No abstract

With the use of a newly introduced technique, the "influx profile analysis," we studied the diffusion of tritiated water in and out of frog ovarian eggs at 25°C. The results show that the rate-limiting step in the exchange of labeled water is not permeation through the cell membrane but diffusion in the bulk of the intracellular water.Eighty-eight years ago Pfeffer postulated that the cell membrane is a universal barrier to the traffic of all solutes and water, i.e. all resistance to water movement lies in the cell membrane, and diffusion of water within the cytoplasm is instantaneous (1). Until recently studies of water movement into and out of living cells were interpreted on the basis of this postulate. In 1959, Dick pointed out that, in spite of the general acceptance of the assumption, there is no evidence in its favor (2, 3). Based on a correlation between the permeability coefficients of a large number of cell types and the surface/volume ratios of these cells, he concluded that cytoplasmic resistance to water movement is not insignificant but actually contributes to the " m e m b r a n e permeability constants" reported in the literature. This finding reopens the basic question of whether the rate of water movement into and out of the living cells is determined by the cell membrane.In this report we shall present the results of our study on the rate of permeation of tritium hydrogen oxide (THO)-labeled water into frog ovarian eggs. This study was made in the hope of obtaining a definitive answer to the above question. For this purpose we made use of a new method called the "influx profile analysis" which has been briefly introduced elsewhere by the senior author of this paper (4).

1. The effluxes of labeled Na+, D-arabinose, and sucrose from normal muscle and muscle poisoned with low concentrations of iodoacetate were studied. The procedure involved repeated loading with isotope, followed by washing of the same muscle while still normal and at different states of dying. 2. The rates of Na+ efflux in both the fast and slow fraction remained either quite constant or showed some unpredictable, minor fluctuations. This was true for both Na+ and the two sugars studied, confirming earlier conclusions that the steady levels of these solutes were not maintained by pumps. 3. In all cases studied, the efflux curves showed at least two fractions. It is the fast-exchanging fraction that steadily and consistently increased in magnitude as the muscles were dying, until finally the concentration of solute in this fraction reached and sometimes surpassed the labeled solute concentrations in the original labeled solutions in which the muscles were equilibrated. The slow fractions showed only a transient increase or none at all. These observations show that it is the fast fraction that represents solute dissolved in cell water and rate-limited by passage through the cell surface and that the partial exclusion of Na+ and the sugars have a unitary cause--a reduced solubility in the cell water which in the presence of ATP exists in the state of polarized multilayers.

A comparison is made between the quantitative predictions of equilibrium ionic distribution in living cells according to the membrane theory (Donnan equilibrium) and according to the association-induction hypothesis. This comparison shows that both theories predict competitive effects of one permeant ion on the equilibrium concentration of another permeant ion; but within the limit of experimental accuracy only the association-induction model predicts quantitatively significant specific competition of one specified ion with the accumulation of another specified ion. The equilibrium distributions of K +, Rb +, and Cs + ions in frog sartorius muscle were studied and quantitatively significant specific competition was demonstrated; these results favor the association-induction hypothesis (adsorption on cell proteins and protein complexes and partial exclusion from cell water). Based on this model we estimated that at 25°C, the apparent association constants for K +, Rb +, and Cs + ion are 665, 756, and 488 (mole/liter) -l. We found that the total concentration of adsorption sites (no less than 240 mmole/kg of fresh cells) agrees with the analytically determined concentrations of t-and "y-carboxyl groups of muscle cell proteins (260 to 288 mmole/kg). I N T R O D U C T I O NIons and other solutes do not, as a rule, distribute themselves within cells at the concentrations anticipated on the basis of simple thermodynamic equilibria (1, 2). Past study of this phenomenon has often been hampered by difficulties in preserving isolated cells in reasonably good condition for a prolonged period of time. Recently, by adapting tissue culture methods, we have overcome this problem and have been able to investigate quantitatively the steady ionic level reached in the muscle cell following changes of the external ionic environment.A priori, a constantly maintained level of an ion or other solute in the cell m a y represent either an equilibrium phenomenon or a steady-state phenomenon. The maintenance of an equilibrium needs no expenditure of free energy. However, in the maintenance of a steady state, which must involve active transport or " p u m p s " of some sort, a continued free energy expenditure is mandatory. Comparison of the total energy available to the muscle cell