Studies On the Physiology of Arenicola Marina L.: III. The Potassium Relations of the Isolated Extrovert (With Remarks On the Effects of Varying the Lever Weight)

The work was done at Bangor, North Wales. The movements of the extroverts were recorded with light isotonic levers, exerting a pull of 0·35 g. on the preparation, except where otherwise stated. The extroverts were mounted in a cylindrical jar of fluid, which was changed by completely siphoning off the old and pouring in new; this operation takes about a minute and causes an upward movement of the lever at the time of change, which is not due to contraction of the preparation.

Salines were made by mixing solutions each isotonic with sea water. Artificial sea water, which was taken as the ‘normal’ starting point, was made up as follows:

After the extrovert had settled down in this mixture, it was exposed to another, in which the amount of potassium, or of potassium and magnesium, chlorides were abnormal; then, after an exposure usually of several hours, the effects of return to artificial sea water were recorded.

It will be seen, as all mixtures are finally made up to 100 with NaCl, that variations in KC1 or MgCl₂ are osmotically compensated by reciprocal variations in the amount of NaCl included. Osmotic pressure, Ca concentration and SO₄ concentration are therefore constant. All solutions were made up in Na bicarbonate N/400 and were thoroughly aerated before use; this kept the pH constant at 8·0–8·2. Experiments were made at temperatures of 11–21°C.; over this range the preparations behave regularly and well. The temperature did not change significantly in the course of any one experiment.

Concentrations of K and Mg are given below as multiples of their concentrations in the artificial sea water; thus K 1 is 0·0108 M and Mg 1 is 0·058 M. We find that there are four physiologically distinct ranges of potassium concentration, which we designate respectively as moderate excess, severe excess, moderate deficit and severe deficit. This diversity of potassium effects is due, as will be shown below, to the complexity of the normal behaviour pattern of the preparation. The effects in each range will be described separately.

On changing from artificial sea water to K 2, 3 or 3·5, the preparation is at first excited; its tone rises and its activity becomes continuous. As the exposure continues, the extrovert accommodates itself to a large extent to the new medium, and the effects become less marked. On return to K 1 there is generally a clear phase of depression (subnormal tone and activity) which passes off as the preparation accommodates itself back again to the original medium (Fig. 1).

The whole picture is extraordinarily like the effect of diminishing the Mg at constant K concentration (Wells & Ledingham, 1940b), for in that case also one gets excitation and accommodation, followed by depression and accommodation on changing back to artificial sea water. It is therefore hardly surprising to find that the effects of moderate K excess can be fully antagonized if the Mg concentration is simultaneously raised. To demonstrate this antagonism, however, the two ions should not be increased in the same geometrical proportion. Change from artificial sea water to K 3 Mg 3 gives a slight Mg excess picture; the best antagonism is seen with K 3 Mg 2·0–2·2 (the exact ratio seems to vary slightly in different preparations). With such a solution, the effects of the two excesses cancel out completely and the preparation behaves as it does in artificial sea water (Fig. 2). Immediately after the change there may be a period of disturbance, lasting for up to 20 min., during which the effects observed resemble neither those of K nor those of Mg; or this stage may be absent, as in the figure, the preparation continuing its activity without even temporary alteration. In either case, the final result is the same.

Very slight excess (K 1·33 or 1·5) gives a small increase in rhythmic activity followed by accommodation, but no evident tone rise; the picture is like that shown in very slight Mg deficiency.

On changing from artificial sea water to K 5–10, there is contracture and inhibition of rhythmic activity (Fig. 3). The contracture can be regarded as an exaggeration of the tone rise seen with moderate K excess, particularly as it is greatest at the beginning of the exposure. The inhibition of rhythmic activity, on the other hand, could not be anticipated from a consideration of the effects of moderate excess, and shows that a qualitatively distinct concentration range has been reached. With K 5 there is very slight continuous activity of low amplitude, and without the usual intermittent pattern; with K 7·5 or 10 here is no rhythmic activity at all. In neither case can any tendency to accommodation, as evidenced by improvement in the rhythm, be discerned, even in an exposure of over $1112$ hr.

On returning from artificial sea water, there is a sharp drop of tone and, if rhythmical activity was still visible under the conditions of K excess, it now ceases. After a period of complete quiescence, gradual recovery occurs.

The effects of great K excess are not substantially modified by simultaneous increase in Mg (e.g. change to K 5 Mg 5 or K 5 Mg 3). Contracture still occurs, although it is somewhat less marked than when Mg is held constant. The rhythmical activity, instead of being improved, is still further inhibited; this is the inhibitory effect of Mg excess added on to the effects of great K excess. Evidently, then, we are here concerned with phenomena of a different mechanism to those seen with moderate K excess. As regards rhythm, at least, the absolute concentrations of the two ions are too high to allow activity, even when they are balanced.

The boundary between moderate and great excess lies in the neighbourhood of K 4. With this concentration there is rhythmic activity of considerably reduced amplitude in which indications of the normal intermittent pattern can nevertheless be distinguished; the latter is seen more distinctly if Mg is simultaneously raised to Mg 3.

Change from artificial sea water to any low K concentration from K 0·75 to 0·25 has the following results: (1) a sharp and persistent tone drop, (2) at first, a period of continuous rhythmic activity, which lasts the longer, the lower is the new K concentration, and (3) a modified form of the normal behaviour pattern then appears, in which the outbursts are separated by unusually long and unusually quiet rest periods, and every now and again an outburst of unusually long duration appears (Fig. 4). These modifications persist for many hours, with no sign of improvement or accommodation.

In some respects, the moderate K deficit picture recalls that given by Mg excess at constant K concentration (Wells & Ledingham, 1940b); in both cases there is a tone drop, a wider spacing of the outbursts, and a depression of activity between the outbursts. On the other hand, the initial excitement following the downward change, the occasional prolonged outbursts, and the absence of any sign of accommodation, differentiate the moderate K deficit picture from that of Mg excess. The result of simultaneously reducing K and Mg was tried in a considerable number of experiments, with various combinadon of concentrations varying from K 0·5 to 0·25, and from Mg 0·75 to 0·25. Full antagonism was never observed. By itself, a drop in Mg concentration has effects like those of moderate K excess; it causes tone rise and raised rhythmical activity, but does not bring the outbursts closer together. The general result of simultaneously decreasing K and Mg is the sum of the effects of decreasing each of the two ions separately; thus in respect of tone level and the amount of activity between outbursts, which are affected in opposite senses by the two ions, there is antagonism between them, whereas the wide spacing of the outbursts and the occurrence of occasional abnormally prolonged outbursts are shown under conditions of moderate K deficit, whether or not there is also a Mg decrease. As regards this particular preparation, K: Mg antagonism seems to be a factor of very limited importance, being complete only in the range of moderate K excess.

On changing from artificial sea water to K 0·05 or 0·00, there is great excitement with partial contracture and continuous activity. The tone level then drops very slowly; the activity continues in a chaotic manner, without any sign of the normal behaviour pattern, for over 12 hr.; during this time there is a steady loss of amplitude. It is remarkable that this activity continues, though abnormal in pattern, for so long a time, even if the K-free bathing medium is repeatedly changed.

The reactions of the extrovert to severe K deficit in no way resemble those to Mg excess, and it is therefore hardly to be expected that they would be antagonized if Mg were simultaneously reduced. In fact, the simultaneous omission of K and Mg from the bathing fluid gives a record showing the sum of the effects of the two deficits. If Mg alone is suddenly withdrawn, there is contracture followed by a considerable degree of accommodation with restoration of the rhythm (Wells & Ledingham, 1940b). If both ions are withdrawn, contracture is still seen; the rhythm, instead of becoming fairly normal again, shows the disturbances resulting from severe K lack.

The effect of changing back from a K-deficient to a normal fluid depends partly on the extent of the deficit, and partly, in certain cases, on the time for which the tissues have been exposed to it. In a slight deficit, the extrovert settles down almost immediately into a condition in which the normal behaviour pattern is present, though modified. On changing back to artificial sea water, the original pattern is promptly resumed, without any noticeable temporary effects of the change (Fig. 4). In very severe deficit, the pattern disappears altogether, although irregular activity persists for some time; on changing back, there is a period of complete relaxation, after which activity is abruptly resumed (Fig. 5). This pause is the well-known ‘potassium paradox’, which has been observed on return from K lack in a great variety of rhythmic preparations (Wells, 1942). The borderline between moderate and severe deficit is, however, not at all clearly defined. On changing from K 1 to 0·25 or 0·20, there is generally an initial period of confused, continuous activity, superposed on a tone rise, and later the intermittent pattern, as modified by moderate K deficit, gradually emerges. The first stage resembles the beginning of an exposure to severe deficit, and the whole picture suggests that an Accommodation process is taking place. The response of different preparations varies very greatly, especially in the duration of the first stage; this is sometimes well marked even with K 0·33. The suggestion that some sort of accommodation process occurs is confirmed by a curious fact; the K paradox appears on returning to sea water if the exposure was short, but not if it was long, e.g. it is shown after 1 hr., but not after 8 hr., in K 0·25. With severe deficit, on the other hand, there is no evidence of accommodation; the intermittent pattern shows no sign of returning, and the paradox is as well marked after 8 hr. exposure as after 1 hr.

A comparative account of the action of K on rhythmic muscles was recently published by one of us (Wells, 1942). On the whole, the Arenicola extrovert fits in pretty well with the general scheme. The peculiar feature of the extrovert is, of course, the elaboration of its performance; superposed on the simple rhythm of alternating contraction and relaxation, it shows a ‘pattern’ of alternating phases of vigorous rhythmicity and comparative quiescence. If for the moment we disregard the pattern, we find that the extrovert agrees in its general K relations with a great range of preparations of diverse functional and histological types; for with severe excess it shows contracture and inhibition of the rhythm, with severe deficit it shows a lasting tone rise and a rhythm which persists for some time, and on return to a fully balanced mixture from severe deficit it gives the ‘potassium paradox’. The pattern is, however, very sensitive, especially to K lack. In the absence of K from the external medium, a snail or crab or frog heart will trace for some time a record which does not differ very greatly from that seen in a fully balanced fluid; with the lugworm extrovert, on the other hand, the pattern disappears altogether from the moment when K is withdrawn (although rhythm continues for many hours), and even slight K deficit is enough to make striking changes in the timing of the outbursts. There is a remarkable similarity between the outbursts of the extrovert and the ‘grouped beats’ which regularly beating muscles, such as hearts, sometimes show under abnormal conditions, and it has been suggested that the pattern represents the stabilization of a condition which occasionally appears in other preparations as a freak (Wells, 1937). If this be true, one would expect the pattern to be more sensitive to environmental factors than the basal features of rhythm and tone upon which it is superposed, and one finds in fact that it only appears in those K concentration ranges which we have designated as moderate. Under conditions of severe excess or deficit, the extrovert loses its distinctive intermittence and behaves like any one of a wide range of rhythmic muscles.

With regard to the mechanism of the intermittent rhythm, three points arise for discussion:

1. From the available data, it is possible to draw certain conclusions about the dependence of the pattern on ion balances. The K concentration range compatible with its appearance falls into two sharply contrasting parts, for the effects of moderate excess are quite different from those of moderate deficit. In moderate excess, the disturbances resemble those produced by Mg lack, and can be completely abolished by an increase in Mg; the extrovert shows considerable powers of accommodation. We seem, then, to have a very perfect case of K : Mg antagonism. In moderate deficit (except for the special case of the borderline concentrations, K 0·25 and 0·20) there is no accommodation; the disturbances are in many respects unlike those produced by Mg increase and cannot be completely antagonized by lowering the amount of Mg; the balance between the two ions is therefore not the only, or even the main, factor. It would, however, appear that the effects of moderate K deficit are due to disturbance of a balance between K and some other, as yet unidentified, constituent of the medium. We showed elsewhere (Wells & Ledingham, 1940a) that the extrovert behaves with great regularity in sea water diluted to as much as four or five times its volume with buffered distilled water. As the total concentration of salts falls, the amplitude of the contractions becomes less, but. the pattern remains apparently perfectly normal. If, therefore, all the ions are decreased together, the pattern is not disturbed; if, on the other hand, K alone is decreased, the characteristic distortions described above are seen. Clearly, then, K is working against some antagonist other than Mg. The identity of this antagonist is not yet known; perhaps the problem will be solved^- by further experimentation.

2. The results obtained with moderate K excess enable us to develop further the discussion in our paper on Mg actions (Wells & Ledingham, 1940b), of the mechanism of accommodation to that ion. The main facts were: (a) a raised Mg concentration depresses, and a lowered one excites, the extrovert, (b) the extrovert slowly accommodates itself to a large extent to a new concentration, (c) after accommodation, return to artificial sea water causes excitement after raised Mg and depression after lowered Mg, and (d) the extrovert then slowly accommodates itself back into the original condition. To these we may now add: (e) the response to moderate K excess is almost exactly like that to lowered Mg, and (f) if K and Mg are simultaneously raised, the extrovert may show no significant response at all, either temporary or permanent.

The mechanism of the response to change in Mg concentration can be envisaged in various, ways. The accommodation process might directly involve the Mg ion; for instance, the performance of the preparation might depend in some way on the relative amounts of Mg inside and outside the cells; a change in external Mg concentration would upset the balance, and Mg might then move slowly across the cell membrane to restore it. On the other hand, the disturbance caused by change in Mg concentration might be corrected by processes in which the ion itself is not involved. The strict antagonism observed between K and Mg makes the former suggestion hard to entertain. To fit the new facts in, awkward additional postulates would have to be made. It therefore seems to us that a hypothesis of the second type, in which the ions do not take part in the accommodation process, is simpler and more satisfactory.

The extrovert itself, being alive, must necessarily contain steady state chains. If we assume that Mg and K act antagonistically on a velocity constant, under conditions comparable to those of the simple system just described, the observed facts receive a ready explanation.

Note that, if the supply reaction is irreversible (S →A), its rate will be constant and will determine that of the whole chain when a steady state is reached—in other words, accommodation to a change in k will be complete. If it is reversible, the concentration A will affect the supply rate and accommodation will be partial. The latter condition is therefore suggested in the case of the extrovert.

(3) As with most rhythmic muscles, the performance of the Arenicola extrovert is affected to some extent by the pull of the writing lever, and the results obtained by varying that factor in the present case are similar in some respects to those got by varying the K or Mg concentration. Fig. 6 shows an extract from one of a series of experiments, in which the tension on the preparation was changed from 0·5 to 0·15 g. and back again, sea water being the bathing medium throughout. It will be noted that, at the higher tension, the extrovert (a) stretches considerably, (b) shows more activity in the rest periods between outbursts, and (c) shows a greater number of strokes, and a less conspicuous tone rise, in each outburst. The number of outbursts in unit time is not significantly affected.

The typical alternation of activity and rest periods is seen even in untied preparations lying in sea water. The fundamental pattern, therefore, is independent of tension, although the details can be considerably modified by varying that factor.

The effects of increasing the tension are very like those seen when K is moderately raised, or Mg moderately decreased (compare Figs. 1 and 6), and it might be thought that the two ions act, over the range in which they antagonize each other, by affecting the sensitivity of the extrovert to the pull of the lever. There is, however, an important difference between the response to a moderate change in the K : Mg balance and that to a change in lever weight. In the latter case there is apparently no accommodation, therefore appears that the two factors, mechanical and chemical, produce similar end-results but in different ways.

Evidently, the working of a steady state system such as the one just described could be disturbed at various points, and the observed result of the chain could be made to change without showing accommodation. If the value of k₀ were suddenly to alter, the rate of A→B would move exponentially to a new value, without any temporary overshoot. If the sensitivity of B were to change (e.g. an alteration of threshold, in the case of a relaxation oscillator), the observed result of the chain would rapidly or immediately assume a new, final value. The effects of a change of tension might be produced in the latter way; the end-result would then be the same as that of a change in the K : Mg balance, but there would be no overshoot and accommodation. At present, this scheme is no more than a plausible explanation of the facts; it does, however, show that mechanical and chemical factors could operate in entirely different ways, even though the final picture is much the same in both cases.

It must be emphasized that the above remarks apply only to K changes within the moderate excess range. The contracture and inhibition produced by great K excess, and the effects of K deficit, are quite different from the results of varying either the Mg concentration or the lever weight.

1. The effects on the isolated extrovert of Arenicola marina L. of varying the potassium concentration of the bathing medium are described. Data are also presented on K: Mg antagonism. Potassium and magnesium concentrations are given as multiples of their concentrations in artificial sea water, which was taken as the ‘normal’ starting fluid.

2. The extrovert normally shows a distinctive pattern of alternating periods of activity and rest, superposed on the more general properties of rhythm and tone. This pattern is very sensitive to changes in potassium concentration. Moderate changes produce modifications of the pattern. Severe changes abolish the pattern and produce effects on rhythm and tone resembling those shown by most rhythmic muscles under like conditions.

3. Moderate K excess (K 1·5–3·5) excites tone and rhythm. Accommodation occurs during long exposure. The effects resemble those of Mg deficit, and can be completely abolished by increasing the Mg concentration.

4. An increase of lever weight has effects on the rhythm resembling those of a moderate K excess or a moderate Mg deficit, but in this case there is no accommodation. It is suggested that the extrovert contains a steady state system such as

   

   where S is a source and the performance of the extrovert depends, through B, on the rate of the process A →B. If change in the K : Mg balance acts on k, its result will be following automatically by accommodation. Change in tension can produce the same end-result, without accommodation, by acting on B.

5. Severe K excess (K 5–10) causes contracture and inhibition of the rhythm. The contracture is partly antagonized by simultaneous increase in Mg, but the inhibition is not antagonized.

6. Moderate K deficit (K 0·75–0·33) causes initial excitement, then a characteristically modified pattern, with widely spaced activity outbursts, and an occasional abnormally long outburst. These effects are not antagonized by simultaneous decrease in Mg. As, however, they are not seen in preparations exposed to sea water diluted to four times its volume, they are due to disturbance of a balance between K and some other constituent of the medium.

7. Severe K deficit (K 0·05 or 0·00) causes partial contracture with chaotic activity, which lasts for many hours. Neither effect can be antagonized by simultaneous decrease in Mg. With borderline deficits (K 0·25 or 0·20) the preparation reacts at first as if to severe deficit, then accommodates itself and gives the pattern characteristic of moderate deficit.

8. The potassium paradox occurs on returning to artificial sea water after severe deficit. With borderline deficits (K 0·25 or 0·20) it is seen after short exposure, but not after long.