order were observed throughout the year. Expression of pump activity and erythropoiesis may vary throughout the seasonal cycle in complex ways that confuse the direct comparison study of cellular properties in a heterogeneous population of cells.

Effect of growth in low-Na+ medium on transport sites in cultured heart cells

1986 ◽  
Vol 250 (1) ◽  
pp. C32-C39 ◽  
Author(s):  
D. Kim ◽  
T. W. Smith
Keyword(s):  
Heart Cells ◽  
Dependent Manner ◽  
Concomitant Increase ◽  
Ouabain Binding ◽  
Na Pump ◽  
Ventricular Cells ◽  
Pump Activity ◽  
Cultured Heart Cells

Increases in intracellular Na+ concentration ([Na+]i) lead to an increase in the number of Na+ pump sites in the cell membrane. To investigate further the role of [Na+]i in the regulation of Na+-K+-ATPase sites, we studied the effect of reduced [Na+]i on the number of Na+ pump sites and Na+-K+ pump activity using spontaneously beating cultured chick ventricular cells. Cells incubated in medium containing 60, 80, 100, or 140 mM Na+ for 24 or 48 h showed an extracellular [Na+]-dependent alteration in cellular Na+ content. The number of Na+ pump sites identified by [3H]ouabain binding binding declined with decreasing levels of Na+ in the medium in a time-dependent manner over 48 h, with a concomitant increase in cellular Na+ content. Verapamil (1 microM) or tetrodotoxin (1 microM) significantly reduced cellular Na+ content by 30 min of exposure and the number of Na+ pump sites by 48 h of incubation. Na+ pump activity determined from the ouabain-sensitive 42K+ uptake rate was significantly reduced in cells grown in low Na+ for 48 h, as was pump capacity, determined in Na+-loaded cells. These results support the view that [Na+]i exerts a long-term modulating effect on the number of physiologically functional Na+ pump sites.

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+.

Increased Na(+)-K+ pump number and decreased pump activity in soleus muscles in SHR

1994 ◽  
Vol 267 (3) ◽  
pp. C836-C844 ◽  
Author(s):  
J. G. Pickar ◽  
R. C. Carlsen ◽  
A. Atrakchi ◽  
S. D. Gray
Keyword(s):  
Fatigue Resistance ◽  
Soleus Muscle ◽  
Functional Capacity ◽  
Spontaneously Hypertensive Rat ◽  
Specific Binding ◽  
Resting Membrane Potential ◽  
Ouabain Binding ◽  
Na Pump ◽  
Age Related ◽  
Pump Activity

We have previously demonstrated electrophysiological and contractile abnormalities in soleus muscles of the spontaneously hypertensive rat (SHR). The age-related decrease in force and fatigue resistance observed in SHR muscles may be produced by alterations in sarcolemmal ion conductance and/or Na+ pump function. The experiments reported in the present paper were designed to assess the functional capacity of the Na+ pump in 6- to 8- and 24- to 28-wk-old SHR and Wistar-Kyoto (WKY) soleus muscles and to correlate pump activity with Na+ pump number and binding affinity ([3H]ouabain binding). Functional capacity was determined by measuring the change in resting membrane potential (RMP) of soleus muscle fibers in response to agents that stimulate (epinephrine and insulin) or inhibit (ouabain) the pump and by measuring maximum ouabain-suppressible 86Rb+ uptake in Na(+)-loaded muscles. Na+ pump number and affinity were quantified by determining the specific binding of [3H]ouabain in soleus muscle slices. SHR soleus muscles contain a greater number of Na+ pump sites (ouabain binding sites) than are present in age-matched WKY muscles but also experience a significant decrease in pump activity with age. SHR may upregulate pump number in response to the significantly higher intracellular Na+ concentration found in soleus muscles at all the ages examined. The apparent reduction in pump capacity with age may play a major role in the observed age-related decrease in SHR soleus force and fatigue resistance.

scholarly journals Ouabain binding and coupled sodium, potassium, and chloride transport in isolated transverse tubules of skeletal muscle.

1979 ◽  
Vol 74 (3) ◽  
pp. 335-349 ◽  
Author(s):  
Y H Lau ◽  
A H Caswell ◽  
M Garcia ◽  
L Letellier
Keyword(s):  
Binding Sites ◽  
Chloride Transport ◽  
Slow Phase ◽  
Ouabain Binding ◽  
Fast Phase ◽  
Highly Active ◽  
Tubular Lumen ◽  
Number Of Binding Sites ◽  
T Tubules ◽  
Low K

The affinity and number of binding sites of [3H]ouabain to isolated transverse (T) tubules were determined in the absence and presence of deoxycholate. In both conditions the KD was approximately 53 nM while deoxycholate increased the number of binding sites from 3.5 to 37 pmol/mg protein. We concluded that the ouabain binding sites were located primarily on the inside of the isolated vesicle and that the vesicles were impermeable to ouabain. ATP induced a highly active Na+ accumulation by the T tubules which increased Na+ in the T tubular lumen by almost 200 nmol/mg protein. The accumulation had an initial fast phase lasting 2-3 min and a subsequent slow phase which continued for at least 40 min. The rate of the initial fast phase indicated a turnover number of 20 Na+/s. The Na+ accumulation was prevented by monensin but was unaffected by valinomycin. Ouabain did not influence Na+ uptake, but digitoxin inhibited it. At low K+ the accumulation of Na+ was reduced 3.7-fold below the value at 50 mM K+. 86Rb, employed as a tracer to detect K+, showed a first phase of K+ release while Na+ was accumulated. After 2-3 min, K+ was reaccumulated while Na+ continued to increase in the lumen. T tubules accumulated Cl- on addition of ATP. This suggested that ATP initiated an exchange of Na+ for K+ followed by uptake of Na+ and K+ accompanied by Cl-.

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.

scholarly journals Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

1977 ◽  
Vol 75 (1) ◽  
pp. 74-94 ◽  
Author(s):  
SA Ernst ◽  
JW Mills
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Salt Gland ◽  
Physiological Data ◽  
Secretory Cells ◽  
Ouabain Binding ◽  
Salt Glands ◽  
Cytochemical Localization ◽  
Secretory Epithelium ◽  
Basolateral Plasma Membrane

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.

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