Stimulation of the sodium pump by azide and high internal sodium: changes in the number of pumping sites and turnover rate.

1. The effects of 5 mM azide on [3H]ouabain uptake and 22Na efflux were determined. Both glycoside uptake and 22Na efflux were enhanced by azide. 2. Azide stimulated the Na pump in muscles whose pumping sites had been inhibited by ouabain and then transferred to a glycoside-free solution. This stimulation was observed before detecting any recovery of the initial pumping activity. 3. When both the resting and the azide-stimulated 22Na efflux had been blocked by ouabain, an additional exposure to azide, in a ouabain-free solution, had no further effects on 22Na efflux. 4. It is concluded that the increase in Na pumping caused by azide is due in part to an increase in the number of pumping sites. 5. [3H]ouabain binding was measured in muscles with different intracellular alkali cation concentrations. Variations in [Na]i from 15 up to 50 mM did not significantly affect the amount of glycoside bound. A substantial increase in binding occurred when [Na]i reached 70 mM. 6. It is proposed that the increase in Na extrusion that occurs during the recovery of Na loaded muscles mostly results from an increased turnover rate of the pump rather than from an increase in number of pumping sites.     

Localization of sodium pump sites in cat salivary glands.

1. Cat submandibular glands were perfused with Locke solution containing [3H]ouabain. In some experiments additional medium was given by retrograde intraductal injection. 2. [3H]ouabain binding sites were localized by light microscopical autoradiography and the findings compared with the electron micrographs. 3. The cells of the striated ducts were heavily labelled with [3H]ouabain, predominantly in the contraluminal parts. 4. At the acinar level moderate amounts of [3H]ouabain were found in relation to the plasma membranes of the demilunar cells. The central acinar cells were found to be virtually devoid of [3H]ouabain. 5. Electron microscopy revealed that the demilunar cells possessed long, tortuous intercellular spaces separated from the secretory canaliculi by junctional complexes. In contrast, the membranes between adjacent central acinar cells were fairly straight. 6. It is concluded that the striated ducts play a dominant role in the ductal reabsorption of sodium, and that the transport is probably mediated by a (Na+ + K+)-activated ATPase. The findings on the acinar level are in agreement with the view that the primary saliva is formed predominantly by the demilunar cells. The role of a (Na+ + K+)-activated ATPase in this process is unclear.     

The connexion between the ion-binding sites of the sodium pump.

1. A study has been made of K influx into human red blood cells in order to determine the influence of internal Na on the affinity of the Na pump for external K. Cells were prepared to contain minimal K and two Na concentrations (about 10 and 30 mueqiv/ml. cells) and incubated in solutions with a range of K concentrations. 2. In choline (Na-free) Ringer, activation of K influx by external K was hyperbolic. The Km for external K increased as the internal Na concentration was raised. The increase was greater than the increase in K influx with saturating external K. The Km for external K increased towards a limiting value as the internal Na concentration was raised. 3. In contrast, in Na-Ringer, the activation by external K was sigmoidal and the affinity for external K was independent of the internal Na concentration. 4. K influx was measured at two submaximal levels of external K with a range of internal Na. The affinity for internal Na fell as external K was raised. 5. The results suggest that in the absence of competition between Na and K on the same side of the membrane, there is a connexion between the ion-binding sites of the Na pump.     

An estimate of sodium/potassium pump activity and the number of pump sites in the smooth muscle of the guinea-pig taenia coli, using 3[H]ouabain

1. Binding of tritiated ouabain to the smooth muscle of the guinea-pig taenia coli showed two components, one saturable at lower glycoside concentrations and the other linear with increasing concentrations.2. The saturable component alone was affected by extracellular potassium concentrations. This component seems to be bound to sodium pumping sites, and when completely saturated binds 1.1 x 10(11) molecules per mg fresh wt. of tissue, or 250-300 molecules per square micron of membrane, assuming a volume:surface area ratio of 1.5 mum.3. Only a fraction of (42)K uptake by the cells can be blocked by ouabain at maximal concentrations. In normal Krebs solution two thirds can be blocked. The remaining one third is presumably passive uptake. The fraction blocked is reduced as the extracellular potassium concentration, and thus passive uptake, is increased.4. The amount of potassium pumped into the cells at various concentrations of extracellular potassium has been calculated. In normal Krebs solution the amount pumped in 45 min was 20.0 m-mole/kg fresh wt., and this was increased at higher potassium concentrations.5. On the assumption of a stoichiometry of 3Na: 2K, the pump sites in normal Krebs solution have a turnover rate of 1320 min(-1).6. Indirect calculations of sodium movements suggest that the sodium permeability may be about 0.9 x 10(-8) cm sec(-1) and the pump may generate a current of 0.9 x 10(-7) A cm(-2). This crossing an Ohmic membrane resistance of 30-60 kOmega cm(2) would be equivalent to a potential difference of 3-5 mV.     

The inheritance of intraerythrocytic sodium level

Intraerythrocytic sodium level (RBC Na) was measured on 1,800 normotensive members of 64 Utah pedigrees ascertained through hypertensive or normotensive probands, sibs with early stroke death, or brothers with early coronary disease. Likelihood analysis provided evidence that RBC Na was determined by four alleles at a single locus. Each allele was recessive to all alleles associated with a lower mean level. The four resultant distributions occurred in the frequencies: 0.8%, 89.3%, 9.7%, and 0.2% with corresponding means (mmol/1 RBC) of 4.32, 6.67, 9.06, and 12.19, respectively. The major locus explained 29.0% of the variance in RBC Na; polygenic inheritance explained another 54.6%. A higher frequency of the genotypes for high RBC Na in pedigrees when the proband was hypertensive than normotensive provided evidence that this major locus increases susceptibility to hypertension.     

“一肾一夹”高血压大鼠肾脏皮质钠泵α亚单位的基因表达

目的:探讨"一肾一夹(1k1c)"高血压大鼠肾脏皮质钠泵α亚单位各异构体基因表达的改变,为高血压发病机制的深入研究提供理论及实验依据。方法:分别应用RT-PCR及免疫组化技术,探讨1k1c高血压大鼠肾脏皮质钠泵α₁、α₂及α₃亚单位mRNA及蛋白水平基因表达的改变。结果: 正常大鼠肾脏皮质主要表达钠泵α₁亚单位,α₂与α₃亚单位表达较弱;在mRNA水平,1k1c高血压大鼠肾脏皮质钠泵α₁亚单位表达明显较强,α₂与α₃亚单位表达无改变,而在蛋白水平各亚单位基因表达与对照组比较均无明显差异。 结论: 1k1c高血压大鼠肾皮质钠泵α亚单位基因表达发生改变,这种改变可能参与该模型的血压升高机制。     

The number of sodium ion pumping sites in skeletal muscle and its modification by insulin.

1. [3H]ouabain binding by frog sartorius muscles shows at least two components: one linked to inhibition of the pump and another not related to transport inhibition. This is suggested by the finding that [3H]ouabain uptake continued to increase when (a) the glycoside concentration was increased beyond that causing maximum transport inhibition, and (b) exposure times longer than those required to produce full inhibition were used. 2. A number of 1600 pumping sites per mum2 of membrane was estimated considering only the cylindrical surface of the muscle. 3. Insulin stimulated the ouabain-sensitive components of 22Na efflux and 134Cs influx. It also increased [3H]ouabain binding to a level of 1-7 times the total resting value. The increases in [3H]ouabain binding and in 22Na efflux followed a similar relationship with respect to insulin concentration. 4. Insulin stimulated the Na pump in muscles whose pumping sites had been inhibited by ouabain and then transferred to a glycoside-free solution. This stimulation was observed before detecting any recovery of the initial pumping activity. 5. When both the resting and the insulin-stimulated 22Na efflux had been blocked by ouabain, an additional dose of insulin, in a ouabain-free solution, had no further effects on 22Na efflux. 6. The effects of insulin were unaffected by cycloheximide or by high concentrations of butyryl derivatives of cyclic AMP and cyclic GMP. 7. We conclude that there are two pools of Na pumping sites in muscle cells: one active and another inactive. Insulin unmasks the inactive pumping sites by a mechanism that is independent of protein synthesis, increases in intracellular [Na] or decreases in intracellular [K].     

The sensitivity of the sodium pump to external sodium

1. When red cells are incubated in potassium-free solutions, ouabain-sensitive sodium efflux is nearly absent with 5 mM-Na externally, but increases as the external sodium concentration is reduced from 5 mM to zero. This increase suggests that the transport mechanism is very sensitive to small amounts of sodium at the outside surface of the cell membrane. Further evidence for such sensitivity has been obtained from the effects of external sodium on the relation between potassium influx and external potassium concentration.2. With 5 mM-[K](o), potassium influx is rather insensitive to [Na](o) but at low potassium concentrations even low levels of sodium inhibit.3. With 140 mM-[Na](o) the potassium influx curve is S-shaped below 1 mM [K](o). At much lower sodium concentrations, the S-shaped region and the value of [K](o) for which potassium influx is half-maximal are both shifted progressively towards zero. At 10 muM-[Na](o), potassium influx is half maximal at 0.14 mM-[K](o) and the curve is close to a rectangular hyperbola down to 22 muM-[K](o); there seems to be a trace of inflexion at about 15 muM-[K](o).4. When [Na](o) is reduced from 5 mM to zero, removal of the inhibitory effect of external sodium ions on sodium: potassium exchange could lead to an increase in sodium efflux into nominally potassium-free solutions if these solutions did in fact contain traces of potassium. Such traces could arise by leakage from the cells, but, in a number of experiments, direct measurements showed that [K](o) was too low to account in this way for all of the observed ouabain-sensitive sodium efflux. A further reason for rejecting this explanation is that ouabain-sensitive potassium loss into nominally (Na+K)-free solutions was unaffected by adding 5 mM-Na. (A slight increase in ouabain-resistant loss was observed.)5. The ouabain-sensitive efflux of sodium into (Na+K)-free solutions therefore seems to represent a mode of behaviour of the transport mechanism distinct both from the sodium: potassium exchange that occurs under physiological conditions and from the sodium: sodium exchange that occurs in K-free, Na-rich media.     

The stoicheiometry of the sodium pump

1. When resealed ghosts containing adenosine triphosphate (ATP), magnesium and sodium were incubated in a medium containing potassium, ATP was hydrolysed vigorously by a ouabain-sensitive mechanism. If the ghosts contained potassium instead of or in addition to sodium, and the external solution contained sodium but no potassium, there was little ouabain-sensitive hydrolysis of ATP. As it is known that the ouabain-sensitive ATPase in fragmented ghosts requires both sodium and potassium ions, these results show that the ATPase is activated by potassium externally and by sodium internally, and suggest that the ions activating the ATPase are the ions that are transported.2. Resealed ghosts containing ATP, magnesium and sodium were incubated in sodium-free media containing potassium, with and without ouabain, and the rate of loss of sodium and rate of hydrolysis of ATP were measured. The hydrolysis of 1 molecule of ATP by the ouabain-sensitive mechanism was accompanied by the ouabain-sensitive loss of about 3 sodium ions.3. (24)Na and (42)K were used to measure sodium efflux and potassium influx in identical batches of fresh red cells under the same conditions and at the same time. Each flux was measured in the presence and absence of ouabain. The ratio (ouabain-sensitive sodium efflux)/(ouabain-sensitive potassium influx) was significantly greater than 1 (1.20 +/- 0.01 and 1.35 +/- 0.01 in two experiments). If a small fraction of the potassium influx represented a ouabain-sensitive potassium: potassium exchange, the ratio of the numbers of ions moved in the sodium: potassium exchange catalysed by the pump must have been even further from unity.4. Resealed ghosts containing [gamma-(32)P]ATP, magnesium, (24)Na and orthophosphate were incubated in balanced salt solutions with and without potassium and with and without ouabain. A comparison of sodium efflux, estimated from (24)Na loss, with ATP hydrolysis, estimated from the formation of [(32)P]orthophosphate, showed that the sodium:sodium exchange in a potassium-free medium was accompanied by little or no ouabain-sensitive hydrolysis of ATP.5. Experiments on intact red cells loaded with (24)Na showed that both sodium:sodium exchange in a potassium-free medium, and sodium:potassium exchange in a medium containing potassium, were partially inhibited by oligomycin (1-10 mug/ml.). Inhibition of the sodium:potassium exchange was not affected by raising the external potassium concentration.     

Induction of transporting sites in a sodium transporting epithelium.

1. Frogs (Rana temporaria) were bathed for 1 week in solutions containing 1-1 mM sodium chloride and either one or both of amiloride (10(-4)M) and spironolactone (10(-5r both of amiloride (10(-4) M) and spironolactone (10(-5) M). This procedure was designed to deplete the sodium transporting compartment of the skin epithelium of sodium, while at the same time antagonizing the effects of endogenous aldosterone. 2. After 1 week the skins were used in vitro to measure the level of sodium transport (short-circuit current) and the density of sodium entry sites in the mucosal surface of the epithelium ([14C]amiloride binding). 3. Sodium deprivation for 1 week caused approximately a doubling of both sodium transport and the density of sodium entry sites in the mucosal surface of the epithelium compared to control skins. 4. When the results for sodium deprived and control skins were pooled there was a highly significant correlation between the density of sodium entry sites and sodium transport. 5. Mechanisms by which sodium deprivation leads to an increase in the density of sodium entry sites are discussed.     

Intracellular sodium activity and the sodium pump in snail neurones

1. Recessed-tip Na(+)-sensitive micro-electrodes were used to measure [Na(+)](i) continuously in snail neurones for experiments lasting up to several hours. The average resting [Na(+)](i) in twenty-two cells was 3.6 mM.2. Inhibition of the Na pump by ouabain caused [Na(+)](i) to increase at an average rate of 0.54 m-mole/min. This corresponds to a passive influx of Na quantitatively similar to that observed in squid axons.3. Changing external K over the range 1-8 mM had little effect on [Na(+)](i), but K-free or 0.25 mM-K Ringer caused a rise in [Na(+)](i).4. Increasing membrane potential by up to 90 mV caused an increased influx of Na, but did not inhibit the pump.5. Reducing external Na caused a decrease in [Na(+)](i) but did not affect the pump rate at a given [Na(+)](i). The pump rate at low [Na(+)](i) was proportional to [Na(+)](i) minus a threshold value of about 1 mM.6. The Na pump appeared still to be electrogenic at subnormal rates of activity.7. It is concluded that, given sufficient external K, the rate of the Na pump depends principally on [Na(+)](i). Changes in external Na or membrane potential appear to affect the pump only indirectly, by changing the Na influx and thus [Na(+)](i).     

Dihydroouabain,a reversible inhibitor of the sodium pump in frog skin

In many studies of the sodium pump in epithelia, a readily reversible analog of ouabain would be most useful. This would enable studies of pump activity to be made under control and experimental conditions on the same tissue. Of three compounds examined on the basolateral membrane of the isolated epithelia of frog skin, dihydroouabain (DHO) had characteristics very similar to ouabain except that it was apparently much more reversible. DHO (1 mmol/l) inhibited short circuit current (Isc) and transepithelial Na flux (J ₁₃ ^Na ) in a fashion similar to ouabain. Isc was inhibited from 17.0±2.5 to 10.2±1.0 A/cm² in 2–4 min whileJ ₁₃ ^Na was decreased from 16.8±1.9 to 4.7±0.8 A/cm² in the same time interval. After 60 min of washout, Isc andJ ₁₃ ^Na recovered to about 70% of control values and were nearly equal. In another set of experiments, the washout of DHO and ouabain were compared directly on the same tissue. Sodium flux recovered four times faster after removal of DHO when compared to ouabain. Pretreatment of tissues with DHO prior to ouabain greatly increased the rate of Na flux recovery after washout of both drugs suggesting that DHO competes for ouabain sites. These data suggest that DHO can be used as a reversible analog for ouabain in studies of the Na pump in frog skin.     

Long-term effect of ouabain and sodium pump inhibition on a neuronal membrane

1. The long-term effects of ouabain on the membrane potential of the Anisodoris giant neurone (G cell) were examined in cells maintained for periods of up to 15 hr at 11-13 degrees C.2. In the presence of ouabain (5 x 10(-4)M), the membrane potential depolarized to a constant level for 1-4 hr, then hyperpolarized for 5-7 hr after which it gradually depolarized again.3. During the hyperpolarizing phase, after 6-8 hr in ouabain, [K](1) fell approximately 50%, [Na](1) increased 50-100% and the P(Na)/P(K) ratio decreased to 25% of its initial value.4. After 8 hr in ouabain the membrane conductance increased two- to fourfold. This increase was independent of temperature and membrane rectification.5. The K permeability (P(K)) was calculated from the constant field equation, and showed a fourfold increase after long-term treatment with ouabain. This rise in P(K) probably underlies the membrane hyperpolarization and the decrease in the P(Na)/P(K) ratio.6. It is suggested that inhibition of the Na(+) pump with ouabain causes a gradual rise in [Na](1) which secondarily leads to Ca(2+) uptake, an increase in [Ca](1), and thereby an increase in P(K).     

The sodium pump: A ghost story

Evidence that the sodium plus potassium activated adenosinetriphosphatase ((Na + K)-ATPase) is present and functions normally in a red blood cell ghost is summarised. The case is then argued that since ghost move neither sodium nor potassium against an electrochemical gradient, the (Na+ + K)-ATPase is not in itself sufficient to generate transmembrane gradients of sodium and potassium ions. If it is not sufficient in ghost, then it cannot be sufficient in intact cells, but most somehow work co-operatively with the cytoplasm. An alternative hypothesis to that of carrier-mediated transported is then proposed, and shown to be consistent with data on intact cells, membrane homogenates, ghosts, and membrane vesicles derived from bacteria.     

The localization of sodium transport sites in a forward pumping secretory system

Summary The standing gradient flow model proposed by Diamondet al. (1967) has been re-investigated mathematically. In sweat gland secretory epithelium the pumps can be distributed theoretically along the entire channel length. At maximal stimulation the concentration of the secreted product depends only on the hydraulic conductivity, on the solute pumping rate and on the outside concentration. The concentration of the secreted fluid is independent of channel length and radius and also independent of the diffusion coefficient. This makes the model more applicable to a great variety of tissue spaces of different shapes. The mathematical treatment also shows that a relationship exists between the magnitude of the hydraulic conductivity and the solute pumping rate. Furthermore, the postulated non-linear decrease in pump activity along the channel wall fits with experimental results.