augmentation of contractions which was followed by depression. Addition of Hajdu & McDowall (1949) showed that when the contractions of the isolated

Transcription

1 225 J. Physiol. (I954) I25, I THE EFFECT OF ADRENALINE ON THE RAT DIAPHRAGM PREPARATION DEPRESSED BY EXCESS POTASSIUM BY KATHARINE A. MONTAGU From the Department of Physiology, King's College, London (Received 27 January 1953) Hajdu & McDowall (1949) showed that when the contractions of the isolated rat diaphragm preparation were depressed by excess potassium in Krebs solution, recovery could be produced by adding 100,ug adrenaline to the 50 ml. bath. Smaller doses of adrenaline were later reported to be effective (Knox, McDowall & Montagu, 1951). This effect has now been investigated in greater detail, using innervated preparations, with the object of elucidating the mechanism of action of adrenaline. METHOD The modification of the method of Biilbring (1946) described by Hajdu, Knox & McDowall (1950) was used. A slip of the rat diaphragm with its phrenic nerve was suspended in a 50 ml. bath containing Krebs solution at C through which a mixture of 95% 02 and 5% C02 was bubbled. The preparation was stimulated through the phrenic nerve by single rectangular pulses of 200 sec duration. Supramaximal stimuli were used at a frequency of 6/min, except where otherwise stated. Adrenaline solutions were prepared daily in distilled water from Parke Davis 'Adrenalin'. RESULTS The effect of potassium Fifty ml. Krebs solution contains the equivalent of 22*25 mg KCl. Hajdu et al. (1950) found that addition of about 29 mg extra KCl/50 ml. produced augmentation of contractions which was followed by depression. Addition of smaller doses produced augmentation only. These results have been confirmed but rather more KCI was found necessary to depress the contractions. A constant level of depression was maintained by adding to the bath 2-6 ml. Krebs solution at 370 C when the contractions had been reduced to about one-half of their initial size by the extra KCI; the volume of fluid in the bath was readjusted to 50 ml. about 5 min later. Such a preparation continued to give a constant but reduced response to nerve 15 PHYSIO. CXXV

2 226 KATHARINE A. MONTAGU stimulation for more than 4 hr, but preliminary experiments showed that the effect of adrenaline became reduced by the prolonged excess of potassium. Therefore, in the later experiments described below, adrenaline was always added within 40 min of the KCl. The initial augmentation produced by extra KCl was usually 35-40% of the initial twitch response, but was sometimes as much as 65 %. In the experiments described below the augmentation produced by adrenaline bore a less constant relation to the initial twitch response than it did to the largest 100 _ 5 80, E o 60 ID C 0.oZ 40 _ u U 20Ill I log (extra KCI), mg/50 ml. Fig. 1. The relationship between twitch height about 40 min after addition of KCl, and log (extra KCl) in two preparations, ( x ) and (A). Contraction heights expressed as percentages of 'KCI stimulant level'. contractions produced by KCl, the 'KCl stimulant level'. All contraction heights were therefore assessed as a percentage of this level. The twitch height in normal Krebs solution was usually about 70 %. In preparations depressed for the first time by KCl, 2 jug adrenaline had a greater effect than in preparations subsequently depressed by KCl. During the recovery produced by 2,ug adrenaline in preparations depressed for the first time, the contractions were sometimes as great as 120% of the 'KCl stimulant level', whereas adrenaline in subsequent KCl depressions produced recovery to only %. The larger adrenaline responses obtained on the first KCl depression were ignored, and the results to be described refer to adrenaline recovery in preparations which had been depressed at least twice by KCI. With a small dose of extra KCl the contractions did not reach a steady level until 40 min or more after its addition; the curves shown in Fig. 1, in which

3 ADRENALINE-POTASSIUM ANTAGONISM the final contraction height is allowing 40 min between each dose. 227 plotted against extra KCI, were obtained by The effect of adrenaline In most preparations in which the contractions had been depressed by the addition of KCI to a steady level corresponding to about 30% of the 'KC1 stimulant level', 0*05,uzg adrenaline produced just perceptible recovery, 0-5,ug adrenaline recovery to about 70%, 2,ug to 85-90% (Fig. 2), and 100lkg adrenaline to %. The time course of the recovery also varied with the dose of adrenaline; the recovery occurred more quickly with the larger doses. Fig. 2. Recovery given by 2jug adrenaline/50 ml. from depression produced by extra KC1. A previous dose of adrenaline reduced the effect of a subsequent one when the preparation was not washed between the two. The reduction was greater the greater the size of the first dose; it was almost complete after 100,ug adrenaline and absent with five successive 0 05,ug doses. Recovery produced by small doses of adrenaline (0.05-2,tg) was maximal within 15 min; it sometimes persisted until the preparation was washed 1-2 hr later. In other experiments the contraction height declined but usually did not quite reach its original depressed level. Persisting recovery was more frequent the smaller the dose of adrenaline. A large dose of adrenaline (5,g or more) has never given prolonged recovery; moreover, persisting recovery produced by a small dose was reversed by a large dose, after this had first produced some further recovery. Antagonism of potassium by adrenaline When a further 2-4 mg KC1 was added to the bath while adrenaline was acting, the effect varied according to the time at which the KCI was added. When added 1-3 min after 2,g adrenaline, the contractions became tempor- 15-2

4 228 KATHARINE A. MONTAGU arily greater than with the adrenaline alone. But when added after about 30 min, the contractions became reduced to the depressed level which existed before the adrenaline was given. At this time, and in the absence of adrenaline, as little as 0-25 mg KCI increased the depression. The antagonistic effect between adrenaline and KCO was further investigated by adding mixtures of adrenaline and KCl to preparations already depressed by KCl. As illustrated in Fig. 3, the addition of a mixture of 10,ug adrenaline Fig. 3. The effect of a mixture of 12 mg KCI and 1,ug adrenaline on a preparation depressed by extra KCI. and 12 mg KCI produced a transient depression (at B). The contractions during the recovery from the depression, as at C, Fig. 3, often became slightly greater than they were before the addition of the adrenaline-kcl mixture. This recovery was more pronounced when the preparation was then left to soak unstimulated in K-free Krebs solution for 5 min and the experiment was repeated, but on subsequent repetitions it became less pronounced. An attempt was made to find a range of mixtures of adrenaline and KCI which would produce a constant effect. The bath was filled with a measured 50 ml. Krebs solution at 37 C. The preparation was stimulated at a rate of 5/min and its contractions were depressed by addition of mg KCI to about one-third of the 'KCI stimulant level'. The stimulation rate was then changed to 18/min; this was usually sufficient to arrest the depression, but when it was not, ml. Krebs solution at 370 C was added and this volume of solution withdrawn about 3 min later. A mixture of adrenaline (0-05 or 0.2,ug) and KCl ( mg) was then added and the maximum response (as at C, Fig. 3) noted. The experiment was repeated several times with the same

5 ADRENALINE-POTASSIUM ANTAGONISM 229 amount of adrenaline but with differing amounts of KCl until a mixture was found which produced such an effect that the maximum contractions (C, Fig. 3) were equal to the initial contractions (A, Fig. 3) in two successive experiments. The preparation was left to soak unstimulated in K-free Krebs solution for 5 min between each experiment. Subsequently, mixtures containing successively increasing amounts of adrenaline as well as of KCI were tested once each on the same preparation. The whole process was then repeated on other preparations until, by successive approximation, mixtures were found which produced the desired effect on three different preparations (equal contractions at A and C). These mixtures are given for one series of experiments in Table 1; in this series, the figures for KCI are slightly below average. TABLE 1. Antagonism between adrenaline and potassium (see text) Amount of adrenaline Amount of KCl (,ug) in mixture (mg) in mixture 0-2 5* F In another series of experiments the antagonism exerted by 0.05 and 100,g adrenaline was determined; it varied from 1-6 to 2-2 mg KCl for 0-05,ug adrenaline, and from 22-5 to 25 mg KCI for 100jig adrenaline in these relatively fresh preparations. DISCUSSION The present experiments show that the ability of adrenaline to antagonize KCI is large in preparations of the rat diaphragm already depressed by extra KCI. Thus 0-05,ug adrenaline can antagonize about 2 mg KCI, i.e. one molecule of adrenaline can antagonize 100,000 potassium ions. This proportion will be very much smaller at the cell surface, because if K, Kb, A, and Ab denote surface and bulk phase concentrations ofpotassium and adrenaline respectively and if KS/Kb is taken as 2 and AS/Ab as not less than 2 x 105 (Danielli, 1944), then K8/A, lies between 4000 V8 and 4000 V8 + 1, where V8 denotes the volume of the surface phase in millilitres. A rough idea of V, may be obtained by assuming that the extramuscular surface phase is created by a shell of albumin molecules forming the outer surface of the muscle fibre. Using Clark's figure (Clark, 1933) for the total surface area of the fibres in 1 mm3 of rabbit's skeletal muscle, 50 cm2, and Danielli's figures for the radius and surface phase volume of an ovalbumin molecule, 2-75 x 10-7 cm and 1-08 x ml. respectively (Danielli, 1941, 1944), V8 would be about 10-3 ml. with a muscle of 0-1 ml. volume. Thus at the cell surface and with these assumptions, the

6 230 KATHARINE A. MONTAGU number of potassium ions antagonized by one adrenaline molecule would be only about four. It has further been shown that 0 5,ug adrenaline produces recovery to about 70% of the 'KCI stimulant level' and antagonizes about 9 mg KCI; removal of this amount of KCl might be expected from Fig. 1 to produce maximum recovery to 100%. But recoveries to more than 70 % are produced only by doses of adrenaline larger than 05,ug and these recoveries do not ordinarily reach 100 %. These facts are probably due to complexity in the mode of action of potassium as well as of adrenaline, for in preparations depressed by extra KCl, removal of some of the extra potassium by dilution with normal Krebs solution has less (and also slower) effects than had its addition 20 min earlier; full recovery is not obtained. Both KCl and adrenaline are known to prolong the duration of the active state of muscle, and this augments the contractions (Goffart & Ritchie, 1952). Each may also alter, probably in opposite directions, the ratio of the concentrations of intramuscular and extramuscular potassium (Goffart & Perry, 1951). Some similarity in the mode of augmentation by KCI and of the recovery, in depressed contractions, produced by adrenaline might explain the finding that the adrenaline recovery is more consistent when compared with the 'KCI stimulant level' than with the contractions in normal Krebs solution. Some difference is probably evidenced by the fact that when a preparation is depressed for the first time by KCI, the recovery then produced by adrenaline may greatly exceed the 'KCO stimulant level'. This larger recovery is not caused by a larger antagonism of potassium, for this antagonism, with small doses of adrenaline, is smaller in the first than in subsequent experiments. Prolonged recovery was most frequently produced by the smallest dose of adrenaline (0.05,ug), and with this dose there was no reduction in the size of the recovery produced by a subsequent 0 05,ug dose. On the other hand, the recovery with a large dose, 100,ug, was transient and no recovery was produced by a subsequent dose. The duration of recovery and the size of a subsequent recovery may therefore be related. SUMMARY 1. The effect of adrenaline on the contractions of the innervated rat diaphragm preparation depressed by extra KCI in Krebs solution has been studied. Adrenaline produces recovery which can be prolonged, but there tends to be an inverse relation between duration of recovery and dose of adrenaline. Increasing the stimulation rate also produces some recovery. 2. The amounts of KCI have been found whose further depressant effect is antagonized by various doses of adrenaline. Using a 50 ml. bath 0)05,ug can antagonize the effect of mgKCl, i.e. about 100,000K ions are antagonized by one molecule of adrenaline. 100,ug adrenaline can antagonize 25 mg KCI.

7 ADRENALINE-POTASSIUM ANTAGONISM 231 REFERENCES BULBRING, E. (1946). Observations on the isolated phrenic nerve diaphragm preparation of the rat. Brit. J. Pharmacol. 1, CLARK, A. J. (1933). The Mode of Action of Drugs on Cells, pp. 9 ff. London: Edward Arnold and Co. DANIELLI, J. F. (1941). On the ph at the surface of ovalbumin molecules, and the protein error with indicators. Biochem. J. 35, DANIELLI, J. F. (1944). The biological action of ions and the concentration of ions at surfaces. J. exp. Biol. 20, GOFFART, M. & PERRY, W. L. M. (1951). The action of adrenaline on the rate of loss of potassium ions from unfatigued striated muscle. J. Physiol. 112, GOFFART, M. & RrrcHirm, J. M. (1952). The effect of adrenaline on the contraction of mammalian skeletal muscle. J. Physiol. 116, HAJDU, S., KNox, J. A. C. & McDowALL, R. J. S. (1950). Potassium and neuromuscular transmission. J. Physiol. 111, HAJDU, S. & MCDOWALL, R. J. S. (1949). Some actions of calcium and potassium in the rat diaphragm. J. Physiol. 108, lop. KNOX, J. A. C., McDOWALL, R. J. S. & MONTAGU, K. A. (1951). The action of adrenaline on the rat diaphragm. J. Physiol. 112, 36P.