Demonstration of an electrogenic Na+-K+ pump in mouse spleen macrophages

The effect of ouabain on the membrane potential of cultured mouse spleen macrophages was examined. Ouabain (10(-3) M) induced a membrane depolarization (6.6 mV) in 18 of 19 cells studied that occurred within several minutes after exposure and was not associated with significant changes in current-voltage relationships. In related studies, cells were placed in K+-free medium in the cold for 2 h to block pump activity. Subsequent exposure to K+ at 37 degrees C resulted in a membrane hyperpolarization. However, addition of K+ also enhanced inward rectification. To differentiate between the effect of K+ on the Na+-K+ pump and its action on inward rectification, two types of experiments were done. Studies performed in the presence of barium, which blocks inward rectification, demonstrated a K+-induced hyperpolarization with no changes in rectification. Additional studies examined the effects of rubidium on Na+-loaded cells. Rubidium, which blocks inward rectification but substitutes for K+ in activating the Na+-K+ pump, induced membrane hyperpolarizations that were reversed by addition of ouabain. These data indicate that macrophages exhibit an electrogenic Na+-K+ pump, which probably contributes to the resting membrane potential under steady-state conditions and can be activated under conditions designed to Na+ load the cells. In addition, they demonstrate that increasing extracellular K+ enhances inward rectification in macrophages.     

An analysis of the effect of adrenaline on electrogenic Na+ pump of visceral nerve fibers in bullfrogs.

The effect of adrenaline (Ad) on the electrogenic Na+ pump of bullfrog visceral nerve fibers was studied, by testing the effect of this drug on the K-activated hyperpolarization (KAH) of these nerve fibers. KAHs were recorded by adding K (0.2--5 mM) to an extracellular K-free solution with which preparations were previously perfused and the actual KAH amplitude (net KAH) was estimated by taking into account the amplitude of membrane depolarization induced by K in the presence of ouabain (0.001 mM). The dose-response relation between K concentrations and net KAHs follows Michaelis-Menten kinetics; the log dose-response relation showed a sigmoid curve and the Lineweaver-Burk Plot of dose-response relation showed a straight line when nH=1.3. The net KAH was augmeted by Ad (0.03--1 mM); facilitative effects of three kinds of catecholamine on the net KAH were Ad greater than noradrenaline greater than isopreterenol. The log dose-response curve was shifted to the left in parallel in the presence of Ad; the Lineweeather-Burk plot showed a straight line when nH=1.3 and this line met the control line at the ordinate point where K concentration is infinitive, while the apparent dissociation constant (Km) decreased to 0.82 from 1.3 mM (control) in the presence of 0.3 mM Ad. Net KAH was augmented by the removal of extracellular Cl or by the action of TEA; the membrane resistance was expected to increase under these experimental conditions. The dose-response relation obtained under these conditions showed an increase of maximum response without changes in Km value. It was concluded on the basis of the present results that the increase in net KAH by Ad was due to an increase in the electrogenic Na+ pump current. The mechanism underlying such an Ad action was discussed, and it was suggested that the rate of the electrogenic Na+ pump was increased by the action of Ad.     

The independence of electrogenic sodium transport and membrane potential in a molluscan neurone

1. The current-voltage relations of the Anisodoris giant neurone (G cell) were studied in the presence and absence of Na pump activity.2. Inhibition of the electrogenic Na pump with ouabain had no effect on either the presence at warm temperatures (10-15 degrees C), or absence at cold temperatures (0-5 degrees C), of inward-going rectification.3. Abolition of inward-going rectification in the warm, by replacement of external K with Rb, did not affect the electrogenic Na pump.4. The current generated by the electrogenic pump was essentially constant between the membrane potentials of - 30 and - 100 mV.5. The potential produced by the electrogenic pump can be predicted by a modification of the constant field equation.6. It is estimated that the energy required to extrude Na was between 3160 and 3700 cal/g-atom, and that uncoupled Na efflux during pump activity was typically between 0.2 and 4.0 p-mole/cm(2).sec.     

Electrogenic sodium pumping in rabbit small intestinal smooth muscle.

Oscillations in the activity of an electrogenic Na pump has been suggested as the ionic mechanism underlying the intestinal control potential (slow wave). We investigated the electrogenicity of this pump in rabbit jejunal smooth muscle. Potassium admission to Na-rich tissues caused a large increase in membrane potential which after 10--20 min decreased toward values comparable with those of normal tissues. This hyperpolarization far exceeded EK and could be prevented by cooling or by ouabain. No hyperpolarization occurred upon K admission to Li-rich tissues in the absence of Na. Thus, the pump in this tissue can operate electrogenically. Goldman's equation was modified so as to account for the pump's contribution to the membrane potential. Using this equation, the calculated contribution of the pump, under normal "steady-state" conditions, is unlikely to exceed a few millivolts. It is concluded that although the pump in this tissue can be electrogenic, its contribution may be smaller than that required if the intestinal control potential resulted from rhythmic turning off and on of the electrogenic Na pump.     

Modulation of periodic cold receptor activity by ouabain

The effect of ouabain on the periodic discharge pattern of feline cold receptors was studied in order to substantiate a possible contribution of Na/K pump activity to signal transduction. Afferent activity was recorded from the cold fibres of an isolated preparation of the tongue. The periodic pattern consisted of beating activity and of grouped discharges and was characterized by two parameters, the oscillation frequency and the number of impulses initiated per cycle. Ouabain (10^–7-10^–6 M) induced in all receptors excitatory responses, consisting of a short vigorous increase of activity followed by inhibition. Thus the receptors never stabilized to or maintained a new static level of activity. The ouabain-induced responses occurred repeatedly in several receptors and were produced by remarkable stereotyped modifications of both the oscillation frequency and the number of impulses per cycle. The oscillation frequency attained peak values which increased monotonically with higher static temperatures and which were considerably greater than peak control values. The data indicate that an electrogenic Na/K pump contributes to the transducer process of cold receptors and that inhibition of this pump evidently gives rise to a depolarizing imbalance of the membrane potential, accelerating the oscillation frequency to a maximum value. Thus the oscillation frequency seems to be controlled by temperature and by membrane potential in cold receptors.     

Parotid acinar cells: ionic dependence of acetylcholine-evoked membrane potential changes

Segments of mouse parotid were placed in a superfusion chamber. Surface acini were impaled by one or two micro-electrodes for measurement of membrane potential and resistance. The acinus under investigation was stimulated by micro-iontophoretic application of acetylcholine (ACh) or adrenaline.Neighbouring acinar cells were electrically coupled. Electrical coupling between acinar cells only occurred within restricted domains probably corresponding to an acinus or a group of acini.Passing direct current through one intracellular electrode, the resting potential of an acinus could be set at desired levels and the dependency of the ACh-evoked potential change on the resting potential investigated. The ACh null potential (initial effect) was about –60mV. A delayed hyperpolarizing effect of ACh could not be reversed.The initial ACh-evoked potential change was sensitive to alterations in extracellular Na, K and Cl concentration. The delayed ACh-evoked hyperpolarization was blocked by ouabain, exposure to Na-free or K-free solutions.It is concluded that ACh increases mainly K and Na membrane conductance causing K efflux and Na influx with a subsequent Na activation of an electrogenic Na pump.     

Temperature dependence of a sodium—potassium permeability ratio of a molluscan neurone

1. The temperature dependence of the membrane potential of a molluscan giant neurone was examined under conditions which block the electrogenic activity of the Na-K exchange pump.2. When the Na pump was blocked by ouabain or the removal of external K, the membrane potential depolarized as temperature was increased.3. This depolarization was prevented by the replacement of external Na with impermeant cations, but was greater when Na was replaced with Li.4. All observed effects of ouabain were attributable to inhibition of the Na pump. The depolarization in response to ouabain at warmer temperatures was completely reversible, and the rate of both onset and reversibility of the ouabain effect was dependent upon temperature.5. Using a modified form of the constant field equation, the internal K concentration and the Na-K permeability ratio, P(Na)/P(K), were calculated from the experimental data.6. P(Na)/P(K) was found to increase from 0.028 at 4 degrees C to 0.068 at 18 degrees C. It is suggested that this increase is due primarily to a change in P(Na).     

Pumped movements of sodium and potassium ions in the isolated epithelium of the frog skin

Summary The action of agents with well known effects on transepithelial Na transport was tested on Na extrusion in epithelial cells of the frog skin. The cells had been previously loaded with Na by incubation in cold, K-free solutions. DNP (5×10⁻⁴ M) totally inhibited Na extrusion and K uptake, while amiloride (10⁻⁵ M) did not show any effect on either of these processes. Ouabain (10⁻⁶M) and absence of K from the medium inhibited completely Na extrusion and K uptake without changing cell water content. Probably the most interesting finding is that K activated Na extrusion along a sigmoid curve, which suggests that, as in other cells, the Na pump of these epithelial cells has 2 sites for K activation. The half-activation concentration of the site with highest affinity was 0.27 mM, the other 1.3 mM. Na extrusion significantly exceeded K uptake either at low K in the medium or during initial recovery in normal K Ringer. This may indicate an electrogenic mode of pump activity.     

Cation exchange and glycoside binding in cultured rat heart cells.

The Na/K-exchange characteristics, ouabain-binding kinetics, and Na pump turnover rates of synchronously contracting monolayers of neonatal rat myocardial cells were studied. The cells exchange Na rapidly (T1/2 = 35 s) with a mean Na flux of approximately 25 (pmol/cm2)/s. The half time (T1/2) of K exchange is much longer (12 min); the mean K flux is 13 (pmol/cm2)/s. Active Na/K transport, as measured by K influx, is relatively ouabain sensitive, and 10(-6) M ouabain produces half-maximal inhibition. Ouabain (10(-2)M) inhibits 60% of the Na efflux and 75% of the K influx. The cells bind [3H]ouabain rapidly (T1/2 = 8 min), but release it very slowly (T1/2 = 11 h), and both the amount bound and the rate of binding were inversely proportional to extracellular K. Specific [3H]ouabain binding demonstrates saturation reaching a maximum of 1.6 x 10(6) molecules per cell at 2 x 10(-7) M [3H]ouabain. From cell surface area and ouabain-sensitive flux measurements, the Na pump density was calculated at 720/micrometer2 with an individual pump turnover rate of 50/s. Thus the studies indicate that despite their neonatal origin, the behavior of the Na pump in these cells is very similar to that in other mammalian tissues.     

A ouabain-sensitive hyperpolarization in rat striatal neurones in vitro

Intracellular recordings were made from rat striatal neurones in vitro. In the presence of intracellular caesium and extracellular tetraethylammonium chloride (TEA) (5 mM) and barium (3 mM), long-lasting plateau potentials developed followed by a prominent voltage independent hyperpolarization which lasted several seconds. A similar afterhyperpolarization was observed when calcium was replaced by barium. The afterhyperpolarization was reduced in a potassium free medium and reversibly abolished in a Na+-free solution or by cooling the slice to 21-24 degrees C. It was also irreversibly blocked by ouabain (50 microM). This hyperpolarization may therefore result from the activation of a Na+,K+-ATPase electrogenic pump.     

Factors related to the low resting membrane potentials of diseased human atrial muscles

We studied the ionic mechanism of low resting potential (RP) of quiescent "diseased" human atrial fibers. The RP was -49.7 +/- 0.8 mV (n = 179) in normal Tyrode's solution (5.4 mM [K]o, 36 degrees C). The changes in RP measured at various levels of [K]o appeared to fit the RP-[K]o relationship predicted by the Goldman-Hodgkin-Katz equation, assuming PNa/PK ratio (alpha) to be 0.102 and [K]i to be 131.9 mM. The alpha far exceeded the normal value (about 0.01) by a factor of 10. Acetylcholine (ACh, 10 microM) led to marked increases in the RP. An application of tetrodotoxin (TTX, 6 microM) and perfusion with low [Na]o (10% of the control) media in the presence and absence of ACh produced considerable hyperpolarizations of the RP. These findings indicate that increased alpha value is due to a combination of decreased PK and increased PNa. Applications of ouabain (5 microM) and a cooling procedure (12.3 degrees C) depolarized the membrane, whereas epinephrine (1 microM) hyperpolarized it. Transient hyperpolarization, which exceeded the steady state levels of RP at 5.4 mM [K]o, was observed with perfusing of 5.4 mM [K]o media following perfusion with K-free media. These findings suggest that electrogenic Na pump current plays a significant role in the maintenance of the RP. In conclusion, partial depolarization of "diseased" human atrial fibers was attributed to both decreases in membrane K+ conductance and increases in Na+ conductance. The electrogenic outward pump current seemed to protect the fibers from severe depolarization produced by the conductance abnormality (increased PNa/PK).     

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.     

Possible involvement of sodium pump in the relaxation of rat aorta induced by alpha-human atrial natriuretic polypeptide

In order to clarify whether the sodium handling of smooth muscle is associated with the relaxing action of alpha-human atrial natriuretic polypeptide (alpha-hANP), we examined the sodium pump-related effects of alpha-hANP on rat aortic smooth muscles. Application of Ca2+ (1.0 to 10.0 mM) to the muscle preincubated in Ca2+-free, and K+-free or 0.5 mM K+ medium for 60 min induced a contraction. Pretreatment with alpha-hANP (1 x 10(-8) M) decreased the contraction evoked in 0.5 mM [K+]o but not that in K+-free medium. After a contraction was elicited by norepinephrine in K+-free solution, an addition of KCl (1.4-5.4 mM) caused a transient relaxation in a concentration-dependent manner, presumably due to the activation of electrogenic Na pump. The alpha-hANP enhanced the relaxation, which was sensitive to ouabain, and the potentiation by alpha-hANP was inversely related to the concentration of K+ added. When alpha-hANP was applied to relax the muscle precontracted by norepinephrine in the varied concentration of external K+, alpha-hANP-induced relaxation was greater in 1.4 or 2.7 mM [K+]o than in 0 or 5.4 mM [K+]o. These results suggest that the vasodilating effect of alpha-hANP is at least partially mediated by the activation of electrogenic Na, K-pump and this effect is prominent when the Na, K-pump is partially suppressed.     

Two modes of Na extrusion in cells from guinea pig kidney cortex slices

Summary Cells from guinea pig kidney cortex slices, which have been loaded with Na and caused to lose K, by leaching at 0.6° C for 2.5 hours, extrude Na with Cl upon rewarming to 25° C in a medium without K. A subsequent rise in the K concentration in the bath at 25° C induces further net Na extrusion, 1 Na being extruded in exchange for 1 K that is taken up. When the leached tissue is rewarmed to 25°C in the presence of K in the bathing fluid (2 or 16 mM), some Na is extruded accompanied with Cl (by a mechanism that is inhibited by ethacrynic acid) and some Na is extruded maintanining a 1:1 ratio with the K that is taken up, (this system being inhibited by ouabain). Thus two modes of Na extrusion are observed, mode A that is accompanied by net Cl efflux, and that is inhibited by 2 mM ethacrynic acid, but not by 1 or 10 mM ouabain and mode B in which one K is taken up for each Na extruded. Mode B is inhibited by 1 mM ouabain and not by ethacrynic acid. DNP and anoxia inhibit both modes A and B. Insufficient doses of ouabain do not explain the refractoriness of mode A to ouabain. Ouabain and ethacrynic acid are known inhibitors of the Na–K-ATPase at much lower doses. It is concluded that both modes may originate in different Na pumps which may have different energy sources. Pump A should be efficient in the volume regulation of the cell. According to experimental procedure, both modes of Na extrusion appear of comparable magnitude. In the steady-state their relative role may be different.     

Passive transepithelial cationic fluxes across the frog skin under short-circuit conditions

The short-circuit current (Isc) across the frog skin in the steady state reflects the active Na+ transport. Inhibition of the Na+-K+ pump by ouabain causes slow decay of Isc. It has been suggested that this slow ouabain effect on Isc could be due to the asymmetric ionic permeations of frog skin. That is, Na+ flux at the apical membrane and K+ flux at the basolateral membrane transiently generate the transepithelial cationic fluxes and are measured as the Isc even under the condition of active Na+ transport arrest. However, this hypothesis on the transient Isc has not been studied experimentally. In the present study, transient inward and outward Isc were observed alternately even after pump arrest by changing the ionic compositions of the bathing solutions in a Ussing's chamber. The time constant of Isc decay was 20-30 min. The Isc responses were quicker and stronger on the isolated epithelia than on the whole skin. Both Isc responses were blocked by amiloride, a Na+ channel blocker. Measurements of the ionic composition of isolated epithelia under experimental conditions indicate that the passive Na+ flux across the apical membrane and the passive K+ flux across the basolateral membrane cause both transient inward and outward Isc under the ouabain-treated condition of frog skin.     

On the temperature dependence of the Na pump in sheep Purkinje fibres

The temperature dependence of cardiac active Na transport is studied in voltage clamped sheep Purkinje fibres by means of simultaneous measurements of the membrane current (I) and the intracellular Na activity (a _Na ⁱ ). During activation of the Na pump a transient outward current (I) anda _Na ⁱ decline exponentially with an identical time constant (). The transient outward current and the decline ina _Na ⁱ are blocked by 10^–4 M dihydroouabain (DHO). Lowering the temperature from 42°C to 17°C prolongs . The electrogenic fraction (e.f.) of the active Na efflux remains unaffected. The Q₁₀ value of the active Na transport derived from the changes of varies within the temperature range studied. The Q₁₀ amounts to 1.2 between 42°C and 35°C, to 2.4 between 35°C and 22°C and to 2.1 between 35°C and 17°C. Correspondingly the activation energy of the active Na transport is not constant between 42°C and 17°C. It is calculated to be 3.4 kcal/mol between 42°C and 35°C, 15.9 kcal/mol between 35°C and 22°C and 12.4 kcal/mol between 35°C and 17°C. Variations in temperature change the maximal rate constant of the active Na transport, whereas the sensitivity of the Na pump towards the extracellular K concentration (K_o) is little affected. The unidirectional active Na efflux of a fibre as a function of the intracellular Na concentration (Na_i) at 35°C and 22°C is derived from the experemental data. The relationship is linear over the narrow Na_i range studied but seems to be more complex when a wider Na_i range is considered.Supported by the Deutsche Forschungsgemeinschaft (SFB 114 Bionach)     

The electrogenic sodium pump in the hyperpolarizing and secretory effects of pancreozymin in the pancreatic acinar cell.

1. Transmembrane potential, effective membrane resistance, and amylase release were recorded simultaneously from acinar cells of isolated rat pancreas perfused with Krebs-Henseleit solution. 2. The hyperpolarizing effect of pancreozymin (Pz) was confirmed by perfusing with a solution containing 5 m-u. Pz/ml. 3. The hyperpolarizing effect of Pz disappeared in the following environments: (a) in low ambient temperature, (b) in K-free medium, (c) in low Na medium, and (d) in the presence of ouabain. In these environments, transient depolarization was frequently observed immediately after stimulation. It is suggested that there are two components in the effect of Pz on the membrane potential of the acinar cells: transient depolarization which coincides with an increase in Na permeability in the initial phase, and continuous hyperpolarization due to an electrogenic Na pump which conceals transient depolarizing phase when the pump is dominant. 4. The secretory effect of Pz was inhibited under conditions that suppressed the electrogenic Na pump. It is proposed that the Na pump activated by Pz maintains the passive Na-influx, increases [Ca2+]i, and, in consequence, uphold the amylase output during continuous stimulation. 5. A medium which was used to bathe the sectioned pancreas with Pz was found to contain a substance which depolarized the acinar cells.     

Potential dependency of the electrogenic Na+-pump current in bullfrog atrial muscles

Experiments were designed to evaluate the concept that the activity of the electrogenic Na+-pump is dependent on the transmembrane potential. Cardiac muscle preparations were used because the electrogenic Na+-pump current can be recorded at different potentials with the voltage clamp method in this preparation. Electrogenic Na+-pump current was identified as the membrane current which was abolished by ouabain (5 microM) and induced by the addition of K+ or an alkali metal cation, such as Rb+, Cs+, or Li+, to the extracellular K+-free solution. Alkali metal cations other than K+ were used to eliminate the possibility that a passive membrane K+ current might be altered by changes in the K+ concentration in the vicinity of the membrane due to activation of the Na+-pump. It was concluded that the activity of the electrogenic Na+-pump current is dependent on the membrane potential.     

Role of the electrogenic Na/K pump in disinhibition-induced bursting in cultured spinal networks

Disinhibition-induced bursting activity in cultures of fetal rat spinal cord is mainly controlled by intrinsic spiking with subsequent recurrent excitation of the network through glutamate synaptic transmission, and by autoregulation of neuronal excitability. Here we investigated the contribution of the electrogenic Na/K pump to the autoregulation of excitability using extracellular recordings by multielectrode arrays (MEAs) and intracellular whole cell recordings from spinal interneurons. The blockade of the electrogenic Na/K pump by strophanthidin led to an immediate and transient increase in the burst rate together with an increase in the asynchronous background activity. Later, the burst rate decreased to initial values and the bursts became shorter and smaller. In single neurons, we observed an immediate depolarization of the membrane during the interburst intervals concomitant with the rise in burst rate. This depolarization was more pronounced during disinhibition than during control, suggesting that the pump was more active. Later a decrease in burst rate was observed and, in some neurons, a complete cessation of firing. Most of the effects of strophanthidin could be reproduced by high K+-induced depolarization. During prolonged current injections, spinal interneurons exhibited spike frequency adaptation, which remained unaffected by strophanthidin. These results suggest that the electrogenic Na/K pump is responsible for the hyperpolarization and thus for the changes in excitability during the interburst intervals, although not for the spike frequency adaptation during the bursts.     

Differential expression of Na+/K+-ATPase α-subunits in mouse hippocampal interneurones and pyramidal cells

The sodium pump (Na+/K+-ATPase), maintains intracellular and extracellular concentrations of sodium and potassium by catalysing ATP. Three sodium pump α subunits, ATP1A1, ATP1A2 and ATP1A3, are expressed in brain. We compared their role in pyramidal cells and a subset of interneurones in the subiculum. Interneurones were identified by their expression of GFP under the GAD-65 promoter. We used the sensitivity to the cardiac glycoside, ouabain, to discriminate between different α subunit isoforms. GFP-positive interneurones were depolarized by nanomolar doses of ouabain, but higher concentrations were needed to depolarize pyramidal cells. Comparison of pump currents in these cells revealed a current sensitive to low doses of ouabain in interneurones, while micromolar doses of ouabain were needed to suppress the pump current in subicular pyramidal cells. As predicted, nanomolar doses of ouabain increased the frequency but not the amplitudes of IPSPs in pyramidal cells. Immunostaining confirmed a differential distribution of α-subunits of the Na+/K+-ATPase in subicular interneurones and pyramidal cells. In conclusion, these data suggest that while ATP1A3-isoforms regulate sodium and potassium homeostasis in subicular interneurones, ATP1A1-isoforms assume this function in pyramidal cells. This differential expression of sodium pump isoforms may contribute to differences in resting membrane potential of subicular interneurones and pyramidal cells.