Hemmung der elektrogenen Na-Pumpe am Meerschweinchenvorhof durch Digitoxigenin

Summary Water content, extracellular space (estimated as inulin space), intracellular Na- and K-concentrations (Na_i; K_i) and membrane potentials of auricles from guinea-pigs were studied before and after cooling in solutions with or without digitoxigenine (2·10^–7 and 3·10^–6 g/ml).The water content—under the conditions employed—was about 800 ml/kg wet wt.The inulin space was about 350 ml/kg wet wt. at 35°C and about 300 ml/kg wet wt. at 4–6°C irrespective of the digitoxigenine concentration used.In control experiments a net sodium transport occurred from the cells at the beginning of rewarming and the membrane potential hyperpolarized beyond the steady-state value recorded at the end of this period. Rewarming in Tyrode solution with 2·10^–7 g/ml digitoxigenine caused little changes of the intracellular Na and K concentration and the membrane potential of the auricles as compared to the control experiments. In Tyrode solution containing 3·10^–6 g/ml digitoxigenine the net Na transport from rewarmed atria was inhibited. The membrane potential did not hyperpolarize, but remained constant during rewarming.These results support the view that after hypothermia the membrane potential is directly affected by an electrogenic sodium pump which can be inhibited by digitoxigenine.

Im Rahmen des SFB 114 Bionach, Bochum.     

The effect of the internal sodium concentration on calcium fluxes in isolated guinea-pig auricles

1. Calcium efflux from guinea-pig auricles followed saturation kinetics when [Ca](o) and [Na](o) were changed while the ratio [Ca](o)/[Na](o) (2) was kept constant. The Michaelis constant, K(m) (Ca+Na) = 40 mM, suggests that a hypothetical carrier system, responsible for sodium-calcium exchange, is far from saturation with the inside concentrations of these ions.2. [Na](i) was altered in the auricles between 12.5 and 60 mM/kg fibre water while total cellular calcium concentration ([Ca](t)) at the beginning of the influx period was not significantly different in the various groups of preparations.3. (45)Ca influx increased appreciably with increasing [Na](i). (45)Ca influx from sodium-poor solution corresponded to an almost equal increase in [Ca](t), while [Ca](t) did not change much in preparations loaded with (45)Ca in Tyrode solution. When the sodium-activated fraction of calcium influx was plotted against [Na](i) (2) the resulting curve indicated saturation with K(m) (Na) = 3500 (mM [Na](i))(2) and maximal influx rate, J(i, max) (Ca') = 1.35 mM/kg wet weight x 10 min.4. When the preparations were re-equilibrated for various times in normal Tyrode solution after [Na](i) had been increased, both the sodium-activated component of calcium influx and [Na](i) (2) decreased with approximately the same rate constants.5. Calcium efflux from auricles with high [Na](i) was increased when it was measured in Tyrode solution while the efflux in sodium-poor solution was inhibited.6. Auricles with increased [Na](i) showed a positive inotropic contractile response.7. The main conclusion reached by these experiments is that calcium influx is affected by [Na](i) in a way which is compatible with a carrier-mediated sodium-calcium exchange system.     

Cell electrical potentials during enhanced sodium extrusion in guinea-pig kidney cortex slices.

1. Experiments were performed on outermost slices of the guinea-pig kidney which are mainly made up of proximal tubular cells. 2. Kidney cells loaded with Na+ by chilling at 0.6 degrees C for 2.5 hr, when subsequently rewarmed to 25 degrees C in a medium containing 16 mM-K+ extrude Na+ at enhanced speed for about 10 min. This Na+ movement is accompanied by efflux of Cl and influx of K+. 3. Measurements of cell potential during enhanced Na+ extrusion show that cells hyperpolarize to values about 30 mV more negative than the K+ equilibrium potential. 4. This hyperpolarization is only partly inhibited by 1 mM ouabain or by 2 mM ethacrynic acid but both agents added together suppress it completely. 5. With 16 mM-Rb instead of 16 mM-K the hyperpolarization is smaller. 6. A diminished extracellular K+ concentration outside of the cells, within the slice, can account for only a small part of the hyperpolarization. 7. The hyperpolarization is proportional to the rate of Na+ pumping. 8. Cl- seems to shunt the hyperpolarization to a greater extent than K+. 9. It is concluded that Na+ extrusion is capable of transferring electric charge across the membrane.     

An effect of the electrogenic sodium pump on the membrane potential in beating guinea-pig atria

Summary The membrane potential of guinea-pig atria was measured in different media before, during and after electrical stimulation of various durations. The stimulus duration and frequency (3 pulses/sec) were kept constant.During short stimulation (<1 min) in Tyrode solution at 35°C the membrane potential depolarized first quickly and later on more slowly. Following cessation of the stimulus train the membrane potential repolarized to the resting value within 1–2 min.Prolonged stimulation (1–7 min) in Tyrode solution at 35°C likewise caused an initial depolarization of membrane potential. Thereafter, the membrane potential repolarized although stimulation continued. After the end of the stimulation period the membrane potential hyperpolarized beyond the resting potential for several minutes. Maximum hyperpolarization was achieved ca. 1 min after the end of the stimulus train.In Tyrode solution at 25°C both the repolarization of the membrane potential during long stimulation and the hyperpolarization after it were diminished. The same effect was observed in Na poor fluids, which contained only 50 or 33% of the Na concentrations in Tyrode solution.Digitoxigenine (3·10^–6 g/ml), when added to Tyrode solution at 35°C, also reduced the repolarization of membrane potential during, and the hyperpolarization after, a long stimulus train.Compared to the resting value the membrane resistance was essentially unchanged 1–3 min after a 2 min stimulation period.From these results it is concluded, that an active, electrogenic Na pump contributes to the membrane potential of beating guinea-pig auricles.     

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).     

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).     

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.     

Contribution of an electrogenic sodium pump to membrane potential in mammalian skeletal muscle fibres.

1. Relationship between the resting membrane potential and the changes in the intraceullar Na and K concentrations ([Na]i and [K]i) was studied in 'Na-loaded' and K-depleted' soleus (SOL) muscles of rats which had fed a K-free diet for 40 and more days. 2. The extracellular space of the muscles was not significantly different between normal and K-deficient rats. The inulin space in both the 'fresh' and Na-rich' muscles can be determined by the same function relating the space to the muscle weight. 3. Presence of 2-5-15 mM-K in the recovery solution hyperpolarized the 'Na-rich' muscul fibres at the beginning of recovery. The hyperpolarized membrane potential exceeded, beyond the measured potential of 'fresh' muscle fibres, the theoretical potential derived from the ionic theory, or even beyond Ek. Then, the measured membrane potential declined progressively during the immersion in a recovery solution and returned to the steady-state value When a considerable Na extrusion and K uptake took place, the measured membrane potential became equal to Ek. 4.he maximal hyperpolarization occurring immediately after immersion in the recovery solution became smaller and had a shorter duration when increasing the external K concentration ([K]o) from 2-5 to 15mM. 5. The K-sensitive hyperpolarization was completely abolished on exposure to 0mM [K]o, on cooling to ca. 4 degrees C, and in the presence of oubain (10(-4) M). The inhibitory effects were reversed on returning to the control conditions. The membrane potential obtained after inhibition of the electrogenic Na-pump with cooling or ouabain agrees well with that predicted by the 'constant-field' equation. 7. The external Cl ions had a short-circuiting effect on the electrogenic Na-pumping activated on adding K ions. 8. The replacement of Na ions in a recovery solution with Li ions resulted in a faster rate of depolarization from the maximal hyperpolarizationp. It is concluded that the resting membrane potential of 'Na-loaded' and 'K-depleted' SOL muscle fibres is the sum of an ionic diffusion potential predicted by either the Nernst equation or the constant-field equation and of the potential produced by an electrogenic Na-pump.     

Membrane potential of smooth muscle cells in K-free solution

1. The changes of the ion content, the membrane potential and of the membrane permeability of taenia coli cells have been studied during exposure to K-free solutions. The relative value of the total membrane conductance was determined by measuring the electrotonic potential during constant current pulses with an intracellular electrode. The P(K) values were calculated from (42)K-efflux in K-free solutions.2. In solutions containing penetrating anions the cells initially depolarize. Thereafter they hyperpolarize to about - 85 mV and again depolarize after 90 min to - 5 mV. These potential changes are much smaller if large anions are used as chloride substitutes. Moreover, the final depolarization is only reached after 4-5 hr. This hyperpolarization is not inhibited by 10(-5)M ouabain.3. These potential changes are accompanied by a progressive exchange of intracellular K by Na. In solutions containing chloride or nitrate the relative value of the total membrane conductance increases to a maximal value, corresponding to the peak value of the calculated P(K). Such changes of the membrane conductance and of P(K) do not occur in K-free solutions containing large anions.4. It is proposed that the initial depolarization is probably caused by an inhibition of an electrogenic Na pump. In chloride or nitrate solution the hyperpolarization is due to an increase of the [K](i)/[K](o) ratio and to an increase of the K permeability. In the presence of large anions the hyperpolarization remains small because this increase of P(K) does not occur.     

The effect of sodium and calcium on the action potential of the smooth muscle of the guinea-pig taenia coli

1. Spontaneous spike activity and action potentials evoked by external field stimulation were recorded, intracellularly and with the double sucrose gap method, from the smooth muscle of guinea-pig taenia coli.2. Replacement of external NaCl with sucrose (leaving 10 mM-Na in the buffer) caused hyperpolarization and stopped spontaneous activity within 10 min. Spikes could, however, be evoked for 2-3 hr. The amplitude, the overshoot and rate of rise of the spike were increased.3. In 10 mM-[Na](o) the intracellular Na concentration was reduced from 35 to 24 mM, shifting the Na-equilibrium potential from +34 to -22 mV.4. Excess Ca (12.5 mM) caused hyperpolarization and increased membrane conductance. The amplitude and the rate of rise of the spike were increased, the threshold was raised and the latency of the spike evoked by threshold stimulation became shorter.5. The effect of reducing the external Ca concentration depended on the Na concentration present, being greater with higher external [Na](o). When the membrane was depolarized and spikes deteriorated in low Ca (0.2-0.5 mM) reduction of Na to 10 mM caused repolarization and recovery of the action potential.6. Mn (0.5-1.0 mM) blocked spontaneous spike discharge after 20 min. Higher concentrations (more than 2.0 mM) were required to block the evoked action potential.7. The results indicate that the smooth muscle spike in taenia is due to Ca-entry and that Na influences spike activity indirectly by competing with Ca in controlling the membrane potential.     

ECS, intracellular pH, and electrolytes of cardiac and skeletal muscle.

The extracellular space (ECS) of muscle from each ventricle of the heart (RV and LV), the atria, diaphragm, and quadriceps was estimated in the anesthetized rabbit from the distribution volumes of [14C]insulin, [14C]sucrose, [51Cr]EDTA, and C1--. Whole-tissue electrolytes were measured and intracellular electrolytes calculated. The ECS of the tissues varied, increasing in the order quadriceps less than LV less than RV less than atria. The volume of distribution of [14C]inulin was always less than that of either [14C]sucrose or [51Cr]EDTA which agreed closely, whereas that of C1-- was always greater. There was no difference in intracellular K+ in muscle from each of the cardiac chambers, whereas intracellular Na+ and C1-- varied, increasing in the order quadriceps less than LV less than RV less than atria. Intracellular pH, measured with [14C]DMO did not differ in any of the tissues studied. It is concluded that, in vivo, the estimated ECS incardiac muscle is lower than that reported in vitro, that [51Cr]EDTA is a satisfactory ECS marker, and that differences in intracellular Na+ and C1-- but not K+ or pH exist between muscle from the cardiac chambers.     

Ionic permeability changes as the basis of the thermal dependence of the resting potential in barnacle muscle fibres

1. The thermal dependence of the resting potential of isolated barnacle muscle fibres was larger (1-2 mV/ degrees C) than predicted by Nernst's equation (about 0.2 mV/ degrees C). A comparative study was made of the influence on thermal dependence of parameters related to (a) passive permeability and to (b) Na extrusion.2. High [K](o) decreased the thermal dependence reversibly. [K(i)], [Na](i) and [Cl](i) were determined by chemical analysis, and Goldman's equation was fitted to data relating V to [K](o) at different temperatures, in the presence and absence of ouabain 5 x 10(-5)M. In both cases the behaviour of V when T was lowered from 20 to 4 degrees C was accounted for by increases in the calculated P(Na/PK) and P(Cl/PK) (from 0.006 to 0.043 and from 0.17 to 0.34 on the average, respectively.)3. Other parameters related to passive permeability (and which caused reversible depolarization): decreased [Cl](o) (methanesulphonate or gluconate substituted), and decreased pH(o) (below 5.0), also decreased the thermal dependence reversibly.4. Inhibitors (ouabain 5 x 10(-5)M, cyanide 2-10 x 10(-3)M, 2,4-dinitrophenol 2 x 10(-4)M) externally applied did not affect either resting potential or its thermal dependence for several hours.5. Increasing [Na](i) three- to fourfold by intracellular injection decreased both resting potential and its thermal dependence.6. Although a small effect by a Na electrogenic pump cannot be excluded, the largest part of the thermal effect on the resting potential is concluded to depend on temperature-induced variations in relative ionic permeabilities to cations and anions. A model is proposed which can account for the data assuming that (a) each permeant ion associates to a separate site in the membrane, and (b) the ion-site equilibrium is temperature-dependent.     

Membrane potential measurement in parotid acinar cells

1. Intracellular recording of membrane potential was made from acinar cells of the isolated mouse parotid gland superfused with physiological salt solutions.2. The mean acinar resting membrane potential was - 68.5 mV during superfusion with Krebs-Henseleit solution. Shift of the superfusion solution to one containing ACh or adrenaline (10(-5)M) always caused a transient hyperpolarization (about 10-15 mV).3. The membrane potential was mainly dependent on the extracellular K concentration ([K](o)). Increasing [K](o) tenfold decreased the membrane potential by 50 mV. This depolarization was not mediated by ACh release from depolarized nerve endings, since it was seen in the presence of atropine (1.4 x 10(-6)M) and not caused by the accompanying reduction in [Na](o) to 40 mM caused only a small depolarization (less than 10 mV).4. When the superfusion solution was shifted, during intracellular recording, from a normal Krebs-Henseleit solution ([K] = 4.7 mM) to a K-free solution, a hyperpolarization of about 8 mV was measured. Reintroduction of the normal K-containing solution after a longer period of K deprivation (30-70 min) resulted in a short-lasting pronounced hyperpolarization (about 20 mV) which could be blocked by Strophanthin-G (10(-3)M).5. In contrast to previous reports, the present findings indicate that the membrane potential of salivary acinar cells is similar, with respect to magnitude and K-dependence, to that of cells of more thoroughly investigated tissues, such as muscle and nerve, and that the membrane Na-K pump is electrogenic, at least when the cells have been loaded with Na.     

Evidence for the genetic control of the sodium pump density in HeLa cells

1. HeLa cells were grown in normal and altered growth solutions; the ion contents, volumes, K sensitive ouabain binding, the Na-K-ATPase and the Na and K transport measured.2. Cells grown in 1 x 10(-4)M ethacrynate or low-K media for 24 hr have a raised [Na](i), a decreased [K](i), and an increased ouabain binding. Those grown in low-K also have an increased Na-K-ATPase activity.3. When cells are put into low-K solutions the [Na](i) initially rises to a high value, and then starts to fall some 8 hours later as the ouabain binding increases, suggesting that these additional sites represent working Na pumps. Flux measurements on low-K cells provide some support for this view.4. Experiments in which sorbitol replaced [Na](o) showed that the increased ouabain binding and Na-K-ATPase was related to the increase in [Na](i) rather than the decrease in [K](i) and was not due to a non-specific effect of [K](o) change.5. The protein synthesis inhibitors cycloheximide and puromycin stopped the effect of ethacrynate and low-K solutions on increased ouabain binding. They also decreased the ouabain binding and K influx in normal cells over 24 hr. Cycloheximide had similar effects on Na-K-ATPase in low-K treated and normal cells. These results suggest that protein synthesis is required for the appearance of more ouabain sensitive sites in the cell membrane, both in response to ethacrynate and low-K treatment and for normal replacement during the cell's life.6. The RNA synthesis inhibitors actinomycin D (AMD) and cordycepin had complex effects on ouabain binding in fresh and ethacrynate treated cells. These inhibitors increased the ouabain binding but decreased the K influx. This discrepancy was due to the appearance of ouabain binding sites with different characteristics from normal sites. A limited investigation of this phenomenon was carried out. Probably AMD stops the normal replacement of sites in the membrane.7. These results are consistent with the hypothesis that HeLa cells have a system for controlling the number of Na pumps in their membranes. This system responds to the level of [Na](i) within the cell and involves protein synthesis. It is not clear to what extent the nucleus is normally involved in this process.     

Hypothermia-induced respiratory arrest and recovery in neonatal rats

To examine the changes in breathing that occur during progressive hypothermia and rewarming in neonatal rats, we cooled and rewarmed rat pups during the first 6 days of life. During cooling, breathing stopped when rectal temperature (Tr) fell below 10.7+/-0.24 degrees C, and recovered spontaneously during rewarming when Tr reached 13.3+/-0.38 degrees C, regardless of age. During cooling, breathing frequency declined progressively, whereas tidal volume increased until Tr fell below 15 degrees C whence it declined to, but never below, normothermic levels. These data support suggestions that failure occurs at the level of the central rhythm generator for breathing and is not due to an inability to sustain the level of motor output. During rewarming, following respiratory arrest, the pattern of change was reversed, but with a significant thermal hysteresis, resulting in slower breathing and cardiac frequencies at any given rectal temperature during rewarming. There were no effects of age observed over the range studied on the changes in respiratory variables associated with hypothermia or rewarming. Breathing restarted spontaneously on rewarming with no evidence that gasping was required to initiate this process. The overall breathing pattern was episodic during the early stages of rewarming, however, suggesting that the respiratory rhythm is only periodically expressed during the initial stages of recovery from hypothermia.     

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.     

Membrane potential and resistance measurement in acinar cells from salivary glands in vitro: effect of acetylcholine

1. Cell membrane potential and input resistance measurements were made on segments of submaxillary glands from mice, rabbits or cats placed in a tissue bath, which was perfused with physiological salt solutions.2. During exposure to a standard Krebs-Henseleit solution, ACh stimulation always evoked a marked decrease in input resistance and time constant. The change in potential evoked by ACh stimulation was either a monophasic hyperpolarization (low resting potential) or a depolarization followed by hyperpolarization (high resting potential).3. Increasing [Ca](o) from 2.56 to 10 mM resulted in an enhanced input resistance. Under this condition it was sometimes possible to obtain current-voltage relations. The relationship was linear in the range -50 to -10 mV. In the absence of extracellular Ca the resting potential was reduced and ACh mostly evoked hyperpolarizations. In those cases when the resting potential remained high biphasic potentials were still observed.4. During exposure to Na-free solutions the resting potential was either unchanged or slightly enhanced. ACh never evoked biphasic potentials, but always large hyperpolarizations.5. In the first period (1 hr) after exposure to a K-free solution ACh normally evoked very large hyperpolarizations, often to more than -100 mV. After several hours of exposure to K-free solution the input resistance gradually increased and ACh evoked a tremendous fall in input resistance and time constant with only a small potential change. Re-introducing control solution, ([K](o) = 4.7) for a short period at this stage, caused a very marked hyperpolarization (about 30 mV) unaccompanied by a change in input resistance and time constant.6. Replacing extracellular Cl by SO(4) hyperpolarized the cell membrane. ACh mostly evoked hyperpolarization under this condition, but occasionally biphasic potentials were observed. Increasing [K] of the sulphate solution depolarized the cell membrane by about 49 mV per tenfold increase in [K]. In the presence of ACh the membrane behaved as a K-selective membrane with a slope of the linear curve relating membrane potential to [K](o) of 59 mV per tenfold increase in [K](o).7. It is concluded that ACh evokes a marked increase in surface cell membrane permeability of salivary acinar cells. The ACh evoked hyperpolarization is due to an increase in P(K): the depolarization frequently preceding the hyperpolarization is probably mainly related to an increase in P(Na). The membrane Na-K pump can act electrogenically at least under conditions of Na loading.     

Contributions of the sodium pump and ionic gradients to the membrane potential of a molluscan neurone

1. The membrane potential of the gastro-oesophageal giant neurone of the marine mollusc, Anisodoris nobilis, was examined during changes of temperature and of the ionic medium.2. The response of the membrane potential to rapid changes in the external K concentration was prompt, stable, and reversible up to 200 mM-K, and was independent of the external Cl concentration.3. Warming the cell produced a prompt hyperpolarization that was approximately 10 times greater than predicted by the Nernst or constant field equations. Electrogenic activity of the Na-K exchange pump was shown to be responsible for this effect.4. At temperatures below 5 degrees C, the relationship between the membrane potential and the external K concentration could be predicted by a constant field equation.5. At temperatures above 5 degrees C, the membrane potential could not be predicted by the constant field equation except after inhibition of the electrogenic Na pump with ouabain or the reduction of internal Na.6. Inhibition of the electrogenic Na pump by low external K concentrations was dependent upon the external Na concentration.7. It is concluded that the membrane potential is the sum of ionic and metabolic components, and that the behaviour of the ionic component can be predicted by a constant field type equation.     

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.     

Contribution of an electrogenic sodium pump to the membrane potential in rabbit sinoatrial node cells

A study has been made of the transient hyperpolarization (K+-induced hyperpolarization) which developed following readmission of potassium after having pre-treated the rabbit sinoatrial node tissue with K+-depleted Tyrode solution for 4--5 min at 35 degrees C. Evidence is presented indicating that the K+-induced hyperpolarization results from the activity of an electrogenic sodium pump: The K+-induced hyperpolarization was inhibited by substituting Li+ for Na+ and by cooling the tissue. The amplitude of the K+-induced hyperpolarization was increased either by increasing K+ concentration in the recovery solution or by decreasing K+ concentration in the pre-treatment K+-depleted solution. By removing Cl- from the perfusates, the amplitude of the K+-induced hyperpolarization increased. In a Cl--depleted solution, the sinoatrial node cell membrane hyperpolarized by approximately 15 mV without a transient depolarization.