Sodium transport by perfused giant axons of Loligo.

The sodium efflux from perfused squid giant axons has been studied using radioactive sodium, and the sufficient conditions for the maintenance of a potassium- and ouabain-sensitive sodium efflux have been established. The following were found.1. Axons extruded and then perfused with their own axoplasm had a sodium efflux which was sensitive to cyanide, potassium and ouabain and was thus similar to the efflux from intact axons.2. A method for replacing natural axoplasm into fibres previously perfused with artificial axoplasm was developed and used to establish an artificial perfusate that was not irreversibly toxic.3. Short perfusion (5 min) with a variety of artificial perfusates was then found to give fibres which had potassium- and ouabain-sensitive sodium effluxes when ATP was present in the perfusate.4. In the absence of ATP the sodium efflux was small and relatively insensitive to both external potassium and to ouabain.5. With ADP in the perfusate, fibres gave a sodium efflux which was ouabain-sensitive but was little affected by the removal of external potassium from the sodium-rich sea water bathing the fibres.6. The perfused fibres differed from intact fibres in having large ouabain-insensitive sodium effluxes.7. After very long perfusions (40-90 min), with the simple media containing ATP, the rate constant for sodium efflux from the fibres tended to be large and was relatively insensitive to potassium or to ouabain.8. Fibres refilled with natural axoplasm after long perfusion showed increased sensitivity to external potassium; refilled with dispersed axoplasm the sodium efflux tended to become very large.9. After very long perfusions with artificial axoplasms containing ATP, a potassium- and ouabain-sensitive sodium efflux was found to persist provided that dextran was present and the total osmotic pressure and the hydrostatic pressure of the perfusate were controlled. Under these conditions the sodium efflux resembled that from briefly perfused fibres. The necessary and sufficient conditions for the maintenance of sodium transport by perfused giant axons are discussed.

The production of adenosine triphosphate in perfused giant axons of Loligo.

1. The light production by squid giant axons perfused with solutions containing extract of firefly tails has been studied to give information about the production of ATP in such axons.2. An initial flash occurs when the perfusion fluid first enters the fibre. There is a secondary production of light, noticeable when the perfusion is halted, providing glutamate or aspartate are present in the perfusion medium.3. There is no secondary light production if glutamate is replaced by sulphate, succinate or alpha-ketoglutarate in the perfusion fluid.4. Ouabain has no effect whereas cyanide and oligomycin both block the secondary light production, the latter irreversibly.5. When the sodium outside the fibre is replaced by lithium or choline the secondary light production is often reduced and occasionally abolished.6. A raised internal sodium does not enhance secondary light production.7. The secondary light production is dependent upon the concentration of AMP in the perfusion solution.8. Fresh axoplasm generates a powerful light on immersion in perfusion fluid whereas dialysed axoplasm, even in the presence of added glutamate, generates no light whatever.9. The evidence, on balance, suggests that, under the conditions of the experiments described, there is no detectable reversal of active transport in perfused nerve fibres but that there is an enzyme system, probably membrane bound, capable of generating ATP from glutamate or aspartate and AMP by oxidative phosphorylation. The enzyme system can be inhibited by the replacement of external sodium.

The circular dichroism of suspensions of frog rod outer segments.

The circular dichroism (CD) of suspensions of frog rod outer segments has been measured. At wave-lengths between 600 and 400 nm1. The suspensions show a large positive CD increasing towards shorter wave-lengths and largely unaffected by bleaching. This probably arises, at least in part, from the preferential scattering of one form of circularly polarized light.2. There is a small reduction in CD on bleaching, slightly larger but similar in sign and wave-length dependence to that shown by pigment extracts. At wave-lengths between 250 and 220 nm3. The suspensions show a CD somewhat similar in sign, magnitude and wave-length dependence to that given by pigment extracts.4. On bleaching there is a reduction of the negative CD at 225 nm again similar to that which takes place when extracts are bleached. The observations suggest that configurational changes, of the kind detected by CD, occur in the bleaching of rhodopsin molecules whether they are present in solutions or in rods.

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

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 mM Na in the external medium, raising the external K concentration from 0 to 100 mM 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 mM 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 mM 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.

An upper limit to the number of sodium channels in nerve membrane?

1. A small volume of artificial sea water containing 300 nM tetrodotoxin (TTX) was applied successively to seven lobster nerve trunks and the cumulative uptake of toxin investigated by bio-assay.2. Light and electron microscopy indicated that the nerve trunks had a total axonal area of 0.7 x 10(4) cm(2)/g.3. Sodium analysis gave a sodium space for the nerve trunks of 30%.4. The amount of toxin taken up by the cells in 1 g of nerve is less than 1.6 x 10(-11) moles.5. It is argued that there are probably fewer than 13 sodium channels/mu(2) axon in lobster nerve.

Cation movements in perfused giant axons.

1. The net movements of Na and K into resting squid giant axons perfused with a K(2)SO(4) artificial axoplasm have been determined. Na enters at a rate of 131 +/- 25 p-mole/cm(2).sec. The K leakage from the fibre is 942 +/- 566 p-mole/cm(2).sec; clearly this latter figure shows how large is the uncertainty in the exact value for the potassium leakage.2. The net entry of sodium associated with activity in such fibres has been measured and is 5.7 +/- 0.7 p-mole/cm(2).impulse.