(Received 22 July 1957) It is now generally accepted that the unequal distribution of ions between cells

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1 190 J. Physiol. (I958) I40, I THE EFFECT OF ALTERATIONS OF PLASMA SODIUM ON THE SODIUM AND POTASSIUM CONTENT OF MUSCLE IN THE RAT By F. 0. DOSEKUN AND D. MENDEL From the Department of Physiology, University College, Ibadan, Nigeria (Received 22 July 1957) It is now generally accepted that the unequal distribution of ions between cells and extracellular fluid is maintained by metabolic activity of the cells, the low cell sodium content in particular being explained by a mechanism of active sodium extrusion (Dean 1941). Although many studies have been made in vitro on the effect of altered environment upon muscle electrolytes (Harris, 1950; Steinbach, 1952), and upon the mechanism of sodium extrusion in various tissues (Hodgkin & Keynes, 1954; Carey & Conway, 1954; Edwards & Harris, 1957), comparatively little is known as to the response of the sodium extrusion mechanism of muscle in vivo when the external sodium concentration is varied extensively. In the present study acute alterations in plasma sodium were produced by water and salt loading in the conscious rat, and the effects of these procedures upon the intracellular content of water, sodium and potassium in cardiac and skeletal muscle were determined, both in normal and adrenalectomized animals. METHODS The principles involved in the calculation of the intracellular concentration of an ion have been given by Boyle & Conway (1941), and by Manery (1954). Muscle water is considered to exist in two phases, namely intracellular and extracellular or interstitial water. In the present experiments inulin space was taken as a measure of extracellular water. Intracellular water is determined by deducting the extracellular water from the total water content of the tissue. The amount of any ion in the intracellular phase can similarly be calculated by deducting from the total amount of tissue ion the amount contained in a known volume of extracellular water. The intracellular concentration is obtained by correcting the intracellular ion to the amount that would occupy 1 1. of cell water. Operative procedure. Wistar strain albino rats of g body weight were used. The method used for determining inulin space was that described by Ledingham (1953). Rats of known weight were lightly anaesthetized with ether, both renal pedicles ligated through a dorsal incision, and an inulin solution (22 %, wlv) was injected into an external jugular vein, in the dose of 0.5 ml./100 g body weight. The animals were deprived of food and water, and after 6 hr blood was collected

2 SODIUM IN MUSCLE 191 from a carotid artery after the administration of heparin (Liquemin, Roche, Products, 500 u./kg body wt.). The heart was then excised, the great vessels, auricles and right ventricle cut away and the left ventricle cut into strips. Surface moisture was removed by the gentle application of blotting paper. Specimens were then placed in tubes of known weight for the determinations described below. The gastrocnemius was used for skeletal muscle determinations. After careful removal of fascia and nerves, the belly of the muscle was similarly divided into pieces, blotted and placed in tubes of known weight. Mu8cle water. Muscle specimens of mg wet weight were weighed to within 0.1 mg, dried for 24 hr at-1000 C, and then re-weighed after cooling to constant weight. Total water content was calculated from the weight loss, in terms of ml./kg wet wt. of tissue. At least two specimens of each type of muscle were used in each experiment. Muscle inulin. Muscle specimens of mg wet weight were weighed to within 0-1 mg in stoppered tubes. Inulin in muscle was determined by the method of Ross & Mokotoff (1951). The method provides for a muscle blank with each experiment and one specimen each of cardiac and of skeletal muscle was used for this purpose. Plasma inulin was determined by the method of Roe, Epstein & Goldstein (1949). The inulin concentration of extracellular water was taken as 1 1 x plasma inulin (Ledingham, 1954). Extracellular water was calculated from the muscle and plasma inulin, and expressed in ml./kg wet wt. of muscle. Muscle Na and K. The muscle specimens used for estimation of total water content were dissolved in 1 ml. 50% (w/v) nitric acid in a boiling water-bath. The solutions were diluted appropriately with ion-free distilled water and Na and K estimated by flame photometry. Results are expressed in m-equiv/kg wet wt. of tissue. Plasma Na and K were measured by flame photometry after appropriate dilution. The concentration of Na in the extracelliilar fluid is taken as 0l96 x plasma Na to allow for the Donnan effect. Adrenalectomy. Bilateral adrenalectomy was carried out through a dorsal incision under light ether anaesthesia. The rats were then maintained on a standard cube diet with 0-5% (w/v) NaCl as drinking water for 3 days before experiment. Water and salt loading. Water loading was effected by an intraperitoneal injection of distilled water (12.5 ml./100 g body wt.) and salt loading by intraperitoneal injection of a 5 % (w/v) solution of NaCl. These volumes were given 3 hr after the inulin injection. Inulin equilibrates in cardiac and skeletal muscle in 3 hr (Ledingham, 1953) and it was considered that had a large volume of intraperitoneal water or saline been given at the same time as the inulin delay in equilibration might occur. The animals were killed 3 hr after the administration of water or saline. In a preliminary investigation, RESULTS carried out to see if the presence of a large volume of intraperitoneal fluid could affect the measurement of inulin space, four rats were given an intraperitoneal injection of normal saline solution (0-9 %, w/v) in the dose 12-5 ml./100 g body wt., 3 hr after intravenous inulin. After a further period of 3 hr, blood and tissues (heart and skeletal muscle) were collected and inulin space determined. The mean value ( ± S.E.) obtained for cardiac muscle was ml./kg wet wt. and for skeletal muscle ml./ kg wet wt. These values were not significantly different from those obtained in a control series of nine experiments in which rats were left for 6 hr after the inulin infusion, the inulin spaces being: cardiac muscle ml./kg wet wt. and skeletal muscle ml./kg wet wt. It was evident that the presence of a large volume of intraperitoneal fluid did not affect the measurement of inulin space.

3 192 F. 0. DOSEKUN AND D. MENDEL The 3 hr period allowed for the equilibration of the injected water or salt load was also considered adequate. In four experiments (two using water and two using saline) blood and tissues were collected 5 hr after intraperitoneal injection of the water or saline. The values obtained for plasma and total muscle Na and K did not differ from the values obtained using the shorter period of 3 hr. The effect of water loading on muscle water and electrolytes The effect of water loading on muscle water and electrolyte was determined in six experiments. Rats were lightly anaesthetized with ether, both renal pedicles ligated and intravenous inulin given. After a 3 hr equilibration period an intraperitoneal injection of water (12.5 ml./100 g body wt.) was given. A further 3 hr period was allowed and the blood was collected from a carotid artery and tissues removed for analysis. The absorption of water from the peritoneal cavity in these experiments was satisfactory, the residual water not being greater than 2-5 ml./100 g body wt. in any experiment. The results given below are compared in each case with nine controls, in which tissues were obtained for analysis 6 hr after inulin injection. Plasma Na and K. The mean plasma Na in the water-loaded group was and that in the control group F0 m-equiv/l., the lowering of plasma Na being highly significant (P < 0-001). The mean plasma K in the water-loaded group was m-equiv/l., a figure not significantly different from the mean value of 5' m-equiv/l. obtained in the control group. Muscle water. Values for total water, inulin space and intracellular water for cardiac and skeletal muscle in the water-loaded and control groups are shown in Table 1. It can be seen that in both types of muscle the total water content increased. With cardiac muscle there was a decrease in the inulin space and an increase in intracellular water amounting to 7.9 % of the initial cell water. The inulin space in skeletal muscle did not differ in the two groups and the increase in intracellular water amounted to 2-1 % of the initial water. Muscle Na. The values for total muscle Na and the extracellular and intracellular Na fractions for cardiac muscle are shown in Fig. 1A and for skeletal muscle in Fig. 1B. From Fig. 1 A it can be seen that with cardiac muscle there was a fall both in total muscle Na and in the extracellular fraction. The intracellular Na however remained unchanged. The results with skeletal muscle differed from those with cardiac muscle. From Fig. 1 B it can be seen that there was a fall in total Na, a small decrease in extracellular Na and a highly significant decrease in intracellular Na. The intracellular concentration of Na in cardiac muscle, calculated from the intracellular Na fraction (Fig. 1A) and the total water content (Table 1), decreased slightly as a result of the increase in cell water; the mean value of m-equiv/l. of cell water was not, however, significantly different

4 SODIUM IN MUSCLE 193 from the control value of m-equiv/l. of cell water (P < 0-7). With skeletal muscle the mean value for intracellular Na concentration of 6f m-equiv/l. of cell water was significantly lower than the control value of 15* m-equiv/1. of cell water (P <0.01), the result of a large loss of intracellular Na and a small increase in cell water. Muscle K. Fig. 2 A shows the total K content for cardiac and skeletal muscle in the water-loaded and control groups. The extracellular K fraction is less than 1 % of the total tissue K. It is therefore not represented in the diagram and intracellular K may be regarded as similar to total K. With cardiac 30- T A B z '20- E~~~~~C T I S.E.± P <0-001 <0-001 <0-8 <0-01 <0-5 <0-01 Fig. 1. The effect of water loading on the Na content of (A) cardiac muscle and (B) skeletal muscle. Black columns controls, open columns water-loaded. T total Na; EC, extracellular Na; IC, intracellular Na (m-equiv/kg wet wt.). 200 fi100-3 Cardiac a3keleta v S.E.± P <0-01 <0.05 < <0-02 Fig. 2. The effect of water loading on the K content of cardiac and skeletal muscle. (A) Total K/kg wet wt. (B) K concn./l. cell water. Black columns controls, open columns waterloaded.

5 194 F. 0. DOSEKUN AND D. MENDEL muscle the loss of K is significant (P <0-01): with skeletal muscle the results were less clear. The differences in intracellular concentrations of K, calculated as already described, are shown in Fig. 2 B. The loss of K from cardiac muscle (Fig. 2A) and in the increase of cell water (Table 1), resulted in a marked lowering of intracellular concentration. With skeletal muscle the small increase in cell water further lowered the K concentration to a level which was probably significant. TABLE 1. Total water content, inulin space and intracellular water of cardiac and skeletal muscle with water loading (ml./kg wet wt.) Cardiac muscle Skeletal muscle Total Inulin Intracellular Total Inulin Intracellular water space water water space water Water-loaded ± ±3-1 Controls 753± ±1-9 55± P < < 0-01 < < < 0-6 < The effect of salt loading on muscle water and electrolytes In seven experiments the effects of intraperitoneal saline upon the intracellular concentrations of Na and K in muscle were determined. Following bilateral ligation of the renal pedicles and a 3 hr period of inulin equilibration, intraperitoneal injection of a 5 % (w/v) solution of NaCl in the dose 5 ml./100 g body wt. was given. After a further 3 hr period blood was obtained from a carotid artery and tissues were removed for analysis. The residual volume of fluid in the abdominal cavity in these experiments was between 6 and 7-5 ml./ 100 g body wt. The results given below are compared with nine controls in each case. Plasma Na and K. A considerable rise in plasma Na resulted from this procedure, the mean plasma Na being m-equiv/l., which was significantly higher than the control value of m-equiv/l. (P < 0-001). The plasma K level of was also significantly higher than the control value of m-equiv/l. (P < 0-001). Muscle water. The values for total water, inulin space and intracellular water are shown in Table 2. It can be seen that with both types of muscle there was a significant reduction in total water content. With heart muscle the inulin space was smaller than the controls although not significantly so. The intracellular water content did not differ from the controls. The reduction of total water appeared to be due therefore to a reduction in the inulin space. With skeletal muscle the inulin space was slightly larger than the controls although not significantly so, and there was a reduction of intracellular water amounting to 6-5 % of the original cell water. Muscle Na. Total Na, extracellular and intracellular Na fractions for cardiac muscle are shown in fig. 3 A and for skeletal muscle in Fig. 3 B. It can be seen that with cardiac muscle there were small increases in each Na fraction,

6 SODIUM IN MUSCLE 195 but that these increases were not significant. The absence of a significant rise in total sodium in spite of high extracellular Na concentration was due to the smaller inulin space found in this group. It was evident, however, that no large increase in the intracellular Na had taken place. With skeletal muscle, however, there was a large increase in total Na and a highly significant increase in the intracellular Na. TABLE 2. Total water content, inulin space and intracellular water of cardiac and skeletal muscle with salt loading (ml./kg wet wt.) Cardiac muscle Skeletal muscle Total Inulin Intracellular Total Inulin Intracellular water space water water space Water Salt-loaded 763±3-1 92± ± ±4-5 66± Controls 753± ± ± ±1-9 55± ±6-8 P <0-001 <0-1 <0-2 <0.001 <0-4 <0-001 P <0-2 <0-8 <0-4 <0-001 <0-1 <0-001 Fig. 3. The effect of salt loading on Na content of (A) cardiac and (B) skeletal muscle. Black columns controls, open columns salt-loaded. Symbols as for Fig. 1. The mean intracellular concentration of Na in cardiac muscle was m-equiv/l. of cell water, a value not significantly different from the controls (P < 0-3). With skeletal muscle the mean intracellular concentration of m-equiv/l. was significantly higher than the controls (P<0-001), the result of an increased intracellular Na content and a los of cell water. Mu4scle K. Fig. 4A shows the total K content of cardiac and skeletal muscle and Fig. 4B the final concentrations in m-equiv/l. of cell water. It can be seen that with cardiac muscle there was a large increase in the total K content, and the final intracellular concentration was also significantly

7 196 F. 0. DOSEKUN AND D. MENDEL higher than the controls. The very small increase in total K in skeletal muscle was not significant. The probably significant increase in final concentration of K is due therefore to the loss of cell water A Cardiac B Skeletal 150 El Cardiac 5 Skeletal S.E.± *2 5.5 P <0.001 <0-3 <0.001 <0-02 Fig. 4. The effect of salt loading on the K content of cardiac and skeletal muscle. (A) Total K/kg wet wt. (B) K concn./l. cell water. Black columns controls, open columns salt-loaded. The effects of water and salt loading on muscle water and electrolytes in adrenalectomized rats Considerable difficulty was experienced in keeping adrenalectomized rats alive for the period required after water or salt loading. The maximum water load which would allow survival for a 3 hr period after tying the renal pedicles was 7 ml./100 g body wt., but this survival time was seriously shortened if operation time was prolonged by venous cannulation. Even when inulin was administered intraperitoneally in the dose 1 ml. of a 22 % (w/v) solution/100 g body wt., a 3 hr period allowed for equilibration, and then a further 3 hr allowed after water loading with the reduced volume, it was found possible to obtain complete data only in three experiments out of a large series. With salt loading complete data were obtained in five experiments by using a reduced volume of 2-5 ml. of a 5 % (w/v) solution of NaCl following intraperitoneal inulin. Blood inulin levels obtained after intraperitoneal inulin were as high as those obtained by the smaller dose intravenously, and sometimes higher. In three experiments using adrenalectomized rats the values for inulin space obtained after intraperitoneal inulin were of the same order as those obtained in a control series given inulin intravenously. A 6 hr equilibration period was allowed in each case.

8 SODIUM IN MUSCLE 197 The effect of adrenalectomy on muscle water and electrolytes. In six rats, used as a control series, muscle water, Na and K were determined 3 days after adrenalectomy. In these experiments an intravenous injection of inulin was given after ligation of the renal pedicles and blood and tissues were collected for analysis after 6 hr. The results given below are compared with nine nonadrenalectomized controls in each case. The plasma Na concentration in the adrenalectomized group was m- equiv/l. and the plasma K m-equiv/l. These values were significantly different (P < 0.01) from the non-adrenalectomized control series. TABLE 3. The cell water, intracellular Na and K concentrations in adrenalectomized rats compared with non-adrenalectomized controls Cardiac muscle Skeletal muscle Cell Na K Cell Na K water (m-equiv/ (m-equiv/ water (m-equiv/ (m-equiv/ (ml./kg) 1.) 1.) (ml./kg) 1.) 1.) Adrenalectomized ± ± ± ±2-9 Controls 641± ± ± ±6-8 15*6± ±4-2 p <0.7 <0.2 <0-2 <0 02 <0-7 <0.01 The mean inulin space in cardiac muscle was ml./kg wet wt. and in skeletal muscle ml./kg wet wt. The inulin space in skeletal muscle was slightly smaller, but not significantly so, than the values obtained in the nonadrenalectomized controls. The values for cell water and intracellular Na and K concentrations in cardiac and skeletal muscle are compared with the non-adrenalectomized control series in Table 3. It can be seen that with cardiac muscle there were no significant differences from the control series in any of these values. With skeletal muscle there was an increase in cell water which was possibly significant, no change in Na concentration, but a significant lowering of intracellular K concentration. The lowering of K concentration was due to both an increase in cell water and a loss of K from the muscle, since the mean values of total K content were: non-adrenalectomized controls f2 m-equiv/kg wet wt., adrenalectomized controls m-equiv/kg. wet wt. These values are significantly different (P < 0.001). The effect of water loading. The results given below, and compared with adrenalectomized controls in each case, represent complete data from three experiments in which values for inulin space were obtained, and from three other experiments in which the renal pedicles were ligated and tissues removed for Na and K estimation 3 hr after water loading. Plasma Na was significantly lowered to a mean value of m-equiv/l., but the mean plasma K of m-equiv/l. did not differ significantly from the controls. The mean inulin space in cardiac muscle was ml./kg wet wt. and in skeletal muscle ml./kg wet wt., values not significantly different from the controls. The mean cell water in cardiac muscle was 657 ml./kg wet wt. and in

9 198 F. 0. DOSEKUN AND D. MENDEL skeletal muscle 720 ml./kg wet wt., representing only small increases in cell water compared with the control values of 646 and 717 ml./kg wet wt. Muscle Na is shown in Table 4 and K in Table 5. The mean inulin space obtained from three experiments was used to calculate the intracellular Na and K content. TABLE 4. Total Na, intracellular Na fraction and intracellular Na concentration in adrenalectomized rats subjected to water and salt loading compared with adrenalectomized controls Cardiac muscle Skeletal muscle Total Intracellular Total Intracellular Na Na Intracellular Na Na Intracellular (m-equiv/kg (m-equiv/kg Na conen. (m-equiv/kg (m-equiv/kg Na concn. wet wt.) wet wt.) (m-equiv/l.) wet wt.) wet wt.) (m-equiv/l.) Water-loaded Controls i ± Salt-loaded ±3i ± TABLE 5. Total K content and intracellular K concentration in adrenalectomized rats with water and salt loading compared with adrenalectomized controls Cardiac musclc Skeletal muscle Total K Intracellular Total K Intracellular (m-equiv/kg K conen. (m-equiv/kg K conen. wet wt.) (m-equiv/l.) wet wt.) (m-equiv/l.) Water-loaded 96± Controls 101± ± ± Salt-loaded 113± ± ±1F9 146±2-7 From Table 4 it can be seen that with cardiac muscle there was a small fall in total Na, but no reduction in the intracellular fraction or the intracellular concentration. With skeletal muscle the values for total Na, intracellular Na and intracellular concentration were not lower than the controls. This is in clear contrast to the significant reduction that occurred in the nonadrenalectomized group subjected to water loading (Fig. 1). From Table 5 it can be seen that there was a loss of total K from cardiac muscle which was probably significant (P < 0.02). There was no similar loss from skeletal muscle. The effect of salt loading. The results of five experiments given below are compared with those for six adrenalectomized controls in each case. The mean plasma Na in these experiments was and the mean plasma K 7l m-equiv/l., both values being significantly higher than the controls (P < 0.001). The mean inulin space for cardiac muscle was ml./kg wet wt. and for skeletal muscle ml./kg wet wt., values not significantly different from the controls. The mean cell water for cardiac muscle was 620 ml./ kg wet wt. and for skeletal muscle 691 ml./kg wet wt. There was a large variation in the values obtained for cardiac muscle and the results are not significantly lower than the controls (P < 0-3). The reduction in cell water of skeletal muscle was possibly significant (P < 0 05).

10 SODIUM IN MUSCLE 199 The values for muscle Na are shown in Table 4. It can be seen that with cardiac muscle there was a small increase in total Na, and smaller but not significant increases in the intracellular fraction and intracellular concentration of Na. With skeletal muscle there were increases in total Na, intracellular fraction and sodium concentration which were probably significant (P <0.01 for total Na and P < 0-05 for intracellular fraction and Na concentration). The results for total K and K concentrations are shown in Table 5. With cardiac muscle there was an increase in both the total and the final K concentration, both increases being highly significant (P <0001). With skeletal muscle no such increases in K occurred. DISCUSSION The present results show important differences in cardiac and skeletal muscle with regard to the transfer of electrolytes when these tissues are exposed to varying concentrations of sodium. In the living rat the present evidence suggests the existence of a homoeostatic mechanism which preserves the intracellular sodium content of cardiac muscle but not of skeletal muscle. Thus the intracellular sodium content of cardiac muscle was always remarkably stable whereas the intracellular sodium content of skeletal muscle varied extensively with changes of plasma sodium. In the experiments with water loading there was no significant reduction in plasma potassium despite the increase in plasma volume that must have occurred. It is clear that the potassium needed to maintain this plasma concentration must have been derived from some reservoir and it is suggested that part of the reservoir may have been the intracellular potassium in muscle. When plasma sodium was raised by salt loading, the plasma potassium also rose. There was evidence of potassium entry into cardiac muscle but not into skeletal muscle, the latter finding confirming the experiments of Eichelberger (1941) and Conway & Hingerty (1948). It would seem therefore that cardiac muscle is more effective than skeletal muscle as a reservoir in potassium. Following adrenalectomy the intracellular sodium content of cardiac muscle was normal and still not affected by water or salt loading. In skeletal muscle, however, although levels of sodium were normal after adrenalectomy, lowering of plasma sodium did not produce the fall in intracellular sodium found in the non-adrenalectomized animal. This suggests the presence of some defect in sodium extrusion. SUMMARY 1. The effect has been studied of alterations of plasma Na on the Na and K content of cardiac and skeletal muscle in conscious rats. 2. With lowering of plasma Na induced by water loading there was a significant loss of cell Na from skeletal muscle but not from cardiac muscle,

11 200 F. 0. DOSEKUN AND D. MENDEL whereas K was lost freely from cardiac muscle and to a less extent from skeletal muscle. 3. Adrenalectomy prevented the loss of skeletal muscle Na at low plasma Na levels but did not affect the loss of K from cardiac muscle. 4. With high plasma Na levels produced by salt loading, Na entered freely into skeletal muscle but not into cardiac muscle, whereas K entered freely into cardiac but not into skeletal muscle. Adrenalectomy did not affect these exchanges. We wish to thank Professor J. Grayson for his advice and help in the preparation of this paper. REFERENCES BOYLE, P. J. & CONWAY, E. J. (1941). Potassium accumulation in muscle and associated changes. J. Physiol. 100, CAREY, M. & CONWAY, E. J. (1954). Comparison of various media for immersing frog sartorii at room temperature and evidence for the regional distribution of fibre Na. J. Physiol. 125, CONWAY, E. J. & HINGERTY, D. (1948). Relations between potassium and sodium levels in mammalian muscle and blood plasma. Biochem. J. 42, DEAN, R. B. (1941). Theories of electrolyte equilibrium in muscle. Biol. Symp. 3, EDWARDS, E. C. & HARRIS, E. J. (1957). Factors influencing Na movement in frog muscle with a discussion of the mechanism of Na movement. J. Physiol. 135, EICHELBERGER, L. (1941). The distribution of body water in skeletal muscle and liver in normal dogs following injection of K salts. J. biol. Chem. 138, HARRIS, E. J. (1950). The transfer of Na and K between muscle and the surrounding medium. Part II. The Na flux. Trans. Faraday Soc. 46, HODGKIN, A. L. & KEYNES, R. D. (1954). Movement of ions during recovery in nerve. Symp. Soc. exp. Biol. 8, LEDINGHAM, J. M. (1953). The distribution of water, sodium and potassium in heart and skeletal muscle in experimental hypertension in rats. Clin. Sci. 12, LEDINGHAM, J. M. (1954). The influence of the adrenal on the water and electrolyte disturbances foliowing nephrectomy, and its relation to renoprival hypertension. Clin. Sci. 13, MANERY, J. F. (1954). Water and electolyte metabolism. Physiol. Rev. 34, ROE, J. H., EPSTEIN, J. H. & GOLDSTEIN, N. P. (1949). A photometric method for the determination of inulin in plasma and urine. J. biol. Chem. 178, Ross, G. & MOKOTOFF, R. (1951). Determination of inulin in muscle. J. biol. Chem. 190, STEINBACH, H. B. (1952). On the sodium and potassium balance of isolated frog muscles. Proc. nat. Acad. Sci., Wa8h., 38,