Calculation of the ionic flux in isolated mammalian muscle at 38° C required a knowledge of internal ion concentration, the cell dimensions, and the kinetics of exchange across the cell membrane. Muscles soaked in Krebs saline showed no indication of fibre swelling or gain of cell water; there was a small fall of intracellular potassium, accompanied by a large rise of sodium. With proper oxygenation, muscle potassium was constant for several hours; anoxia rapidly produced a fall in potassium and gain of sodium. The use of radioactive tracers showed that potassium was completely exchangeable. The mean half-time for exchange of potassium between tissue and saline was 45 min. Initially the rate was somewhat more rapid, but it finally became steady. There was no significant difference between the rates of entry and exit. Potassium exchange was apparently slowed by diffusion through the inter­spaces; the calculated exchange rate across the cell membrane had a half-time of 36 min. The mean potassium flux, after correcting for diffusion, was 21 x 10 -12 equiv. cm -2 s -1 . Fibre sodium exchanged with a half-time of about 10 min, and the outward sodium flux was 28 x 10 -12 equiv. cm -2 s -1 . High values were found for the intercellular space, being 26 ml./100 g in soaked diaphragm muscles as measured by inulin. This was confirmed by a method involving radioactive sodium, and the inulin space in vivo was similar. In their passage through the intercellular fluid, inulin, potassium and sodium appear to follow simple diffusion kinetics, and their apparent diffusion coefficients have been estimated.

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SUMMARY1. Thin diaphragm muscles in dialysed serum maintained their total sodium at values similar to those found in vivo. The fibre sodium exchanged with a half-time of 5 min, and the flux was 1O p-mole.cm-2. sec-'. The internal sodium was less than 10 ,umoles/g fibre water and the ratio of exterr-al to internal sodium was at least 15. The calculated energy expenditure for sodium extrusion was less than 2 % of the resting metabolism.2. In physiological saline the half-time for exchange of fibre sodium was similar to that in serum, but in saline there was an increase in sodium permeability and a raised total and fibre sodium. 3. Muscles treated with insulin (0-02 u./ml.) showed a more rapid exchange of sodium compared with muscles in saline, with no change in the permeability to sodium. Insulin also affected sodium movements in the absence of external glucose.4. In saline the fraction of sodium which exchanged rapidly occupied 0-33 by volume of the muscle, after corrections for diffusion, and this value was similar to the mannitol space.5. Fibre diameters were measured in frozen sections. Muscles prepared by fixation and embedding showed marked shrinkage.

Isolated tissues are employed for a variety of biological studies, and it is usually found that considerable changes in their ion content have occurred following immersion in physiological saline. The present investigation was commenced in an endeavour to maintain mammalian muscle at body temperature with the same sodium content as that found in the normal animal.After attempts with other preparations rat diaphragm was selected, as this muscle is not too thick for oxygenation and it can be rapidly dissected and immersed in the medium. It was known from previous work that the potassium could be maintained at normal values (Creese, 1954), though in these experiments there was a large rise in sodium content. In the present study it was found possible to maintain the sodium of the muscle at its normal low value provided the dissection was rapid, the tissue was well oxygenated and the medium was fortified with serum or certain proteins. Some initialresults have been reported earlier (Creese, D'Silva & Northover, 1958). METHODSRats. Male albino rats of 110-130 g were used and were maintained on a rat cake diet. The range of weights for any one set of results did not exceed 8 g.Saline. The composition except where otherwise stated was (mM): Na+ 145, K+ 5-0, Ca2+ 1-3, Mg2+ 1*2, Cl-125, HCOO 25, S02-1-2, HPO2-plus H,PO; 1-2. The glucose content was 200 mg/100 ml.; the temperature was 380 C. Double-glass-distilled water was used for the solutions.G6la&wnare was cleaned with the use of a detergent, and boiled in distilled water before use. No chromic acid was used.Ditsection and immersion. It was necessary that the muscle should be dissected, attached to a holder and suspended in a small volume of medium in a time short enough to prevent deterioration. The rats were stunned and decapitated. The left hemidiaphragm was rapidly exposed, two cotton threads were inserted around the rib with a needle, and parallel cuts were made about 1 cm apart in the diaphragm in such a way that a minimum number of fibres was damaged. The holders were made of glass rod with a plastic cross-piece (Perspex), and a platinum hook was attached to the lower end of the glass rod (Fig. 1). A hole was made