The oxygen consumption of mammalian non-myelinated nerve fibres at rest and during activity

1. A study has been made of the oxygen consumption of non-myelinated nerve fibres of rabbit desheathed cervical vagus nerves at rest and during activity.2. The average resting oxygen consumption (Q_r) was 0·0924 μmole/g. min at 21° C. Stimulation for 1-3 min at 3/sec caused an extra oxygen consumption (Q_s) of 816 p-mole/g.shock.3. When the frequency of stimulation was increased, to 10/sec and 30/sec, Q_s fell. When the frequency was decreased, to 1/sec and 0·3/sec, Q_s increased slightly.4. When the temperature was decreased, Q_r fell; when the temperature was increased, Q_s also increased. Temperature similarly affected Q_s with high frequencies of stimulation, but had relatively little effect on Q_s at low frequencies of stimulation.5. An isolated single shock seemed to produce an increase in oxygen consumption of about 1200 p-mole/g, and this value was largely independent of temperature.6. When part of the sodium in the Locke solution was replaced by barium, Q_r decreased (by 12%) whereas Q_s increased (by 87%).7. Veratrine (1 μg/ml.) increased both Q_r (by 142%) and Q_s (by 361%).8. Acetylcholine (1·7 mM) increased Q_r (by 32%).9. When nerves were transferred to potassium-free solutions there was little change in Q_r, and Q_s fell slightly (by 8%).10. When the potassium concentration in the Locke solution was increased 4-fold, Q_r increased (by 27%).11. Salicylate (1-10 mM) increased Q_r (by 24%) and abolished Q_s.12. When the sodium of Locke solution was replaced by lithium, Q_r decreased (by 19%) and Q_s was abolished.13. In sodium-Locke solution ouabain (100 μM) decreased Q_r (by 26%) and abolished Q_s. In lithium-Locke solution ouabain also decreased Q_r (by 28%).14. All or nearly all of the oxygen consumed at rest or during activity seemed to be used to pump potassium ions into, and sodium ions out of, the axoplasm.15. The K/O₂ ratio during pumping was about 5·0.     

On the electrogenic sodium pump in mammalian non-myelinated nerve fibres and its activation by various external cations

1. A study has been made of the hyperpolarization that follows a period of electrical activity (the post-tetanic hyperpolarization) in mammalian non-myelinated nerve fibres.2. Evidence is presented that under certain circumstances this postetanic hyperpolarization is a result of activity of an electrogenic sodium pump that normally is absolutely dependent on the external presence of potassium.3. When the external chloride is replaced by sulphate or by isethionate the post-tetanic hyperpolarization, which in normal Locke solution is only a few millivolts in amplitude, is increased usually to about 20 mV, and on occasion to 35 mV.4. This effect of removing the chloride takes several minutes to develop and is consistent with the idea that the increase in the post-tetanic response is the result of removing the short-circuiting effect of internal chloride ions (by their being washed out into the chloride-free bathing medium).5. Small anions, such as chloride, nitrate, iodide, bromide, and thiocyanate can short-circuit the electrogenic pump, whereas larger anions such as sulphate and isethionate cannot. The bicarbonate ion, which is larger than chloride, short-circuits the pump but less effectively.6. In Locke solution containing 5 mM potassium the post-tetanic hyperpolarization declines exponentially, with a time constant of about 1-3 min. The time constant is inversely related to the external potassium concentration.7. However, when the external potassium concentration is zero the hyperpolarization declines rapidly to a very small value. Subsequent addition of potassium to the bathing medium causes a marked redevelopment of the hyperpolarization.8. This potassium-activated response declines exponentially with a time constant that is inversely related to the potassium concentration. When the added potassium concentration is 5 mM, the time constant is 1.9 min.9. The amplitude of the potassium-activated response increases with increasing concentrations of potassium.10. Other cations can produce this activated response. Thus, thallium is more effective than, rubidium as effective as, caesium and ammonium about 1/10 as effective as, and lithium ions about 1/30 as effective as potassium in producing the activated response. Choline is quite ineffective.11. The size of the post-tetanic response is little affected by changes in the duration of the period of stimulation. However, increasing the duration definitely increases the time constant of recovery.12. Reducing the external sodium concentration increases the size of the post-tetanic hyperpolarization (by about 25%), but the effect is complex and requires further study.13. Reducing the calcium of the Locke-solution from 2.2 to 0.2 mM has no appreciable effect on the post-tetanic response, nor has increasing the pH of the Locke from 7.2 to 9.2.14. When the membrane potential is increased or decreased, by externally applied currents, there is relatively little change in the post-tetanic response.15. A mathematical model of the electrogenic pump, devised to mimic the experimental results, was analysed with an analogue computer. A satisfactory agreement between model and experiment was achieved by a model in which: (1) the rate of extrusion of sodium ions depends on the degree to which a pool of carrier molecules on the inside surface of the membrane is combined with sodium; (2) each carrier molecule transfers three sodium ions at a time; (3) the rate constant for extrusion of sodium ions also depends on the presence externally of potassium ions, which combine with some sites on the external surface of the membrane that are half-saturated when the external concentration of potassium is 2.8 mM.     

The interaction at equilibrium between tetrodotoxin and mammalian non-myelinated nerve fibres

1. As a preliminary to chemical studies an estimate has been made of the equilibrium dissociation constant (K) for the interaction of tetrodotoxin (TTX) with the non-myelinated fibres of the rabbit desheathed vagus nerve.2. TTX causes a parallel shift to the right of the curves obtained when either the height or the conduction velocity of the compound action potential are plotted against the logarithm of the external sodium concentration.3. A model has been formulated based on the independence principle and the Hodgkin-Huxley theory, and the experimental results shown to be consistent with it. On this basis, and on the assumptions that one TTX molecule blocks one sodium channel, and that binding is Langmuir, K was estimated to be about 3-5 nM at about 20 degrees C. Other simple non-Langmuir models gave essentially similar low values for K.4. An alternative method of computing K that makes rather different assumptions gives a similar low value.5. Despite the low value for K, a TTX concentration of at least 100 nM is needed to block conduction completely and this seems to be related to the fact that conduction is not completely blocked until the external sodium concentration falls below 7% of its value in normal Locke.6. The minimum sodium concentration needed to support conduction increased with temperature.     

The binding of tritiated ouabain to mammalian non-myelinated nerve fibres

1. A study has been made of the binding of radioactively labelled ouabain by desheathed rabbit vagus nerves, which consist mainly of non-myelinated fibres. The corresponding inhibition of the electrogenic sodium pump was also measured.2. By varying the ouabain concentration and the external potassium concentration two kinds of binding sites could be distinguished: a first site specifically associated with pumping and whose ability to bind ouabain is dependent on the external presence of potassium; and a second site not associated with pumping and unaffected by external potassium.3. Just complete inhibition of the sodium pumping mechanism is associated with a specific binding of ouabain of about 4.3 p-mole/mg dry nerve.4. This gives an upper limit for the density of sodium pumping sites of about 750 per square micron.5. The turnover rate (i.e. (cation pumped)/(number of sites)) at 20 degrees C is about 22 sec(-1).     

The ionic content of mammalian non-myelinated nerve fibres and its alteration as a result of electrical activity

1. A study has been made of the ionic content, and of the fibre water, of rabbit desheathed vagus nerves at rest and after activity in various modified Locke solutions.2. In normal Locke solution the intracellular sodium amounted to 86.1 mumole/g dry, and the intracellular potassium 186.0 mumole/g dry. Since the fibre water amounted to 1.124 g/g dry the intracellular sodium and potassium concentrations were 76.6 and 165.5 m-mole/kg fibre water, respectively.3. In potassium-free Locke solution the intracellular sodium and potassium concentrations were 111.0 and 121.7 m-mole/kg, respectively. The intracellular chloride concentration was 40.5 m-mole/kg.4. In normal Locke solution, a brief period of stimulation followed by a 10 min recovery period produced no significant change in the intracellular contents of potassium, sodium, or water.5. However, in chloride-Locke solution from which the potassium had been omitted, or to which ouabain (1 mM) had been added, stimulation caused a loss of potassium per impulse of about 20 p-mole/mg dry, i.e. about 4 p-mole/mg wet. A similar loss was found in isethionate-Locke solution that was potassium-free. There was no significant change in any of the other quantities measured (sodium, chloride, and fibre water).6. The efflux of radioactively labelled potassium was measured in potassium-free Locke solution at rest (k(r)) and during activity (k(s)). At 24 degrees C, k(r) was 0.0084 min(-1) and k(s) was 0.000178 impulse(-1).7. When the chloride of Locke solution was replaced by isethionate there was an increase in both k(r) (about 12%) and k(s) (about 21%).8. Ouabain (1 mM) increased k(s) (by about 70%). This increase seemed to be independent of whether the anion present was chloride or isethionate.9. It is argued that these results indicate that the electrogenicity of the pump plays a relatively small role in maintaining the ionic balance in mammalina C fibres.     

The effects of external cations and ouabain on the intracellular sodium activity of sheep heart Purkinje fibres

1. The intracellular Na activity of sheep heart Purkinje fibres has been measured using recessed-tip Na(+)-sensitive glass micro-electrodes.2. The internal Na activity was 7.2 +/- 2.0 mM (mean +/- S.D., n = 32) at the normal external Na concentration, [Na](o), in these experiments of 140 mM (equivalent to an external Na activity of 105 mM). The equilibrium potential for Na across the fibre membrane was therefore approximately + 70 mV.3. When the [K](o) was altered the internal Na activity changed, reaching a new level within about 20 min. Increasing the [K](o) from 4 to 25 mM decreased the internal Na by approximately 30%, while decreasing the [K](o) from 4 to 1 mM increased internal Na by 20%.4. The removal of external K produced an easily reversible increase in the internal Na with an initial rate equivalent to a concentration change of 0.24 +/- 0.07 m-mole/min (mean +/- S.D., n = 8).5. Ouabain produced increases in the internal Na activity that were only very slowly reversible. The threshold concentration for producing an increase was approximately 10(-7)M.6. When [Na](o) was reduced the internal Na activity fell rapidly with a single exponential time course (time constant 3.3 +/- 0.8 min, mean +/- S.D., n = 16) to a new, relatively stable level. The recovery of internal Na on return to the normal [Na](o) did not have a simple time course. It was normally complete within 10-30 min.7. The relationship of the stabilized level of the internal Na activity to the [Na](o) was approximately linear over the range 140-14 mM-[Na](o). When [Na](o) was reduced from 140 to 14 mM the internal Na activity fell by 72 +/- 5% (mean +/- S.D., n = 21).8. When the [Na](o) was reduced, the decrease in the internal Na activity was partially inhibited by Mn or by removal external Ca.9. When the [Ca](o) was altered over the range 0.2-16 mM the internal Na activity was reduced by approximately 50% for a tenfold increase in the [Ca](o).10. The relationship between internal Na and contractility is discussed.     

Optical studies on the kinetics of the sodium pump in mammalian non-myelinated nerve fibres

1. A study has been made of the changes in the fluorescence of desheathed rabbit cervical vagus nerves that occur during and after electrical stimulation of its non-myelinated fibres.2. Stimulation for 5 sec at 30 shocks/sec produces a maximal decrease, of about 1% of the resting fluorescence. Stimulation for less than 0.5 sec fails to produce responses visible above the inherent noise in the recording system.3. A pharmacological dissection (with ouabain, metabolic inhibitors, and calcium) has revealed four phases of fluorescence change:(a) under conditions where the sodium pump is functioning, there is a prolonged decrease in the fluorescence following electrical activity;(b) even in the absence of pumping the mere entry of sodium into the nerve causes an initial decrease in fluorescence;(c) the entry of calcium ions with electrical activity also causes an initial rapid decrease in fluorescence;(d) following these phases of decreased fluorescence there is a phase of increased fluorescence.4. These changes in fluorescence are related to changes in the NADH concentration in the nerve resulting from:(a) the splitting of ATP during sodium extrusion;(b) the initial binding of sodium to the sodium- and potassium-dependent ATPase, which is the sodium pump;(c) the stimulation of mitochondrial respiration by calcium that has entered during the spike; and(d) an increased glycogenolysis as a result of the calcium entry during activity.     

The origin of the initial heat associated with a single impulse in mammalian non-myelinated nerve fibres

1. A study has been made of the temperature changes associated with the passage of a single impulse in rabbit desheated vagus nerves.2. The initial changes consist of an evolution of positive heat followed by a reabsorption of most of it; i.e. there is a phase of positive and a phase of negative heat production.3. The size of the positive heat, its time of onset, and the time of onset of the negative heat have been measured by an analogue method of analysis. In addition, these parameters, together with the size of the negative heat and the duration of both phases of initial heat, have been studied with the aid of a computer, and also by conventional heat block analysis.4. At about 5 degrees C the measured positive heat is 7.2 mucal/g. impulse. It starts as soon as the compound action potential reaches the thermopile and lasts for about 107 msec.5. This positive heat decreases with increasing temperature, the ratio of heat at 4 degrees C to that at 14 degrees C being 1.86.6. The measured negative heat at about 5 degrees C is 4.9 mucal/g. impulse. It starts 102 msec after the onset of positive heat, and lasts for about 240 msec.7. When the sodium of Locke solution is replaced by lithium the positive heat is reduced by 19%, but the negative heat is increased by 22%.8. In potassium-free solutions the positive heat is hardly affected (increase of 5%), but the negative heat is more than doubled. As a result the nerve may become briefly colder than its initial temperature by about 2 mu degrees C.9. The effect of sodium-deficient solutions on the positive heat is somewhat variable, but the negative heat is consistently diminished.10. Replacement of the chloride of Locke solution by sulphate or nitrate has little effect on the positive heat. The negative heat is reduced in size by 26% and in duration by 22%.11. Replacement of most of the sodium of Locke solution by barium reduces or abolishes the negative heat, and increases the measured size of the positive heat nearly threefold.12. Veratrine (10(-5) g/ml.) produces a nearly tenfold increase in the net positive heat.13. Ouabain (1 mM) and antimycin A (1 mug/ml.) applied for 30-60 min have little effect on the initial heat production.14. Over the temperature range 5-15 degrees C, and for the ionic solution changes described above, there is close agreement in timing between the positive heat and the rising phase of the action potential, and between the negative heat and the falling phase.15. Because of the inevitable temporal dispersion of the action potential over the face of the thermopile, the observed temperature changes are smaller than those which occur at a single point in the nerve close to a stimulating electrode. The corrected value of the positive heat at 5 degrees C is 24.5 mucal/g. impulse, while that of the negative heat is 22.2 mucal/g. impulse.16. The heats of mixing of the ions in solution that interchange during the action potential are much too small to account for the observed initial heats, but an exchange of ions associated with fixed charges might make a significant contribution to the heats.17. The condenser theory, according to which the positive heat represents the dissipation of electrical energy stored in the membrane capacity, while the negative heat results from the recharging of the capacity, appears unable to account for more than half of the observed temperature changes.18. It seems probable that the greater part of the initial heat results from changes in the entropy of the nerve membrane when it is depolarized and repolarized.