382 J. Physiol. (I950) III, 382-387 6I2.817.I*546.32 POTASSIUM AND NEUROMUSCULAR TRANSMISSION BY S. HAJDU, J. A. C. KNOX AND R. J. S. McDOWALL From the Physiology Department, King's College, University of London (Received 14 December 1949) In the course of experiments made in another connexion, it was observed that isolated preparations of the rat diaphragm which had been denervated were more sensitive to the depressant action of extra potassium in the Ringer solution than normal preparations. This has now been studied in detail. METHODS The methods were those described by Hajdu & McDowall (1949) with the modification that in many instances the muscle was stimulated directly while totally immersed in Krebs's solution, the formula of which has already been given (Hajdu & McDowall, 1949). To obtain the following results the temperature must be kept strictly at 380 C. for reasons given below. The nerve was stimulated with currents of 200 psec. duration at 25 V., the muscle directly by currents of 2000 ssec. at 75 V. delivered by an electronic rectangular wave stimulator. The relatively large currents were used because of the total immersion of the nerve and muscle in the fluid. This is found to give the most constant results and avoids any effects of bubbles of oxygen, but calculation based on the responses of a curarized muscle with the bath empty and full indicates that only about one-tenth of this current passes through the muscle. When desired, denervation of one side of the diaphragm was carried out by section of the phrenic nerve in the neck under ether anaesthesia and checked by the finding after 10 days of no response to nerve stimulation, and a contracture response to acetylcholine which was not present in normal innervated diaphragms (Gasser & Dale, 1926; Dale & Gaddum, 1930). RESULTS A small increase in the potassium concentration of the bath fluid causes an increase of the contraction of the preparation stimulated through its nerve with single maximal shocks. Larger doses cause an initial augmentation followed by a depression of the twitch. Thus, if the potassium concentration of the bath fluid is increased from 5-92 m.mol. (i.e. the concentration in Krebs's solution) to 7 m.mol., an augmentation occurs and a depression with 13-72 m.mol. In a denervated preparation, depression is brought about by 12-64 m.mol. and the early augmentary effect is slight (Fig. 1). This may seem a very small increase in sensitivity, but it has become apparent that the muscle is extremely
POTASSIUM AND NEUROMUSCULAR TRANSMISSION 383 sensitive to minute changes in the concentration of potassium in and around this amount, while the response is materially affected by temperature (see below). A B Fig. 1. The response to potassium (24 mg. in 50 c.c. bath) of (A) normal diaphragm, and (B) the denervated other half diaphragm of the same animal. Time in sec. A A B A C A Fig. 2. The response of denervated diaphragm to acetylcholine (100 u. Ach) at A before and after a depressant dose of potassium (24 mg., at B) which renders it inexcitable to electrical stimulation. The response to A was abolished by 100 a. D-tubocurarine, added at C. Time in sec. If the muscle is left in the high-potassium-concentration bath fluid its contractions continue to decline until it ceases to respond, even to stimuli previously maximal or which earlier produced a tetanus. This is due not to failure of contractility but to a very great change in excitability, for by greatly increasing the stimulus by reducing the amount of fluid in the bath, the muscle can be made to contract as before. The reduction cannot be due to the addition of an electrolyte. Calculations based on the response of a curarized muscle partially and completely immersed in fluid indicate that a voltage of 50 times the strength previously required may fail to excite when excitability
384 S. HAJDU, J. A. C. KNOX AND R. J. S. McDOWALL becomes depressed. If more potassium is added, the muscle eventually becomes inexcitable to all strengths of stimuli. If a denervated preparation is used similar changes are produced, but in spite of the great loss of excitability to electrical stimulation, the response to acetylcholine is not reduced (Fig. 2). This response is a sluggish contraction but relaxation may occur in two stages (Dale & Gaddum, 1930), one rapid and the other slow and often incomplete, leaving a contraction remainder or contracture. It is of interest to note that after such a dose of acetylcholine in the absence of excess potassium a subsequent dose was ineffective unless the preparation was washed in fresh solution and this although the response to electrical stimulation after a temporary depression returns to normal. This point has been observed by Buchtal & Lindhard (1939) in the striated muscle of the lizard and by Rosenblueth & Luco (1937) in the cat, and is referred to subsequently in the discussion. ~ ~ ~._ Fig. 3. Effect of adding glucose at G in restoring the nerve response to a preparation which has been depressed by lack of glucose and excess potassium. The nerve and muscle were stimulated in alternate groups at M and N. In the early part of the record the onset of the block is seen. Time in sec. Effect of neuromuscular block. Production of neuromuscular block by either D-tubocurarine or by lack of glucose (Hajdu & McDowall, 1949) increases the depressant effect of raised potassium concentration in a normally innervated preparation. If the neuromuscular transmission has been completely blocked by glucose lack, 'the addition of glucose brings about recovery although the extra potassium is still present (Fig. 3). The response to acetylcholine, however, is not sensitized by lack of glucose, e.g. acetylcholine in doses which cause contraction of a denervated preparation is still without effect on the innervated preparation. When the neuromuscular transmission is blocked by the addition Of D-tubocurarine to the bath, there is a similar increase in the sensitivity of the muscle to potassium. Much the most striking effects are, however, produced by adding the potassium first in doses which are known to depress denervated
POTASSIUM AND NEUROMUSCULAR TRANSMISSION 385 muscle but which have no effect on the innervated preparation. Now if curare is added, not only is the response to nerve stimulation with a given stimulus abolished but all response of the muscle to a stimulus originally maximal is lost (Fig. 4). '1' ' -1' -E' 1- -I- A B C D E F Fig. 4. At A, a test dose of D-tubocurarine was given, the muscle and nerve being stimulated alternately in groups. The nerve response only ceases. At B, the fluid in the bath was replaced by normal Krebs s solution. At C, 24 mg. potassium was added with negligible effect but now the addition of D-tubocurarine at D causes a cessation of response of both nerve and muscle. At E, the additional potassium only was removed by changing the fluid and the muscle recovered; and at F, the D-tubocurarine was removed and the nerve recovered. Time in sec. A Fig. 5. Effect of temperature on the response to nerve stimulation. The depression produced by extra potassium (added at A) is relieved by the reduction of the temperature at the arrow from 370 to 320 C. Time in sec. The effect of temperature. The response of the rat diaphragm to amounts of potassium around the concentration used above is extremely sensitive to change of temperature. Thus an increased concentration of potassium at 370 C. produces a typical augmentation followed by a depression, but the depression is recovered from if the temperature is reduced to 320 C. (Fig. 5). Direct and indirect stimulation are affected equally. Similar effects are produced on a denervated preparation. The relation of the action ofpotassium to temperature
386 S. HAJDU, J. A. C. KNOX AND R. J. S. McDOWALL has already been referred to by Quilliam & Taylor (1947) in connexion with its action on curare. Even at room temperature similar results are obtained but an increased amount (5 or 10 mg. in the bath) of potassium may be necessary. At this lower temperature it is easy to demonstrate that large doses of potassium depress neuromuscular transmission before muscle is affected, though stimuli of frequency high enough to produce a tetanus may still be effective. DISCUSSION It has been shown that denervation or a procedure which interferes with the acetylcholine liberated at nerve endings sensitizes the rat diaphragm to the depressant action of potassium. From this we may conclude that the normal acetylcholine liberated when nerve is stimulated antagonizes the depressant action of potassium, and this is particularly well seen in the ability of higherfrequency stimuli to pass a block sufficient to stop stimuli of low frequency. How it may do so is a matter of some interest. We have already seen that potassium in small doses stimulates muscle especially at temperatures below normal, while larger doses, on the other hand, depress, commonly after an augmentation. Since it is known that potassium antagonizes curare, it may be suggested that the augmentary phase of potassium action is at least in part through an acetylcholine mechanism and this is supported by the finding that eserine augments this action (Wilson & Wright, 1936). This, however, cannot be the whole explanation, for it has been pointed out by Magladery & Solandt (1942) that potassium will actually stimulate skeletal muscle and especially when it is denervated. It may be that potassium stimulates the synthesis or liberation of acetylcholine, for it has been shown by Feldberg (1944) that potassium increases such synthesis in brain slices, and this view is supported by the observation that the depression is less at lower temperatures, which also increase the synthesis of acetylcholine (Feldberg, 1944). It is more difficult to explain why the response to acetylcholine in the denervated muscle during potassium depression of excitability to electrical stimulation remains unaltered. This observation, taken together with the observation that a second dose of acetylcholine may be ineffective while the response to electrical stimulation returns to normal, supports the suggestion of Buchtal & Lindhard (1939) that there may be two modes of access to the end-plate. The depressant action of potassium, we have seen, is enhanced by denervation, by neuromuscular block and by eserine, depending on the dose. With these considerations it is now possible to explain the apparent depressant action of curare on muscle after a stimulating dose of potassium, thus. In normal muscle if the dose of potassium is suitably chosen, the augmentary effects through acetylcholine and the depressant effects on muscle nearly balance each other. When, therefore, the augmentary effect through acetyl-
POTASSIUM AND NEUROMUSCULAR TRANSMISSION 387 choline is removed by curare, denervation or other means, the depressant effect on muscle is unopposed. These findings open up a still more important question as to whether the normal activity of acetylcholine is the antagonism of the potassium of muscle as has been suggested by Verzar (1943). This is being made the subject of a subsequent investigation. SUMMARY 1. Extra potassium first increases and, in large doses, then depresses the excitability of muscle at body temperature in Krebs's solution. 2. At room temperature neuromuscular transmission is depressed by extra potassium, and eventually a neuromuscular block is produced which, like that produced by curare, can be antagonized by more rapid stimulation. 3. The depression of the muscle is enhanced by denervation and it is suggested that the acetylcholine liberated normally antagonizes potassium depression. We are indebted to Dr J. E. Hall for confirming the observations on the effect of temperature, and Fig. 5 is from his work. REFERENCES Buchtal, F. & Lindhard, J. (1939). J. Phy8iol. 95, 59P. Dale, H. H. & Gaddum, J. H. (1930). J. Physiol. 70, 109. Feldberg, W. (1944). J. Phy8i"l. 103, 367. Gasser, H. S. & Dale, H. H. (1926). J. Pharmaol. 28, 287. Hajdu, S. & McDowall, R. J. S. (1949). J. Phy8iol. 108, 502. Magladery, J. W. & Solandt, D. W. (1942). J. Neurophy8l. 5, 356. Quilliam, J. P. & Taylor, D. B. (1947). Nature, Lond., 160, 603. Rosenblueth, A. & Luco, J. V. (1937). Amer. J. Phy8iol. 120, 781. Verzar, F. (1943). Theorie und Mu8kelkontraction. Basel: Schwabe. Wilson, A. T. & Wright, S. (1936). Quart J. exp. Phy8iol. 26, 128.