1. Exchangeable Ca in guinea-pig auricles and ventricular trabeculae of sheep and calf hearts was labelled with (45)Ca and the loss of radioactivity into inactive rinsing solutions of different ion composition was measured for periods up to 6 hr. At no time did the decrease of radioactivity in the muscle follow a single exponential course, while the rate coefficient k (= fraction of (45)Ca lost per minute from muscle into rinsing solution) decreased slightly with time.2. On the basis of the temperature-sensitivity of Ca efflux from auricles the activation energy has been calculated to have a value of 5900 cal/mole, corresponding to a Q(10) of 1.35. 2,4-Dinitrophenol (5.5 x 10(-5) - 5.5 x 10(-4)M) had either no effect on Ca efflux or increased it slightly.3. Compared to control efflux in 1.8 mM [Ca](o) Ca efflux decreased to 70% in Ca-free solution, to 20% in Ca-free, Na-free solution and to 65% in Ca-containing, Na-free solution, NaCl being replaced by either sucrose or LiCl. Quantitatively, Ca efflux from auricles has been shown to depend to a large extent on the ratio [Ca(2+)](o)/[Na(+)](o) (2). The affinity for Na of the activation site for Ca efflux (carrier) is much less than for Ca.4. The efflux from muscles soaked for about 2 hr in Ca-free solution was not linearly related to Ca-concentration in the tissue but followed a square law.5. While Ca content in auricles increased in Ca-containing, Na-poor solution it decreased again when Tyrode solution was readmitted indicating a Na-sensitive Ca net transport in cardiac muscle.6. The results are interpreted in terms of a modified exchange diffusion mechanism (Ussing, 1947) which is responsible for Ca extrusion from mammalian cardiac muscle.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1968.sp008467

The dependence of calcium efflux from cardiac muscle on temperature and external ion composition

Obligatory, coupled cotransport of Na+, K+, and Cl− by cell membranes has been reported in nearly every animal cell type. This review examines the current status of our knowledge about this ion tra...

Sodium-Potassium-Chloride Cotransport

1. At high internal K concentrations the efflux of Na from red cells increases with internal Na concentration following an S-shaped curve. As internal K is reduced the S-shaped region and the value of internal Na for which the Na efflux is half-maximal are both shifted progressively towards zero.

2. The effects of internal Na on the shape of the Na efflux curves can be quantitatively accounted for if it is assumed that the rate of Na efflux is linearly related to the number of pump units having three identical and non-interacting sites occupied by Na.

3. The effects of internal K on the shape of the Na efflux curves are fully explained if it is assumed that the inner sites for Na of the Na pump also behave as identical and non-interacting sites for internal K, being the K-carrier complexes unable to promote Na translocation. The apparent affinity of the Na pump for internal K is about 50 times less than for internal Na.

4. Internal K not only alters the apparent affinity of the Na pump for Na, but also affects its turnover rate. The turnover rate of Na: K exchange increases with internal K following a curve which saturates at about 30 m M internal K. The turnover rate for Na:Na exchange increases linearly with internal K.

5. The linear dependence of the rate of Na:Na exchange on internal K explains why, when internal Na is increased at the expense of internal K, the rate of Na:Na exchange progressively decreases after passing through a maximum.

6. The effects of external Na on the rate of Na:Na exchange can be satisfactorily explained assuming that they are due to the occupation by external Na of three identical and non-interacting sites on each pump unit. The apparent affinity of the Na pump for external Na is about 160 times less than the apparent affinity for internal Na.

7. Under all the experimental conditions tested, it was found that the relation between flux and cation concentration at one of the surfaces of the cell membrane is altered only by a constant factor by changes in the cation composition at the opposite surface of the cell membrane. This fact strongly suggests that there are no interactions between the inner and outer sites of the Na pump.

8. The effects of inner and outer cations on both the Na:K and the Na:Na exchanges catalysed by the Na pump suggest that cation fluxes are proportional to the number of pump units having its inner and outer sites simultaneously occupied by the relevant cations. It seems therefore that sequential models for ion transort do not provide an adequate description of the molecular mechanism of active transport in red cells.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1973.sp010234

The interaction of sodium and potassium with the sodium pump in red cells

The (Na+ + K+)-activated ATPase. Enzymatic and transport properties.

1. Fifty to ninety per cent of the Na efflux from axons of Loligo forbesi is inhibited by ouabain. The properties of the ouabain-sensitive component of the Na efflux are different from those of the ouabain-insensitive component.

2. In unpoisoned axons with an average Na content of 75 m-mole/kg axoplasm the bulk of the ouabain-sensitive Na efflux is dependent on external K.

3. In the presence of 460 m M Na in the external medium, raising the external K concentration from 0 to 100 m M increases the ouabain-sensitive Na efflux along a sigmoid curve which shows signs of saturating at high K concentrations.

4. The curve relating ouabain-sensitive K influx to external K concentration is similar in shape to that for the ouabain-sensitive Na efflux. At all K concentrations examined the ouabain-sensitive K influx was less than the ouabain-sensitive Na efflux.

5. Potassium-free sea water acts rapidly in reducing the Na efflux. There is no appreciable difference between the rates of action of K-free sea water on the Na pump and Na-free sea water on the action potential.

6. Caesium and Rb can replace external K in activating the ouabain-sensitive Na efflux. Both the affinity and maximum rate of the Na efflux mechanism are lower when Cs replaces K as the activating cation.

7. Isosmotic replacement of external Na by either choline or dextrose, but not Li, increases the affinity of the ouabain-sensitive Na efflux mechanism for external K without appreciably affecting the maximum rate of pumping. External Li behaves like external Na and exerts an inhibitory action on the Na efflux.

8. There is a large ouabain-sensitive Na efflux into K-free choline or dextrose sea waters. Addition of either Na or Li to the external medium reduces this efflux along a section of a rectangular hyperbola. The properties of this efflux suggest that there is a residual K concentration of up to 2 m M immediately external to the pumping sites in the axolemma.

9. Over the range of internal Na concentrations studied (16-140 m-mole/kg axoplasm) the ouabain-sensitive Na efflux increased linearly with Na concentration.

10. Tetrodotoxin (10−6 g/ml.) reduces the Na influx by about half, but does not affect the ouabain-sensitive Na efflux.

11. Isobutanol (1% v/v) reversibly decreases both the ouabain-sensitive and ouabain-insensitive components of the Na efflux.

12. Application of 2 m M cyanide to axons immersed in K-free sea water produces a transient rise in the Na efflux. This rise is not seen if ouabain is included in the sea water. The rise in efflux occurs at a time when the axons are partially poisoned and contain adenosine triphosphate (ATP) but no arginine phosphate (ArgP). A similar, but maintained rise can be obtained after application of dinitrophenol (DNP) at pH 8·0. The increased Na efflux in these partially poisoned axons is also inhibited by ouabain.

13. Under conditions of partial-poisoning by alkaline DNP, there is a ouabain-sensitive Na influx from K-free sea water. The ouabain-sensitive Na influx is of similar size to the ouabain-sensitive Na efflux. These results show that in partially-poisoned axons immersed in K-free sea water intracellular Na exchanges with extracellular Na in a one-for-one manner by a ouabain-sensitive route. External Li cannot replace external Na in maintaining this process.

14. Axons partially poisoned with alkaline DNP are not insensitive to external K. In the absence of external Na their response to external K is essentially the same as that seen in unpoisoned axons.

15. Possible mechanisms are discussed for the appearance of Na-Na exchange in partially poisoned axons.

The ouabain-sensitive fluxes of sodium and potassium in squid giant axons.

1. When red cells loaded with 24Na are incubated in balanced glucose salt solutions, the removal of external potassium reduces sodium efflux by about one third. Ouabain reduces the residual sodium efflux by about one half.

2. The ouabain-sensitive efflux of sodium into potassium-free solutions is accompanied by an equal ouabain-sensitive influx of sodium.

3. If sodium in the external potassium-free salt solution is progressively replaced with choline, both the ouabain-sensitive sodium efflux and the ouabain-sensitive sodium influx are reduced until, with only 5 m M-Na externally, both become very small.

4. At sodium concentrations intermediate between 140 m M and 5 m M, ouabain-sensitive sodium influx and ouabain-sensitive sodium efflux remain equal within the limits of experimental error. The relation between the magnitude of the ouabain-sensitive sodium exchange and the external sodium concentration is roughly linear.

5. As the external sodium concentration is decreased beyond 5 m M towards zero, ouabain-sensitive sodium efflux increases again.

6. In the presence of 5 m M-K, the ouabain-sensitive sodium efflux is scarcely affected by replacing most of the external sodium with choline.

7. In the presence of ouabain, sodium efflux is unaffected by external potassium and is little affected by replacing external sodium with choline.

8. The results suggest that in the absence of external potassium the ouabain-sensitive transport mechanism catalyses a one-for-one exchange of sodium ions across the cell membrane. The relation between this exchange and Ussing's classical ‘exchange diffusion’ is discussed.

9. The exchange does not occur in the presence of external potassium at physiological concentrations.

10. Calculations of sodium efflux were based on the specific activities of total cell sodium: separate experiments confirmed that these were the same as the specific activities of sodium lost to sodium-free solutions. No evidence of a slowly exchanging sodium fraction was found.

P. J. Garrahan

Papers

The behaviour of the sodium pump in red cells in the absence of external potassium