The Effect of Ouabain on the Efflux of Sodium Ions in the Nerve Cords of Two Insect Species (Periplaneta Americana and Carausius Morosus)

Recent studies have shown that the inorganic ions in insect central nervous tissues can be separated into two fractions on the basis of the kinetics of their exchange with the haemolymph (Treherne, 1961b, 1962, 1965a). In these studies the rapidly exchanging ions were identified as those situated in the extracellular spaces, the slowly exchanging fractions representing the intracellular ions. The efflux of the slowly exchanging sodium was found to be reduced in the presence of dilute metabolic inhibitors, suggesting that the relatively low intracellular concentration of this cation is maintained by the presence of secretory processes directed into the extracellular fluid. As with cells from other animal groups (cf. Harris, 1954; Hodgkin & Keynes, 1954) the outward movement of this cation appears to be achieved by a linked-ion pump, for sodium efflux was found to be substantially reduced in the absence of external potassium ions (Treherne, 1961a).

The sodium and potassium movements taking place in a wide variety of animal cells have been shown to be reduced in the presence of various cardiac glycosides, an effect which appears to result from the inhibition of the carrier system involved in active transport rather than to any uncoupling of the energy supply involved in this process (cf. Glynn, 1964). In insects, however, the uptake of potassium by the Cecropia mid-gut (Haskell, Clemons & Harvey, 1965) and the movements of sodium in Malpighian tubules (Maddrell, unpublished observations) have been found to be unaffected by the presence of ouabain. It is of some interest, therefore, to know whether the extrusion of sodium from insect nerve cells is affected by this substance or whether they differ from vertebrate excitable cells in this respect, perhaps as a consequence of a different carrier-mechanism associated with the specialized ionic environment of the nerve cells in some insect groups. The present investigation was undertaken to determine the effect of this cardiac glycoside on sodium movements in the nerve cords of the cockroach (Periplaneta americana), which has a normal, relatively high, sodium content in the haemolymph, and in the stick insect (Carausius morosus), which has an abnormal haemolymph containing a very low concentration of sodium ions.

In these experiments the efflux of sodium was measured in nerve cords which had been made radioactive by soaking them in a solution containing ²²Na at a concentration similar to that of the haemolymph. To do this individual insects were decapitated, the dorsal integument was cut away and the viscera were removed. The body cavity was then filled with the radioactive solution so as to cover the nerve cord completely. With Periplaneta the nerve cord was exposed to the experimental solution for 30 min. and with Carausius for 1 hr. The nerve cord was then removed, freed of adhering tissue and carefully blotted on filter paper. With Periplaneta only the abdominal nerve cord was used, with Carausius the whole ventral nerve cord. The isolated radioactive nerve cord was then washed in successive aliquots of non-radioactive physiological solution, the radioactivity of the washings being measured using a Labgear counter.

The physiological solution used in the experiments with Periplaneta was that devised by Van Asperen & Esch (1956) and had the following composition: Na 157·0 IDM/I. ; K, 12·3 mM/1.; Ca, 4·5 HIM/I.; Mg,4·5mM/l.; Cl, 184·1 mM/1.; HCO₃,2·1 mM/1.; H₂PO₄, 0·1 mM/1.; glucose 133·0 mM/1.). The solution used with Carausius was that of Wood (1957) and was made up as follows: Na. 15·0 mM/1.; K, 18·0mM/l.; Ca, 7·5 mM/1.; Mg, 50·0 mM/1.; H₂PO₄, 6·0MM/I.; HPO₄, 4·5 mM/1.; Cl, 133·0 mM/1.; the osmotic concentration of the fluid was maintained by the addition of 204·2 mM/1. glucose.

All the experiments were carried out at a temperature of 25·0° C.

As with normal preparations (Treherne, 1961b, 1962, 1965a) the efflux of sodium from the nerve cord was found to occur as a two-stage process in the presence of ouabain (Fig. 1). The cardiac glycoside was, however, found to reduce greatly the efflux of radiosodium in the slowly exchanging fraction. Fig. 2 illustrates the effect of three concentrations of ouabain on the escape of sodium from the nerve cord of Periplaneta. In the presence of 10⁻⁵ M ouabain the rate constant for sodium extrusion was reduced from a mean value of 3·94 × 10⁻³ sec.⁻¹, with normal preparations, to a mean value of 2·23 × 10⁻³ sec.⁻¹. Increasing the concentration of ouabain to 10⁻⁴ M reduced the exchange constant to a mean value of 1·8o × 10⁻³ sec.⁻¹, the further increase to 10⁻³ M producing no further appreciable effect on sodium efflux.

Ouabain was also found to affect the slowly exchanging sodium fraction in the nerve cord of Carausius. Fig. 3 illustrates the effect of the addition 10⁻⁴ M ouabain to the bathing solution on the escape of ²²Na in this preparation. The normal exponential decline in radioactivity (K_out = 1·14 × 10⁻³ sec.⁻¹) was reduced in the ouabain-treated preparations to a mean value of 6·18 × 10⁻⁴ sec.⁻¹.

The addition of dilute cyanide to ouabain-treated preparations was found to produce very little effect on sodium efflux. Fig. 4 illustrates these observations in which 10⁻³ M KCN was added to a bathing solution containing 10⁻⁴ M ouabain.

It is clear from the above results that the intracellular efflux of sodium ions was appreciably reduced in the presence of ouabain in the nerve cords of both insect species. In Periplaneta addition of 10⁻⁴M ouabain to the bathing medium caused a reduction in the rate constant (K_out) from 3·94 × 10⁻³ sec.⁻¹ to 1·80 × 10⁻³ sec.⁻¹ and in Carausius from 1·14 × 10⁻³ sec.⁻¹ to 6·18 × 10⁻⁴ sec.⁻¹ in the presence of this substance. These figures indicate that about half of the sodium efflux was blocked in the presence of the cardiac glycoside, the ouabain-sensitive fractions corresponding to 0·54 and 0·45 respectively. Uncoupling of the energy supply to the sodium pump, by the addition of cyanide, did not further reduce the sodium efflux from ouabain-treated axons. This suggests that the greater part of the active extrusion of sodium from the axoplasm is effected by a single ouabain-sensitive carrier mechanism.

The effect of ouabain on sodium movement in these insect preparations was much less than that observed with squid giant axons, in which the sodium efflux of normal preparations was as much as 8 times greater than that measured in the presence of ouabain (Caldwell & Keynes, 1959). The relative insensitivity of the insect axons to ouabain can, as a first approximation, be related to the concentration gradient of sodium maintained between the intracellular and extracellular compartments in the nerve cord. Fig. 5 summarizes some of the available data, showing the relation of the ouabain-sensitive fraction of sodium efflux to the concentration gradient maintained across the cell membrane for several cell types. These results show that the very large ouabain-sensitive fraction of sodium efflux described for the squid axon can be related to the low concentration of this cation in the axoplasm relative to the bathing medium. Similarly, the cells showing a lower sensitivity to ouabain tend to exhibit a lower concentration gradient of sodium across the cell membrane.

The above estimated value for the outward movement of sodium in cockroach axons implies that their degree of insensitivity to applied ouabain does not result from the presence of a relatively feeble sodium pump, but from the large passive component in sodium efflux which exists in this rather leaky axon membrane. From the data illustrated in Fig. 2 it can be calculated that the ouabain-sensitive fraction in sodium efflux is approximately 67·0 × 10⁻¹²M cm.⁻² sec.⁻¹ as compared with a value of around 25·7 × 10⁻¹²M cm.⁻² sec.⁻¹ which can be estimated for the squid giant axon from the data of Keynes (1951) and Caldwell & Keynes (1959). It also follows from this and from the data illustrated in Fig. 5 that the concentration gradient of sodium across the insect axon membrane can as a first approximation be related to the combined effects of the activity of the sodium pump and the passive permeability of the membrane to sodium ions.