Investigation of the changes in concentration of the other cations. The. small they need not be considered now. (Received November 7, 1935.

Transcription

1 :577.I74.5 THE ACTION OF ADRENALINE ON SERUM POTASSIUM. By JOHN L. D'SILVA. (From the Department of Physiology, King's College, London.) (Received November 7, 1935.) IN a previous paper [1934] it was shown, and it has since been confirmed by Marenzi and Gerschman [1935], while Schwarz [1935] has demonstrated a similar effect in the rabbit, that the concentration of potassium in the serum was very promptly affected by adrenaline. Following the intravenous injection of 0.05 mg. into cats under chloralose anaesthesia, it was increased by as much as 50 p.c. within a minute, only to fall within the succeeding few minutes. A possible mechanism for its control was suggested. Two problems arising from this work have now been investigated with a view to determining, (a) whether the concentration of the commoner cations was affected simultaneously, and (b) what the source might be from which the additional potassium was derived. Investigation of the changes in concentration of the other cations. The work to be described deals only with sodium and calcium. The other cations, e.g. magnesium, occur in blood in but small amounts, and as their contribution to the economy of the total base of the body must be small they need not be considered now. Sodium. The possibility that sodium as well as potassium might be affected by the administration of adrenaline must not be passed over lightly in spite of the many physiological differences between them. It seems probable, however, apart altogether from any specific action of adrenaline on the sodium of the serum, that the disturbance of the concentration of the sum of the bases in the blood, when that of potassium is increased, might result in sodium passing out into the tissues. The action of adrenaline on the concentration of sodium in the serum was therefore investigated. A blood sample (about 8 c.c.) was collected from the femoral artery of a cat under chloralose and allowed to clot. Adrenaline, 0 05 mg., was

2 220 J. L. D'SIL VA. injected intravenously and after 1 min. a second sample (8 c.c.) was collected. In each sample the concentrations of potassium and sodium were determined in the serum. Sodium was determined by Butler and Tuthill's modification [1931] of the method of Barber and Kolthoff [1928]. The accuracy of the method in many analyses carried out on aqueous solutions of sodium chloride was + 2 p.c. Potassium was determined by Kramer and Tisdall's [1921] cobaltinitrite method with an accuracy of p.c. Large increases were observed in the concentration of potassium in the second sample in every case. With regard to the sodium it is clear that no change in concentration detectable by the analytical method occurred (Table I). But an error of + 2 p.c. in the amount of sodium TABLE I. Effect of 005 mg. of adrenaline on the concentrations of sodium and potassium expressed in mg. and millimols (mmol.) per 100 c.c. serum. Sodium Potassium Before After Diff. Before After Diff.,, ~~~~~~~in,- N in Exp. mg. mmol. mg. mmol. mmol. mg. mmol. mg. mmol. mmol * * * * present, about 350 mg., would make a variation of less than 7 mg. undetectable, that is about 0-3 mmol., and the amount that may have left the blood to compensate for the observed increase of potassium is only about half that amount. So it is not possible to say whether that occurred. Calcium. This was determined by the method of Trevan and Bainbridge [1926] as modified by Patterson [1930]. The results of consecutive analyses on inorganic solutions agree always within +2 p.c., and frequently are much nearer. In every case the total calcium was estimated. The changes observed are very small and probably within the experimental error. In every case potassium was determined in addition and served as a control. The technique of obtaining the blood samples was the same as above. Serum anions. From the results of other investigators it seems certain that adrenaline exerts no specific action on the chloride content of serum. The concentration of bicarbonate and the carbon dioxide tension of blood are somewhat lowered. It is impossible to assess the significance

3 ADRENALINE AND POTASSIUM IN SERUM. of these variations because of the undeterminable amount of proteinate ion which may be present. A possibility which presents itself is that adrenaline may lead to an increase of the ammonia in the blood. If this were so, the concentration of potassium would appear to be increased, because ammonia forms an insoluble compound with sodium cobaltinitrite analogous to that formed by potassium. But no differences have been observed between determinations of potassium carried out on ashed and unashed samples of serum. As adrenaline apparently exerted a specific effect on the serum potassium, it was of interest to determine the site from which the base was mobilized. That it was not the alimentary canal was shown in a previous [1934] paper; evisceration did not abolish the response to adrenaline. A connection between the pancreas and this reaction was suggested, but adrenaline still mobilized potassium in pancreatectomtzed animals. The following evidence was obtained for regarding the liver as the source of potassium. (1) Exclusion of the portal area from the circulation. The normal response of the animal was determined as described previously (blood samples of about 5 c.c. from the femoral artery) and the animal was then allowed to rest for 3 hour in order that the potassium should have regained its original level [D'Silva, 1934]. The anterior and posterior mesenteric arteries, the coeliac axis and the portal vein were ligatured, so as to avoid engorgement of the abdominal viscera without disturbing the animal more than was necessary, and after a few minutes a normal blood sample was obtained. Then 005 mg. of adrenaline injected intravenously caused no change in the concentration of potassium in the serum after 1 min. as shown in Table II. Incidentally this indicates that the muscles contribute nothing to the potassium changes of short duration which are under investigation. TABLE II. Effect of adrenaline after exclusion of the liver from the circulation in cats. Normal Potassium Normal Potassium serum po- 1 min. after after he- 1 min. after Exp. tassium adrenaline patectomy adrenaline Remarks * * Starved for 2 days There is an objection to this experiment, as the operation not only renders the blood-pressure unstable (and often causes an increase in the 221

4 222 J. L. D'SIL VA. " normal" concentration of potassium), but frequently causes a rapid fall. The mobilization of potassium by adrenaline has been observed over a wide range of blood-pressure (not below about 80 mm.) in many cats, but the records do not indicate a correspondence between it and the potassium response to adrenaline. (2) Comparison of the potassium in bloodfrom the hepatic vein with that in bloodfrom thefemoral artery. Blood from the hepatic vein was obtained as follows: An incision was made in the mid-line just below the diaphragm and extended laterally just below the course of the lowest rib and parallel to it on the right side of the cat, far enough to give easy access to the hepatic vein when the muscles were retracted and the liver pushed gently aside. Blood samples of 5 c.c. were withdrawn easily during 20 sec. by means of a 10 c.c. syringe fitted with a No. 13 needle. When the needle was withdrawn from the vein there was no haemorrhage, and subsequent saniples were got by inserting the needle into the vein through the original wound. Successive samples obtained from this vein on analysis showed concentrations of serum potassium identical with those obtained from the femoral artery, within the limits of experimental error. (3) Comparison of the effects of injections of adrenaline into a systemic vein and directly into the portal system. A cannula was fixed into one of the veins from the spleen. This did not interfere to any extent with the venous return from that organ and was convenient. The spleen was brought to the ventral surface of the body and the cannula fixed in position by suitably stitching the wound through which the spleen was reached. The results obtained are detailed in Table III. From Exps. 2 and 3 it is observed that the response to adrenaline injected into the portal system is several times as great as that when the drug is injected into the TABLE III. Time in minutes after injection in brackets. Adrenaline Serum potassium injected, A Exp. mg. Site of injection Normal After adrenaline Remarks Splenic vein (1) Arm vein (0.5) Same animal 0*01 Splenic vein 18* (05) j 2nd 1 injection hour after Arm vein (0.5) 0*01 Splenic vein (0.5) Arm vein 21* (1) Arm vein *4 (1) (Same animal Splenic vein (1) 2nd injection after I.0.5 hour

5 ADRENALINE AND POTASSIUM IN SERUM general circulation. Approximately the same increase in the concentration of potassium is obtained by injecting 0.01 mg. into the splenic vein as is given by injection of 0-05 mg. into the vein of the forelimb. This is taken as strong evidence of the liver being a source of potassium. It is important to notice that the blood samples in these experiments were withdrawn 0-5 min. after injection of the drug. The results are not so conclusive, especially with small doses, when samples are taken a longer time after injection, as, for example, 1 min. in Exp. 5. Here equal doses (0.005 mg.) injected into the arm vein and the splenic vein resulted in increases of 4-2 and 6*4 mg. per 100 c.c., respectively. In several experiments large doses (e.g. 0*02 mg.) were injected into the splenic vein. In nearly every case, of which one is recorded, there was an increase of p.c. in the potassium in the serum of blood withdrawn from the hepatic vein after 1 min. During the past two years in very many experiments doses even as big as 0 05 mg. were injected into the arm vein and caused an increase of no more than 70 p.c. in the serum of blood drawn from the femoral artery, often considerably less. (4) The action of adrenaline on the perfused liver. The object of the experiment was to determine directly, without the complications of the muscles of the body, whether or not adrenaline caused any liberation of potassium in the liver. First of all both adrenals were excised under chloralose, in order to prevent the discharge of adrenaline when the animal was bled. A ligature was then passed, but not tied, round the inferior vena cava below the diaphragm but above the entry of the renal veins. The portal vein was dissected out as far from its entry to the liver as possible and two ligatures were passed round it, one slightly distal to the other. Usually it was necessary to tie a branch of the vein which enters it in this region. During the succeeding few minutes, the temperature of the perfusion fluid was brought to 380 and the cannulae prepared. The distal ligature on the portal vein was tied and the perfusion cannula, dripping slowly, was tied into the vein. Immediately the chest was opened and an incision made in the inferior vena cava near the heart. The perfusion rate was increased greatly to wash the blood out of the liver, and simultaneously the ligature on the vena cava, posterior to the diaphragm, was tied. After about 2 min. most of the blood had been washed from the liver, and then a cannula was inserted into the inferior vena cava and the perfusate collected through it. The liver was kept warm by means of a heater and the closure of the mid-line incision through which the structures posterior to the diaphragm were reached.

6 224 J. L. D'SIL VA. Hitherto, in these experiments, the hepatic artery has not been perfused or even tied for two reasons: (a) it was considered essential to maintain the natural oxygen supply of the organ to the last possible moment, and (b) the relative blood supplies from the hepatic artery and the portal vein are such that there is unlikely to be any appreciable back-leak through the artery, especially with the low perfusion pressure (about cm. of water) used. Any other collateral circulation is negligible. The perfusion fluid used was a modified Ringer's fluid in which the concentration of potassium was reduced to between 17 and 19 mg. per 100 c.c., and which to improve the buffering was made more alkaline; it was necessary to bring the ph up to 7-8 in order to obtain an outflow of ph 7-3 or 7-4. The fluid used by van Dyke and Hastings [1927-8] has proved far more satisfactory. Adrenaline, 0-1 or 0*2 mg., was injected into the perfusion fluid, and samples of the perfusate were collected during successive periods of 1 min. (Table IV). The reaction, a marked increase in concentration of TABLE IV. Effect of adrenaline on the perfused liver. Potassium in mg./100 c.c. of perfusate. Time in Adrenaline Exp. min. in brackets. Injection at 0 dose (mg.) (-1); 0; 27-3 (1); 18-8 (2); 17-5 (3) *2 (- 1); 0; 26-2 (1); 19 0 (2); 16-8 (3) (- 1); 0; 25-4 (1); 20-8 (2); 20-4 (3) (-1); 0; 24X7 (1); 21*1 (2); 17.2 (3) 0-2 potassium in the perfusate, was very rapid, nearly all the potassium liberated being found in the fluid collected during the first minute. The figures obtained are far outside the experimental error, +2 to 3 p.c., and establish the liberation of potassium by the liver as a result of the action of adrenaline. Connection between the liver glycogen stores and the power of the animal body to mobilize potassiumr in response to adrenaline. In one experiment a cat was first starved for 2 days and then under ether and chloralose the injection of adrenaline 0 05 mg. into a systemic vein caused the potassium concentration to go up from 23*1 to 37-0 mg./100 c.c. after 1 min. It is apparent, therefore, that the phenomenon occurs even when the amount of glycogen in the liver is greatly diminished. To obtain further evidence on this rats were used because of the ease with which they can be rendered almost free from hepatic glycogen and because of the convenient size of their livers which can be taken entire for analysis. In the first experiment six rats were chosen at random

7 ADRENALINE AND POTASSIUM IN SERUM. from stock and divided into two groups. The controls were killed by a sharp blow on the head and the livers were excised as soon as possible, washed in saline, dried between filter paper, and then for hours in a steam oven to constant weight. After 5-8 hours the tissue was broken up to facilitate drying. Dry ashing, followed by digestion with about two drops of concentrated nitric acid, gave a colourless residue easily soluble in 0-5 c.c. of concentrated hydrochloric acid. The solution was transferred quantitatively to a measuring flask and the potassium was determined. The experimental animals were starved for 54 hours and the liver tissue was analysed in the same way. In another series a batch of eight rats was starved for 24 hours and then given a solution of glucose in water, 10 mg./g. body weight, 4 hours after which four rats were killed and their livers analysed for potassium. These served as controls and were assumed to have considerable liver glycogen stores, as Cori, Cori and Buchwald [1930] found. The remaining rats were starved for 24 hours, at the end of which time their glycogen stores were almost completely depleted, before their liver tissue was analysed for potassium. In both series, the livers of the starved animals contained slightly larger amounts of potassium, but this is probably due to some secondary cause (Table V). It seems clear that the potassium content of the liver is independent of the glycogen stores in the organ. TABLE V. Effect of starvation on the potassium in the liver. SERIES 1 SERIES 2 Percentage Percentage pota#sium potassium Exp. in liver Mean Exp. in liver Mean ' 1 1*48' *70 p.c p.c ) controls 3 lost F controls ~~ ~~~~~~~4 1.40) Op.c stape 6 158[ 1-52 p.c starved The action of insulin on the concentration of potassium in the serum. This has been found by several experimenters to lower the concentration of potassium in the serum. Large doses were given to cats under chloralose with little result (Table VI). It is possible that chloralose may retard the hypoglyceamic effect of the drug. Relatively large doses of insulin were necessary. Doses as large as 5 units per kg. have no effect when

8 226 TABLE VI. J. L. D'SIL VA. Effect of insulin in cats under chloralose. Potassium concentrations. Time in min. after Exp. injection in brackets Insulin, units 1 21*3 (0); 215 (15); 22-0 (20); 20-3 (25) 10 subcut (0); 16-8 (45); 16-6 (90); 17-0 (135); 15.4 (180) JiS subcut. starved "~~ 's"~~~~ ~ ), ''~ ~for 17 hours (0); 18-2 (30); 17-5 (60); 16-8 (120) 17-5 intraven. No insulin (0); 20-4 (30); 20-5 (60); 21-7 (90) 6 c.c. of 10 p.c. glucose intraven. injected subcutaneously and but a small effect when injected intravenously. Decreases of nearly 50% have been observed in unanessthetized dogs [Kerr, 1928; Briggs, Koechig, Doisy, and Weber, 19234] and in rabbits [Harrop and Benedict, 1924]. In Exp. 4 glucose was injected intravenously in an attempt to study the effect on the serum potassium of the insulin which the animal would secrete in response to the rise of blood-sugar level. No effect was observed. In a previous paper [1934] a marked decrease in the concentration of potassium in the serum was described as following the large initial increase after intravenously injected adrenaline. This was thought to be due to a secretion of insulin resulting from the hyperglyeamia induced by adrenaline. The idea was supported by the fact that pancreatectomy abolished the "after fall ". In view of the experiments now described on the influence of insulin on the serum potassium, it seems difficult to reconcile the two experiments unless, indeed, the pancreas is capable of secreting massive quantities of insulin or, alternatively, the secreted hormone is in a highly active condition. The concentrations of serum potassium following the injection of adrenaline, 0.05 mg., into a systemic vein have now been followed from blood samples withdrawn from the hepatic vein. The results in Table VII show that an "after fall" definitely does exist. TABLE VII. The "after fall" in the concentration of potassium occurs in the hepatic vein following 0-05 mg. of adrenaline intravenously. Potassium concentration in mg./100 c.c. Time in min. after Exp. injection in brackets Remarks (0); 24-2 (0.5); 16-9 (5.5); 17.3 (12.5) From hepatic vein (0); 27-6 (0.5); 19-5 (5.5); 18-2 (13.5); 20-3 (88.5) From femoral artery (0); 41 5 (1); 21-3 (7); 17-9 (13) From hepatic vein (0); 17-7 (7); 16-5 (14); 17-3 (21) From hepatic vein

9 ADRENALINE AND POTASSIUM IN SERUM. 227 DISCUSSION. In 1934 the remarkable correspondence between the changes that occur in the blood sugar and blood potassium under any given set of conditions was recorded [D'Silva]. It was suggested that the two phenomena were closely correlated in some way. That suggestion receives support in the present work, inasmuch as the liver is shown to be the source from which both substances are mobilized by adrenaline. The liver does not contain abnormally large quantities of potassium, but it is possible that the base, or a part of it, is disposed in the cells in some way peculiar to that organ. If smears of liver tissue are examined by Macallum's [1905] method the distribution of potassium in the cells will be observed to be different from that in skeletal muscle, for example. Apart from the result of the action of adrenaline there appears to be no difference between the concentration of potassium in the serum of hepatic and systemic blood. If any, the differences would be very small and within the limits of analytical error, added to which, any excess of potassium in the blood so rapidly disappears that the concentration of potassium cannot be " built up " in the serum, as sugar can, the threshold of which is so much above its normal concentration. SUMMARY. 1. Adrenaline when injected intravenously into cats under chloralose ancesthesia is without any specific effect on the calcium of the serum. Any change in the sodium that may occur is too small to detect. The effect on potassium is specific. 2. The liver is the source from which the potassium is mobilized following an injection of adrenaline because: (i) exclusion of the liver from the circulation abolishes the effect; (ii) blood from the hepatic vein contains more than that from the femoral artery; (iii) injection of adrenaline into the portal vein produces a much more marked effect on the concentration of potassium in the hepatic vein, than injection into an arm vein; (iv) adrenaline liberates potassium from a perfused liver. 3. The potassium content of the liver tissue of rats is independent of the glycogen stores of the organ. 4. Only very large doses of insulin diminish the concentration of potassium in the serum of cats under chloralose. This research was carried out during the tenure of a Sir Halley Stewart Fellowship. My thanks are due to Prof. R. J. S. McDowall for his criticism, and to Mr J. McWhan for his help with some of the experiments.

10 228 J. L. D'SIL VA. REFERENCES. Barber, H. H. and Kolthoff, I. M. (1928). J. Amer. chem. Soc. 50, Briggs, A. P., Koechig, I., Doisy, E. A. and Weber, C. J. (1923-4). J. biol. Chem. 58, 721. Butler, A. M. and Tuthill, E. (1931). Ibid. 98, 171. Cori, G. T., Cori, C. F. and Buchwald, K. W. (1930). Ibid. 86, 375. D'Silva, J. L. (1934). J. Phy8iol. 82, 393. van Dyke, H. B. and Hastings, A. B. (1927-8). Amer. J. Phy8iol. 83, 563. Harrop, jun., G. A. and Benedict, E. M. (1924). J. biol. Chem. 59, 683. Kerr, S. E. (1928). Ibid. 78, 35. Kramer, B. and Tisdall, F. F. (1921). Ibid. 48, 223. Macallum, A. B. (1905). J. Phy8iol. 32, 95. Marenzi, A. D. and Gerschman, R. (1935). Biochem. Rev. 4, 303. Patterson, J. (1930). Biochem. J. 24, 355. Schwarz, H. (1935). Arch. exp. Path. Pharmak. 177, 628. Trevan, J. W. and Bainbridge, H. W. (1926). Biochem. J. 20, 423.