establishing perfusion and of collecting and analysing the effluent fluid 1934]. Comparable increases in serum potassium were obtained when

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1 I74.5:612.I26 ACTION OF ADRENALINE ON THE SERUM POTASSIUM BY J. L. D'SILVA From the Department of Physiology, King's College, London (Received 24 March 1937) IN a previous communication it was shown [D'Silva, 1936b] that the liberation of potassium by adrenaline in the perfused cat's liver was facilitated by the presence of oxygen. Larger quantities of the base were set free when the perfusion was carried out with blood than when van Dyke & Hastings' fluid was used. Evidence was adduced to show that asphyxia of the tissues attendant on the use of the inorganic fluid was not responsible for the results. Further, before the organ was capable of responding to a second dose of the drug, a period of recovery (oxidative) was necessary. In the present investigation various factors which might be concerned in the liberation of potassium have been studied further. METHODS Cats anaesthetized with chloralose after ether were used. In all cases the animals were allowed to rest after the induction of anesthesia, for large amounts of ether have a tendency to increase the serum potassium [D'Silva, 1934]. Potassium was estimated by the method of Kramer & Tisdall [1921] as modified by Hubbard [1933]. Some of the experiments were carried out on the perfused liver. The technique of establishing perfusion and of collecting and analysing the effluent fluid has been described previously [D'Silva, 1936a]. RESULTS Response of the intact animal to successive doses of adrenaline The injection of 0 05 mg. of adrenaline into an arm vein resulted in a large increase in the concentration of potassium in the serum [D'Silva, 1934]. Comparable increases in serum potassium were obtained when

2 304 the same dose was injected at intervals of 10 min. five times (Table I, Exps. 1 and 2). The minimum amount of blood (5 c.c.) was withdrawn from the femoral artery after each injection, to minimize the effects of loss of blood. As the animals appeared to be capable of dealing with the drug administered under the above conditions, the interval between successive injections was shortened and the dose increased in another series of experiments. After withdrawing 5 c.c. of blood from the femoral artery, successive injections of 0.1 mg. of the drug were administered every 3 min. 1 min. after each injection further blood samples were obtained, four in all. The blood was allowed to clot overnight and potassium was determined in the serum. In each case there was a definite increase in the concentration of the base. There was appreciable hemolysis in the third and fourth samples, probably a result of the haemolytic action of the drug. 45 min. later, injections of 0 1 mg. of adrenaline again increased the concentration of potassium in the serum to levels identical with those after the first series of injections (cf. Table I, Exps. 3 and 4). TABLE I. J. L. D'SILVA Effect of successive injections of adrenaline on the intact animal Concentration of potassium in serum in mg./100 c.c. Interval in min. between injections; Exp. Before adrenaline After adrenaline and dose in mg , 26-5, 26-8, 25-2, ; , 24*9, 240, 24-5, , 25-3, 25-3, ; 01 Interval of 45 min , , 25-5, 24-9, ; 0.1 3; 041 Interval of 45 min , ; , -, 23-8,-, 20-4,-, ; 041 Interval of 1 hr ,-, ; ,, 24-5,, 22-9,,20-0 3; 01 Interval of 1 hr , -, ; 01 In other experiments, the injections of adrenaline were given as before, but blood samples were taken 1 min. after the first, third, fifth, and seventh doses in order to minimize the effects of loss of blood, which increases the concentration of potassium in the serum [D'Silva, 1934]. In this series also, the third and fourth blood samples showed definite signs of haemolysis. There was but a feeble mobilization of the base in

3 ADRENALINE AND SERUM POTASSIUM response to the seventh injection. Within an hour, however, the animal had recovered completely its capacity to liberate the base (Table I, Exps. 5 and 6). Experiments described later (cf. Table V) show that the increase in the concentration of the base cannot be ascribed solely to the transference of potassium from the corpuscles into the plasma, as the whole blood potassium of cats, in contrast with that of many other animals, is not very much greater than the concentration of the base in the serum. Response of the perfused liver to successive doses of adrenaline In a previous communication [D'S ilva, 1936b] evidence was obtained that injections of adrenaline, separated by periods of 5-15 min., into the affluent fluid of a liver perfused with blood did not mobilize the same amounts of potassium. Less of the base was liberated in response to the second and third injections. It was considered possible that the massive doses (0.2 mg.) injected, which tended to increase the resistance of the liver to perfusion, may have been responsible for the results obtained. Another series of experiments was performed in which eight injections of 0.1 mg. of adrenaline were made at intervals of 3 min. into the inflowing blood. The effluent fluid was collected during two successive periods of 12 min. After centrifugation, potassium was determined in the plasma which showed scarcely any visible signs of hamolysis. The amount of the base liberated in the second period of 12 mi. was much less than that liberated during the first (Table II), but the latter figure TABLE II. Effect of successive doses of 0 1 mg. of adrenaline, injected at intervals of 3 min., on the perfused liver Average rate of perfusion c.c./min. Mg. of potassium liberated Exp. First 12 min. Second 12 min. First 12 min. Second 12 min * * * *0 8*0 (13-2 mg. in Exp. 1) was variable and was usually loss than that obtained after one dose of 0-2 mg. of the drug [D'Silva, 1936b]. A slow perfusion rate was chosen to ensure as far as possible that the conditions favoured the "fixation" of the injected adrenaline by the liver cells. At the same time the amount of oxygen brought to the tissues for their metabolic recovery was diminished. Besides, with successive injections, the liver vessels tended to shut down and diminish further the perfusion rate, PH. XC

4 306 J. L. D'SILVA an effect which was to some extent counterbalanced by increasing the perfusion pressure. This did not necessarily imply that a larger area of the liver was irrigated and may have led to the results described. Effect of insulin on the recovery process Subcutaneously injected insulin (up to 25 units) was without apparent effect on the response of the intact animal to successive injections of adrenaline. It did not antagonize the power of the drug to mobilize potassium. Perfusion of the liver with blood to which insulin had been added, or the injection of insulin at any stage in the experiment, had no effect on the capacity of the organ to liberate the base in response to repeated injections of adrenaline. Effect of a single dose of adrenaline on the recovery process An injection of adrenaline (0.1 mg.) was made into the affluent blood of a perfused liver and thereafter for consecutive periods of 10 and 6 min. the effluent blood was collected. In the first sample, the amount of potassium in the plasma always exceeded that in the affluent plasma by mg. whereas in the second sample this difference was negligible. Since the liberation of potassium following a single dose of adrenaline is completed in about 6 min., the results of this experiment indicate that the liver cells, deprived of potassium by the drug, were incapable within the next 10 min. of extracting the base to any extent from the perfusion fluid. Effect of starvation Food (except water) was withheld from cats for 2 days, but the response of the blood potassium to intravenously injected adrenaline (0.05 mg.) resembled that in the intact animal, as did the capacity of the isolated liver to mobilize the base when perfused with blood containing adrenaline (0.1 mg.) (Protocol 1). Protocol : serum potassium was 19-7 mg./100 c.c. Injected intravenously005 mg. adrenaline : serum potassium was 25 mg./100 c.c : serum potassium was 19-4 mg./100 c.c. Injected intravenously 0-05 mg. adrenaline : serum potassium was 24 6 mg./100 c.c : established perfusion of liver with defibrinated blood : 0.1 mg. adrenaline mobilized 12*5 mg. potassium. Effed of phloridzin Food (except water) was withheld from the animals (which were not allowed access to their excreta) for 3 days and phloridzin dissolved in

5 ADRENALINE AND SERUM POTASSIUM warm 1-25 p.c. sodium bicarbonate solution was injected subcutaneously in doses of 1 g. three times a day. The animals were then anaesthetized with chloralose administered after ether. Adrenaline (0.05 mg.) injected intravenously caused an increase in the concentration of potassium in the serum. The animals recovered the power to liberate more of the base after mi. (Table III). TABLE III. Effect of 005 mg. of adrenaline on the phloridzinized cat Serum potassium Interval mg./loo c.c. between A & 5 injections Exp. Before After min. 1 20x i '4 23-0i X0 [ 307 Effect of iodoacetic acid Iodoacetic acid (0.05 mg. per 1 g. wt. of animal) in water containing enough sodium bicarbonate to neutralize the acid, when administered subcutaneously to cats before the induction of anaesthesia, had no noticeable effect on the mobilization of potassium by 0.1 mg. of adrenaline administered intravenously. Moreover, the power of recovery of the animal to mobilize a further quantity of the base in response to a second dose of the drug given 20 min. later was unimpaired. Protocol 2 shows Protocol : injected subcutaneously iodoacetic acid (005 mg./l g. wt. of animal) in water containing enough NaHCO3 to neutralize the acid. Anasthetized with chloralose after ether : 0.1 mg. of adrenaline given intravenously increased the serum potassium from 18*8 to 27-0 mg./100 c.c. in 1 min : 0.1 mg. of adrenaline given intravenously increased the serum potassium from 18-4 to 26-4 mg./100 c.c : established perfusion of the liver with oxygenated blood : 0.1 mg. of adrenaline mobilized 10X8 mg. potassium in 6 min : injected 30 mg. of iodoacetic acid as the sodium salt : 0-1 mg. of adrenaline mobilized 9-8 mg. potassium in 6 min. that the effect of iodoacetic acid in the perfused liver was very small, as the base liberated by a single dose of adrenaline was comparable with its effect on the normal liver (cf. Table IV). A further large dose of iodoacetic acid had no effect on the liberation of potassium by the isolated organ. When iodoacetic acid (0.1 mg. per 1 g. body wt.) was administered subcutaneously after the induction of anaesthesia 0.1 mg. of adrenaline injected intravenously usually proved fatal, although only after a few 20-2

6 308 J. L. D'SIL VA (less than 10) minutes. The increase in concentration of potassium in the serum 1 min. after the injection was always observed although the increase was rather less than that observed in a control experiment performed before iodoacetic acid was injected. In each experiment, post-mortem examination revealed the ventricles in a state of severe contraction. The quantitative liberation of potassium by adrenaline in the perfused liver Perfusion was established as described previously [D'Silva, 1936a] with freshly drawn, defibrinated ox blood diluted with van Dyke & Hastings' [1927-8] fluid so as to bring the blood cell volume to 45 p.c. In no case did the plasma show visible evidence of haemolysis. Adrenaline was injected into the ingoing fluid and the potassium liberated during 8 min. was determined in the effluent fluid. After a period for recovery (10 min.) a second injection of the drug was given and the potassium output of the organ was again determined as a check on the first determination, but was invariably lower. This was not unexpected in view of the experiments described previously. Table IV shows that 0.1 mg. of the drug liberated mg. of potassium. TABLE IV. The quantitative liberation of potassium by adrenaline in a liver perfused with blood Potassium liberated Average Adrenaline Potassium per 0.1 mg. adrenaline perfusion rate injected liberated (calculated) Exp. c.c./min. mg. mg. mg The variation observed in the results from the mean value (13-8 mg.) is not large considering the nature of the experiment. The amount of potassium liberated by a second dose of the drug has usually been lower (up to 30 p.c.) than that liberated by the first dose. Potassium content of cat's blood 1 c.c. of oxalated blood was evaporated to dryness, the major portion of the carbonaceous matter removed by heat, and the residue gently ignited with one or two drops of concentrated nitric acid. The colourless ash was dissolved in the minimum amount of hot dilute hydrochloric acid and transferred quantitatively to a centrifuge tube, in which the

7 ADRENALINE AND SERUM POTASSIUM 309 TABLE V. Concentration of potassium in cat's blood Mg. per 100 c.c. Exp. Blood Serum analysis for potassium was carried out. Table V which records the results of several analyses and the corresponding concentrations of potassium in the serum shows that the red cells do not contain a very much higher concentration of the base than the plasma. SUMMARY 1. The intact cat and the isolated liver appear capable of responding equally (by mobilizing potassium) to 0.1 mg. of adrenaline injected intravenously at intervals of 3 mn. 2. Adrenaline mobilizes potassium in the intact animal as well as from its perfused liver, after (a) starvation, (b) treatment with phloridzin. 3. lodoacetic acid is without effect on the power of adrenaline to mobilize the base. Insulin apparently plays no part in the process mg. of adrenaline liberates mg. of potassium in the perfused liver, and other doses proportional amounts. 5. The whole blood potassium of cats is less than twice the concentration of the base in the serum. I am indebted to Prof. McDowall for his interest in this work and to the Sir Halley Stewart Trust for a Fellowship during the tenure of which these experiments were carried out. REFERENCES Cori, C. F. (1931). Physiol. Rev. 11, 156. D'Silva, J. L. (1934). J. Phy8iol. 82, 393. D'Silva, J. L. (1936a). Ibid. 86, 219. D'Silva, J. L. (1936b). Ibid. 87, 181. van Dyke, H. B. & Hastings, A. B. (1927-8). Amer. J. Phy8iol. 83, 563. Hubbard, R. S. (1933). Ibid. 100, 557. Kramer, B. & Tisdall, F. F. (1921). J. biol. Chem. 48, 223.