Studies on terminal differentiation of rat renal proximal tubular cells in culture: ouabain-sensitive K and Na transport

We have studied the ontogeny of Na-K ATPase-mediated Na and K transport in rat renal proximal tubular cells using electron probe analysis. The cells were cultured from kidneys of 10-day-old, young (Y), and 40-day-old, adult (A) rats. Before an experiment cells were Na-loaded and K-depleted by incubation in K-free medium. The maximum rate of ouabain-sensitive Na and K transport was measured after reactivating the Na-K pump by transferring the cells from K-free medium to medium containing 5 mM K. In cells cultured for 2 days, ouabain-sensitive Na and K net initial transport rates were significantly higher in A than in Y cells. Between 2 and 4 days in culture there was a significant decrease in ouabain-sensitive Na and K transport rates in both Y and A cells. From 2 to 4 days of culture there was, in Y but not in A cells, a significant decrease in K/Na ratio. The decrease in K/Na ratio was due to a significant increase in Na content. After incubation in K-free medium, net intracellular solute accumulation was observed in A and Y cells cultured for 4 days but not in A and Y cells cultured for 2 days. In conclusion, maximal Na- and K-pump-mediated transport increases during terminal differentiation. This increase can be measured in cells cultured for 2 days. With longer time in culture, Na-K pump activity decreases and the difference between A and Y cells is not measurable.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of ouabain and ethacrynic acid on the intracellular sodium and potassium concentrations in renal medullary slices incubated in cold potassium-free ringer solution and re-incubated at 37 degrees C in the presence of external potassium.

1. The cells in slices cut from the renal outer medulla of normally hydrated adult rats were loaded with Na and depleted of K by incubation for up to 100 min in cold iso-osmolal K-free Ringer containing 180 mM-Na. There was a continuous net cellular water loss during this time; an inverse linear relationship existed between water content and intracellular Na concentration. 2. The original intracellular Na and K concentration were restored following 60 min re-incubation in warm Ringer (37 degrees C) containing 5-9 mM-K. Restoration of cellular water content was incomplete after re-incubation for up to 120 min. 3. During incubation in cold K-free Ringer the presence of 1 mM ouabain did not affect cellular Na uptake or K and water loss. Ethacrynic acid, 1 mM, completely blocked cellular Na uptake and water loss, without affecting the intracellular K concentration at 100 min. When ouabain and ethacrynic acid were present together water loss was also prevented but intracellular Na concentration rose slightly by 100 min. 4. During re-incubation in warm K-containing Ringer 1 mM ouabain inhibited Na extrusion completely for up to 60 min while only partially preventing K uptake and further depressing the level of cellular hydration. Ouabain in the presence of 1 mM ethacrynic acid had similar effects on intracellular Na and K concentrations, but raised the level of intracellular water above that of cells in control slices. 5. Ethacrynic acid alone, 1 mM, did not interfere with Na extrusion or K uptake, but also raised intracellular water above control values. 6. The results obtained are discussed in relation to (a) the nature of the preparation used, (b) the possible membrane transport processes occurring and their known or suggested sensitivity to ouabain and ethacrynic acid, (c) the mechanisms which may be responsible for cell volume maintenance in the medulla.     

Involvement of Na and Cl in ouabain-induced cell swelling in thick ascending limb of rat kidney

Changes in the volume of isolated segments of rat medullary thick ascending limb (MAL) were studied by a photographic technique, after tubule incubation in isotonic solutions in the absence or presence of ouabain and/or K. When segments were incubated at 30°C in NaCl solution, their volume increased by 75% after removal of external K, and by 170% after removal of external K plus addition of 1 mmol/l ouabain. At steady state, tubular volume was a function of the external K concentration. Resting volume was obtained with external K concentrations higher than 0.1 and 1.0 mmol/l in the absence and presence of ouabain respectively. When MAL samples were incubated in isotonic K-free Na₂SO₄ or K-free choline Cl solution, their volume per unit of length was similar to that determined in NaCl medium, but there was no swelling after the addition of ouabain. The ouabain-induced swelling was shown to depend on both the Na and Cl concentrations in the incubate (apparentK _m of 87 and 80 mmol/l for Na and Cl respectively). Swollen tubules recovered their resting volume when ouabain, Na or Cl was removed from the incubation medium. Recovery of resting volume was also observed after addition of K into the incubation medium. These observations indicate that rat MAL cell volume is the result of coupled passive net fluxes of Na and Cl, which depend on the respective electrochemical gradients for Na or Cl across the cell membranes and the Na-pump activity which continuously extrudes Na.     

Effect of sodium and sodium-substitutes on the active ion transport and on the membrane potential of smooth muscle cells

1. The changes of the ion content and of the membrane potential of taenia coli cells have been studied during prolonged exposure to Na-deficient solutions containing either Li or choline.2. A K-free solution containing either 71 mM-Na-71 mM-Li or 71 mM-Na-71 mM choline causes a slower loss of cellular K than a 142 mM-Na solution. In both these Na-deficient solutions the membrane hyperpolarizes to about -100 mV for periods up to 6 hr. This hyperpolarization is partially abolished by 2 x 10(-5)M ouabain.3. Replacing all extracellular Na by Li and maintaining 5.9 mM-K causes a fast loss of all Na and a progressive replacement of K by Li. These changes of the intracellular ion content are accompanied by a depolarization of the cells, suggesting that intracellular Li cannot substitute for Na in activating the ion pump.4. Exposing K-depleted cells to a K-free 71 mM-Na-71 mM-Li solution results in a ouabain sensitive transport of Na and Li against their electro-chemical gradient.5. The K-uptake by K-depleted cells from a solution containing 0.59 mM-K is increased by reducing [Na](o) to half of its normal value. This finding indicates that external Na inhibits the active Na-K exchange.6. In Na-enriched tissues half of the Na efflux is due to a ouabain insensitive Na-exchange diffusion. If Li is used as a Na substitute, the Na-Li exchange compensates for the diminution of the Na-exchange diffusion unless ouabain is added.     

Changes of intracellular sodium and potassium ion concentrations in isolated rat superior cervical ganglia induced by depolarizing agents

1. Na and K contents of isolated rat superior cervical ganglia were measured by flame photometry, and intracellular Na and K concentrations ([Na](i) and [K](i)) calculated using Li and (35)SO(4) to determine extracellular space (e.c.s.).2. Resting concentrations after 1-2 hr incubation at 25 degrees C in normal Krebs solution were: [Na](i), 19.8 +/- 0.9 m-mole (kg cell water)(-1); [K](i), 192.7 +/- 2.8 m-mole (kg cell water)(-1) (mean +/- S.E. of mean of thirty-five ganglia). Correction for losses during e.c.s. measurement gave 22 mM [Na](i) and 207 mM [K](i) as probable fresh concentrations.3. Carbachol (180 muM for 4 min) increased [Na](i) by 47.8 +/- 2.9 m-mole (kg cell water)(-1) and decreased [K](i) by 54.6 +/- 4.3 m-mole (kg cell water)(-1). Maximal exchange with carbachol or nicotine (at approximately 1 mM for 4 min) amounted to 80-100 m-mole (kg cell water)(-1). On washing with Krebs solution containing 2.5 mM hexamethonium recovery of ionic concentrations occurred with a rate constant of 0.3-0.4 min(-1).4. Restitution of ganglionic Na and K after carbachol was inhibited by washing with K-free solution, and slowed by ouabain (0.14 mM), cyanide (2 mM) or cooling (Q(10) 2.7 between 17 and 27 degrees C).5. Equilibrium potentials for Na and K (E(Na), E(K)) at rest were calculated to be +49 and -88 mV. At a membrane potential (E(m)) of -70 mV, the permeability ratio P(Na):P(K) was calculated at 0.04:1 (assuming P(Cl):P(K) < 0.1).     

Microcalorimetric determination of energy expenditure due to active sodium-potassium transport in the soleus muscle and brown adipose tissue of the rat.

1. The resting heat production rate (E) of soleus muscles from young rats and brown adipose tissue from adult rats was measured by means of a perfusable heat flux microcalorimeter in the absence and presence of ouabain. In the soleus muscle, the acute response of E to ouabain was compared with the ouabain-suppressible components of 22Na-efflux and 42K-influx. 2. In standard Krebs-Ringer bicarbonate buffer, ouabain (10(-3)M) induced an immediate but transient decrease in E of around 5%. Both in muscle and adipose tissue this was followed by a progressive rise in heat production rate. 3. When the medium was enriched with Mg (10 mM), ouabain produced a sustained decrease in E of the same magnitude as in the standard medium and the secondary rise was less marked or abolished. Under these conditions, in the soleus muscle, ouabain inhibited E by 5% (i.e. by 1-76 +/- 0-22 mcal.g wet wt.-1.min-1), 22Na-efflux by 58% (0-187 +/- 0-013 micronmole. g wet wt.-1.min-1) and 42K-influx by 34% (0-132 +/- 0-028 micronmole. g wet wt.-1.min-1). 4. When the muscles were loaded with Na by pre-incubation in K-free Mg-enriched medium, the addition of K (3mM) induced an immediate ouabain-suppressible increase in E of 2-98 +/- 0-33 mcal. g wet wt.-1.min-1 and a concomitant stimulation of 22Na-efflux of 0-388 +/- 0-136 micronmole. g wet wt.-1.min-1. 5. Maximum Na/ATP ratios for the active Na-K transport process were computed, with no assumption as to the in vivo free energy of ATP hydrolysis. These were 2-1, 1-9 and 2-3 under the conditions described in paragraphs (2), (3) and (4) respectively. 6. The calculated reversible thermodynamic work associated with active Na-K transport corresponded to 34% of the measured ouabain-induced decrease in E. On the premise that the maximum efficiency of the cellular energy conservation processes is 65%, this estimate indicates that the minimum energetic efficiency of ATP utilization by the active Na-K transport process in mammalian muscle is 52%.     

Uptake of ( 3 H)ouabain and Na pump turnover rates in cells cultured in ouabain

1. The binding of [(3)H]ouabain to fresh Girardi and Hela cells and to those cultured in low concentrations of ouabain for 24 hr has been measured.2. Fresh cells bind 1.6-2.2 x 10(6) molecules of ouabain(*) per cell from K-free Krebs, but less than 0.3 x 10(6) molecules from 15 mM-[K](o) Krebs. The ouabain(*) binds with a t((1/2)) of about 8 min from K-free 2 x 10(-7)M ouabain(*) and is released with a t((1/2)) of about 20 hr. Cells in a poor condition probably exchange ouabain more quickly.3. Cells incubated in ouabain(*) for 24 hr bind ouabain in amounts dependent on the [ouabain] and the external [K]. At the highest [ouabain] used the total amount bound exceeds that bound by fresh cells. Lowering [K](o) in the medium increases the maximum ouabain which is bound.4. Cells incubated in ouabain(*) for 24 hr bind an additional amount of ouabain when exposed to 2 x 10(-7)M ouabain(*) in K-free Krebs.5. There is a close relationship between the% of the total ouabain bound in 24 hr and the% inhibition of the Na efflux suggesting that this ouabain is bound to the Na pumps.6. Radiochromatography of the counts recovered from the cells showed that it migrated to the same place as the applied [(3)H]ouabain. The wash-off rate of ouabain bound to cells during incubation is similar to that from fresh cells, both tests suggesting that the ouabain exists in the same state in the cells.7. The number of Na ions extruded per pump is constant at about 60 sec(-1) in fresh cells and those pre-incubated in ouabain.8. The total ouabain bound by the cells is closely related to the [Na](i) in cells pre-incubated in ouabain but is unaffected by it in fresh cells where [Na](i) is raised acutely.9. These results are compatible with the hypothesis that partial blocking of Na pumps leads to the production of more pumping sites by the cell.     

Nucleotide requirements for sodium-sodium exchange catalysed by the sodium pump in human red cells

1. Resealed red cell ghosts containing a variety of nucleoside triphosphates, or a mixture of tri- and diphosphates, were allowed to lose (24)Na into a 10 mM-K medium or a K-free high-Na medium in the presence and absence of ouabain.2. Only ATP supported a ouabain-sensitive Na:K exchange in the 10 mM-K medium, or a ouabain-sensitive Na:Na exchange in the K-free medium.3. Because of ATPase and adenylate kinase activity, it is difficult to control the levels of ATP and ADP inside resealed red cell ghosts. Some control was achieved by incorporating into the ghosts an ATP regenerating system consisting of creatine phosphate and creatine kinase.4. Measurements of (24)Na efflux from ghosts prepared in this way showed that ADP is required for the ouabain-sensitive exchange of Na that occurs in K-free high-Na media, but not for the normal exchange of Na for K that occurs in K-containing media.5. The significance of these findings is discussed.     

Ouabain-sensitive ion fluxes in the smooth muscle of the guinea-pig''s taenia coli.

1. Tissues with raised intracellular Na levels, produced by incubation in K-free media, were used throughout. The uptake of 42K by these Na-loaded tissues was followed for 10 min in the presence and absence of 1-37 X 10(-4) M ouabain, this being sufficient to inhibit Na pumping maximally. Subtraction of the uptake seen in the presence from that seen in the absence of ouabain gave estimates of the pumped ouabain-sensitive K uptake. 2. In Na-free (MgCl2) medium this depended on the [K]0 in a sigmoidal fashion with a half maximal [K]0 for activation of some 4mM. The maximal uptake of K was 3 m-mole/kg.min corresponding to a transmembrane flux of some 12-5 p-mole. cm-2.sec-1. 3. In the presence of Na the K activation curve became more obviously sigmoid and higher concentrations of K were needed to achieve a given active K influx. The results were well fitted by assuming that Na and K competed for two identical, non-interacting sites on the external pump face. 4. Addition of K during the efflux of 24Na into a Na-free (MgCl2) medium led to an increased rate of tracer loss. The magnitude of this increase depended on the [K] used in a hyperbolic fashion and it was abolished by addition of ouabain. The [K] causing half-maximal activation of ouabain-sensitive Na efflux was in the order of 1-2 mM. 5. When the [K] in the uptake media was 1-5 mM; Na, Li, Rb and Cs all inhibited ouabain-sensitive K uptake, the order of effectiveness being Rb greater than Cs greater than Na greater than Li. With a E1TKA10 OF 0-15 MM low concentrations of Cs and Rb were shown to stimulate K uptake. Such an effect is predicted by assuming two ion binding sites on the pump's outer face, and that the pump can translocate mixtures of K and either Rb or Cs...     

A note of the mechanism by which inhibitors of the sodium pump accelerate spontaneous release of transmitter from motor nerve terminals.

1. The actions of 0-1 mM ouabain and of K-free Ringer have been examined at the frog neuromuscular junction. 2. After a delay of more than 30 min, ouabain produces an increase in the miniature end-plate potential (m.e.p.p.) frequency. This increase occurs unchanged in Ca-free Ringer containing 1 mM-EGTA and is therefore unlikely to be due to an entry of Ca into the motor nerve terminals. 3. If the nerve to the preparation is stimulated repetitively in Ca-free Ringer containing 0-1 mM ouabain and 1 mM-EGTA the response of the m.e.p.p. frequency depends on the timing of the tetanus relative to the beginning of the ouabain treatment. 4. During the first 30 min of exposure to ouabain, the tetanus produces a small, transient increase in the m.e.p.p. frequency similar to that which occurs before ouabain is present. After about 30 min the same tetanus produces large, irreversible increases in the m.e.p.p. frequency. 5. Superfusion of an end-plate with K-free Ringer causes an immediate exponential rise in the m.e.p.p. frequency that is unaffected by the presence of external Ca ions. On replacing the normal K of the Ringer (2 mM) the m.e.p.p. frequency recovers quickly to its original value. 6. Late in an exposure to 0-1 mM ouabain the m.e.p.p. frequency becomes extremely sensitive to changes in the external Na concentration, [Na]o. Reducing [Na]o increases the m.e.p.p. frequency. The sensitivity to [Na]o is independent of external Ca ions or whether the isotonic substitute for NaCl is LiCl or sucrose. 7. It is suggested that the spontaneous release of transmitter is facilitated, in some way, by the changes in the monovalent cation content of the nerve terminals that result from blocking the Na-K exchange pump. The Na sensitivity of the m.e.p.p. frequency that develops simultaneously can be explained if a Na-dependent Ca efflux system is present in the membrane of the presynaptic terminals.     

An electrogenic component of resting potential in rabbit ventricular muscle?

The resting potential and the intracellular Na and K concentrations (Nai, Ki) were determined at several extracellular K concentrations (Ko) between 0.5 and 18 mM and after inhibition of the sodium pump with 0.5 microM ouabain. Exposure to low Ko (0.5 mM) produced a transient hyperpolarization (from -80 to -100 mV) followed by a depolarization that led to a stable potential of -60 mV within 25 min. Similar potential levels were observed in the presence of ouabain regardless of the Ko/Ki ratio. Intracellular sodium increased at Ko < 5 mM, whereas Ki rose at Ko less than or equal to 1.0 mM. Because of the large decrease of Ki at Ko = 0.5 mM, Ko/Ki was the same at 0.5 and 1 mM. However, the resting potentials at the steady state differed by 50 mV at these concentrations. A PNa/PK of 0.032 for the control conditions was obtained with the Mullins-Noda equation using 2.5 as the Na-K coupling ratio. This PNa/PK value yielded a Goldman potential of -69 mV; so we estimated that electrogenic sodium extrusion contributed -10 mV to the resting potential. The size of the electrogenic potential increased as Ko was lowered from 5 to 1 mM. This finding suggests that the control of the Na-K coupling ratio may be independent of the mechanism that controls the pumping rate.     

Alterations in monovalent cation transport in Sindbis virus-infected chick cells

Influx experiments using the potassium tracer 86Rb+ indicated that the activity of the Na+K+ ATPase, or sodium pump, was reduced 40-50% as a consequence of Sindbis virus infection of avian fibroblasts. The inhibition of this ouabain-sensitive, active transport system temporally correlated with a decrease in the intracellular K+ concentration and the termination of cellular protein synthesis. By contrast, the rate of influx facilitated by the furosemide-sensitive (Na+K+Cl-) cotransport system was only slightly depressed. Efflux experiments indicated that no alterations in the relative rate of nonspecific permeability or "leakage" of K+ could be detected in chick cells infected by Sindbis virus. The amount of [3H]ouabain bound to Sindbis virus-infected cells paralleled the reduction in Na+K+ ATPase activity. These binding studies revealed no difference in the number of Na+ pump sites. The Km of ouabain binding, however, increased approximately 3.5-fold in the virus-infected cells. No change in the apparent affinity of the Na+ pump for K+ could be detected, yet the Vmax for ouabain-sensitive K+ transport was decreased. These experiments suggest that a reduction in Na+K+ ATPase turnover results in the altered intracellular monovalent cation levels found in Sindbis virus-infected chick cells.     

The distribution of chloride ions in the smooth muscle cells of the guinea-pig's taenia coli

1. The intracellular Cl concentration of taenia coli cells, determined by an analytical procedure and by an extrapolation procedure, has a value between 60 and 73 m-mole/l. cell water.2. This concentration is too high to be explained by a passive distribution. The discrepancy could be due to a binding of Cl in the intracellular or extracellular compartment or to an active uptake of Cl by the cells.3. Determination of the activity coefficient for Cl in homogenates of smooth muscle did not support the hypothesis of binding of Cl ions.4. The efflux of (36)Cl from taenia coli cells was not affected by foreign anions. After 1 hr exposure to a Cl-free solution, the tissues contained less than 1 m-mole of Cl/kg wet wt., even if Cl had been replaced by a slowly penetrating anion. Because the intracellular cation concentration remained constant, it has to be assumed that new anionic groups can be formed in the cells.5. The intracellular Cl concentration decreases during exposure to ouabain or to K-free solution. The uptake seems therefore to be linked to the uptake of K through the Na pump.6. Exposure to K-free solution increases the K permeability of the membrane. Under the same experimental conditions the Cl permeability of the membrane increases as long as K is leaking out of the cells.7. The anions in the external solution exert an important influence on the K permeability of the membrane. NO(3) and I cause a small increase of the permeability and large anions such as benzenesulphonate, propionate or pyroglutamate cause a pronounced decrease of this permeability.     

ATP hydrolysis associated with an uncoupled sodium flux through the sodium pump: evidence for allosteric effects of intracellular ATP and extracellular sodium

1. A method has been developed for regenerating [gamma(32)P]ATP of constant specific activity within resealed red cell ghosts, and for measuring its hydrolysis. The method may be used to follow the hydrolysis of ATP at concentrations down to 1 muM, and for periods long enough for the ATP at these very low concentrations to turn over several hundred times.2. Using this method we have been able to show that the ;uncoupled' efflux of Na caused by the Na pump when resealed red cell ghosts are incubated in (Na + K)-free media is associated with a hydrolysis of ATP. The stoicheiometry is roughly 2-3 Na ions expelled per molecule of ATP hydrolysed.3. Measurements of ATP hydrolysis and Na efflux as functions of intracellular ATP concentration have shown that uncoupled Na efflux, and its associated ATP hydrolysis, are saturated at intracellular ATP concentrations in the region of 1 muM.4. Measurement of ATP hydrolysis as a function of ATP concentration in resealed ghosts incubated in a K-containing medium gave a complicated activation curve suggesting the involvement of high-affinity (K(m)ca. 1 muM) and low-affinity (K(m)ca. 100 muM) sites.5. When resealed ghosts containing about 1 muM-ATP were incubated in a Na-free or in a high-Na medium, the addition of K to the medium reduced the rate of ouabain-sensitive ATP hydrolysis.6. Ouabain-sensitive ATP hydrolysis in resealed ghosts incubated in K-free choline media was inhibited by external Na at low concentrations (K(i) < 1 mM), but this inhibition was reversed as the external Na concentration was further increased.7. The results show that uncoupled Na efflux may be thought of as the transport mode associated with Na-ATPase activity, just as Na-K exchange is the transport mode associated with (Na + K)-ATPase activity. The significance of the differences between uncoupled Na efflux and Na-ATPase activity, on the one hand, and Na-K exchange and (Na + K)-ATPase activity, on the other, is discussed.     

Vanadate stimulates the pumped movements of Na in skeletal muscle

We have measured the effects of concentrations of vanadate ranging between 0.01 and 10 mM on the²²Na efflux of frog sartorius muscles. The addition of vanadate had no effects when concentrations lower than 0.5 mM were used; higher concentrations increased Na efflux. The increase was abolished by the addition of ouabain (10^–5M). In muscles pretreated with ouabain vanadate did not modify Na efflux. The stimulatory effects of vanadate on Na efflux were also observed in Na-free solutions indicating that the effux of vanadate was not caused mainly either by an increase in the exchange of Na for Na or by an increase in Na entry into the muscle. We also examined the effects of vanadate on muscles immersed in solutions containing 20 mM K⁺; both vanadate and increased K⁺ produced stimulations of Na efflux that were additive. Similarly when the effects of vanadate and insulin were measured on the Na efflux of the same muscle, additive effects were found. As the ouabain-sensitive Na efflux in frog muscle is generally agreed to be due to the activity of the Na-K ATPase, our findings suggest that the net effect of vanadate in intact muscle cells is an increase in the activity of the Na pump. Since vanadate affects many enzymes it is quite possible that the stimulatory action is not due to a direct effect on the Na-K ATPase but may be mediated through an intermediary step. Regardless of the specific mechanism, it is evident that, our results as well as other findings in the literature, strongly indicate that Na pumping by intact cells can be increased by vanadate administration. Hence it is not justified to attribute the physiological modifications caused by vanadate administration to blockade of the Na-K ATPase unless the attribution is justified by specific experimental evidence.     

Functional Na+/K+ pump in rat dorsal root ganglia neurons.

Steady-state Na+/K+ pump current (Ip) in isolated adult rat dorsal root ganglia neurons was studied to determine if the plasma membrane Na+/K+ pump activity is uniform across the population of dorsal root ganglia neurons. Cells were voltage-clamped at -40 mV and holding current (Ih) was recorded using whole-cell patch-clamp techniques under conditions that stimulate the Na+/K+ pump (60 mM intracellular Na+ and 5.4 mM extracellular K+). Ip was defined as the 1 mM ouabain-sensitive fraction of Ih. Data suggest the existence of three subpopulations of dorsal root ganglia neurons having mean steady-state Ip densities of 1.6+/-0.1, 3.8+/-0.2 and 7.5+/-0.4 pA/pF. Neurons with small Ip had an average soma perimeter of 95+/-3 microm, while neurons with medium and large Ip density had significantly larger soma sizes (131+/-8 and 141+/-7 microm, respectively). Neurons with a large Ip density had a significantly lower specific membrane resistance (Rm; mean 4.0+/-0.3 kohm x cm2) than neurons with medium or small Ip density (19+/-6 and 31+/-6 kohm x cm2, respectively). Regardless of these differences, in all groups of neurons Ip had a low sensitivity to ouabain (Ip half inhibition by ouabain was observed at 80-110 microM). These data suggest that the Na+/K+ pump site density and/or its activity is not uniform throughout the dorsal root ganglia neuron population; however, this non-uniformity does not appear to relate to the functional expression of the different alpha isoforms of the Na+/K+ pump. The major functional Na+/K+ pump in the dorsal root ganglia neuron plasma membrane appeared to be the low ouabain affinity (alpha1) isoform.     

Movements of labelled sodium ions in isolated rat superior cervical ganglia

1. Isolated rat superior cervical ganglia were incubated in Krebs solution containing (24)Na and carbachol for 4 min at 25 degrees C. They were then washed at 3 degrees C for 15 min to remove extracellular (24)Na and the efflux of residual intracellular (24)Na stimulated by warming to 25 degrees C.2. During the 15 min wash at 3 degrees C desaturation curves became exponential with a rate constant of 0.012 +/- 0.001 min(-1) (n = 24). This was assumed to represent loss of intracellular (24)Na, and initial uptake of (24)Na was calculated therefrom by back-extrapolation to zero wash-time. After 4 min in (24)Na + 180 muM carbachol intracellular [(24)Na] so calculated was 61.6 +/- 3.1 mM (n = 18), representing 83% labelling of intracellular Na. In the absence of carbachol intracellular [(24)Na] was 10.0 +/- 0.5 mM, representing 49% labelling. Extracellular Na was labelled by > 90% after 4 min in (24)Na. The apparent rate constant for washout of extracellular (24)Na was 0.6 min(-1) at 3 degrees C and 0.95 min(-1) at 25 degrees C.3. The loss of the residual intracellular (24)Na during temperature stimulation was interpreted quantitatively in terms of an exponential decline of the bulk of intracellular (24)Na with an extrusion rate constant of 0.39 +/- 0.1 min(-1) (n = 18), efflux being delayed by passage through the extracellular space with an effective rate constant of 0.8-1.2 min(-1).4. The peak rate constant (k(C)) for the desaturation curve at 25 degrees C was 0.35 +/- 0.01 min(-1). An Arrhenius plot of log k(C)/T degrees K(-1) yielded a two-stage linear regression with a transition at 20 degrees C. Activation energies of 8 and 31 kcal. mole(-1) were calculated above and below this transition respectively.5. Omission of K from the 25 degrees C temperature-stimulating solution reduced k(C) by 62%. The K-sensitive component of extrusion rate constant was a hyperbolic function of [K](e) with half-saturation at 5.6 mM-[K](e) and maximum k(C) of 0.58 min(-1).6. Cyanide (2 mM), 2,4-dinitrophenol (1 mM) and ouabain (1.4 mM) reduced k(C) by 50-90%. The half-maximally inhibiting concentration of ouabain was about 60 muM.7. Substitution of sucrose, Li or choline for external Na did not reduce the extrusion rate of (24)Na in either 6 mM-[K](e) or 0 mM-[K](e). Li stimulated (24)Na extrusion in Na-free, K-free solution.8. The properties of the ganglionic Na pump deduced from rates of temperature-stimulated (24)Na extrusion accord with the view that the ganglion hyperpolarization observed after Na loading by exposure to nicotinic depolarizing agents results from electrogenic Na extrusion. A comparable hyperpolarization is observed after temperature stimulation following Na loading.     

Sodium and rubidium fluxes in rat red blood cells

1. The Na content of rat red cells was found to be 4.40 m-mole/l. cells. When incubated in K(Rb)-free Na this value was doubled in 1 hr, whereas in K(Rb)-free choline it was reduced to about 35% in the same period of time.2. In cells with elevated Na (13.70 m-mole/l.) the activation curve of Rb influx by external Rb reached the same V(max) in sodium as in choline. The shape of the curve was sigmoid in the first case (K(m) about 1.05 mM) and hyperbolic in the second (K(m) about 0.20 mM).3. The activation curve of rubidium influx by internal sodium was linear at least up to 12 m-mole/l. cells with a slope of 0.84. From this concentration it could increase more steeply, though the data is insufficient to assure it.4. In normal cells the efflux of Na in K(Rb)-free Na Ringer was 5.64 m-mole/l. cells. hr, and it was reduced to 4.32 m-mole by 10(-4)M ouabain. This was accompanied by a reduction of Na influx by 4.14 m-mole, representing then a Na-Na ouabain-sensitive exchange mechanism.5. At a concentration of 5 mM, external Rb increased Na efflux in 2.32 m-mole/l. cells. hr above the K(Rb)-free levels, and reduced Na influx by 2.13 m-mole.6. It is proposed that the Na pump is able to operate even in the absence of external K(Rb), though at reduced rate and on a Na-Na exchange basis (Na is the only monovalent cation in the bathing solution). External K(Rb) would have two actions: to increase the rate of shuttling of the carrier (catalytic effect) and to switch the Na-Na to a Na-K(Rb) exchange.7. These results raise a question of the real significance of the Na/K(Rb) ;coupling' ratio and the K-free effect on the Na pump mechanism.     

Effect of thyroid hormone and serum on the development of Na+, K+-adenosine triphosphatase and associated ion fluxes in cultures from rat brain

The effect of culture conditions, serum supplementation or chemically defined medium and the influence of thyroid hormone were studied on the development of the Na+, K+-adenosine triphosphatase (Na+,K+-ATPase) and on the intracellular content of K+ and Na+ ions in cultures which either were greatly enriched in a neuronal cell type, the cerebellar granule cells, or contained a mixed population of cells (brain reaggregates). Foetal rat brain reaggregates displayed lower Na+,K+-ATPase activity when cultured in chemically defined medium than in the presence of serum. Supplementation of the serum-free medium with thyroid hormone resulted in a rise in the Na+,K+-ATPase activity and [3H]ouabain binding to levels similar to those found in the cultures grown in the serum-containing medium. Thyroid hormone had no significant effect on the Mg2+-ATPase activity and on the intracellular content of Na+ and K+ ions. In the granule cell-enriched cerebellar surface cultures the Na+,K+-ATPase activity was lower when the cells were grown in chemically defined medium compared with the serum-containing medium, and the intracellular Na+ to K+ ratio was higher. Thyroid hormone had no effect on the Na+,K+-ATPase activity, [3H]ouabain binding or Mg2+-ATPase activity. The hormone also failed to influence ATPase activities in cerebellar astrocytes maintained in chemically defined medium. Although thyroid hormone had no effect on the Na+,K+-ATPase activity of cultured cerebellar granule cells, treatment with the hormone resulted in a decrease in the ratio of intracellular Na+ to K+ ion content. The effect of the hormone on the Na+,K+-pump activity in live cells was therefore tested by estimating ouabain-sensitive 86Rb uptake. This was regulated as in other cell types, by the rate of Na+ entry: the Na+-ionophore monensin trebled the rate of 86Rb uptake, which was also increased (+30-100%) by 10% foetal calf serum, the maximal response being obtained by about 20 min exposure to serum. The effect was completely blocked by the Na+/H+ exchange inhibitor amiloride. The factor(s) in the serum responsible for the regulation of the Na+,K+-pump were, however, not the thyroid hormones, which failed to affect 86Rb uptake. On the basis of comparing thyroid hormone effects on the different cultures studied it was concluded that not every type of neural cell is target of the hormone action during development.     

Effect of prolonged ouabain treatment of Na, K, Cl and Ca concentration and fluxes in cultured human cells

1. Girardi and Hela cells (derived from human heart and cervix respectively) were grown as monolayer cultures in B.M.E. (Eagles basal medium) containing concentrations of ouabain up to 5 x 10(-8)M for periods ranging up to 5 days. The cell sizes, numbers, Na, K, Cl, and Ca concentrations and fluxes were then measured.2. Twenty-four hours incubation in ouabain concentrations equal to or less than 5 x 10(-8)M caused a rise in [Na](i) and an almost equal fall in [K](i) to new steady levels. The concentrations so reached were linearly related to the ouabain concentrations, such that in 5 x 10(-8)M ouabain [Na](i) rose to 124 m-mole/l. intracellular water and [K](i) fell to 55 m-mole/l. i.c. water in Girardi cells. In Hela cells the changes were smaller at any particular ouabain concentration. These levels were maintained constant for at least 5 days.3. In cells in the logarithmic phase of growth, raising [Na](i) and lowering [K](i) by ouabain caused a slowing of growth rate proportional to the ouabain concentration used. In cells in the stationary phase there was no change in the cell numbers over 24 hr. The volume of the cells was not directly affected by the treatment.4. Reducing [K](o) from the normal value of 5.4 to 2.5 mM increased the effect of any ouabain concentration, whereas increasing [K](o) to 7.5 decreased the effect of ouabain.5. Reduction of [K](o) to 2.5 mM had no effect on the [K](i) or [Na](i) but halved the cell numbers, probably by a reduction in the growth rate. The mechanism of this effect is obscure.6. In Girardi cells raising [Na](i) and lowering [K](i) by prolonged treatment increases the total Na fluxes and decreases the total K fluxes but keeps the total Na + K flux constant. High-Na, low-K cells had a reduced Na:K exchange compared to fresh cells and also had a Na:K pumped ratio nearer 4:1 than the 3:2 normally found.7. These cells also show ouabain-sensitive and ouabain-insensitive Na:Na exchanges. In high-Na, low-K cells the ouabain sensitive Na:Na exchange is the same as in fresh cells. The effect of treatment on the ouabain insensitive Na:Na exchanges has not been elucidated.8. The Cl content and fluxes are not altered by prolonged ouabain treatment. From this it is inferred that the membrane potential in high-Na, low-K cells is the same as in normal cells.9. High-Na, low-K cells have the same calcium content and fluxes as fresh cells. From this it is concluded that there is no Na:Ca coupling in these cells.