A simple model is proposed to account for large increases in transporter-mediated ion flux across cell membranes that are elicited by small fractional changes of cell volume. The model is based upon the concept that, as a result of large excluded volume effects in cytoplasm (macromolecular crowding), the tendency of soluble macromolecules to associate with membrane proteins is much more sensitive to changes in cell water content than expected on the basis of simple considerations of mass action. The model postulates that an ion transporter may exist in either an active dephosphorylated state or an inactive phosphorylated state and that the steadystate activity of the transporter reflects a balance between the rates of phosphatase-catalyzed activation and kinase-catalyzed inactivation. Cell swelling results in the inhibition of kinase relative to phosphatase activity, thereby increasing the steadystate concentration of the active form of the transporter. Calculated volume-dependent stimulation of ion flux is comparable to that observed experimentally. Effect of Crowding on the Binding of Macromolecular Ligands to Surface SitesMacromolecular reaction rates and equilibria inside living cells are not governed, in general, by mass-action rate and equilibrium expressions. Both theory and experiment have shown that the thermodynamic activity of each macromolecular species in a highly volume-occupied, or crowded, solution exceeds the activity of that species at an identical concentration in an uncrowded fluid (9). The activity coefficient of the ith species, defined as the ratio of thermodynamic activity ai to concentration ci, is denoted by y,. For a fluid containing a mixture of molecular species that may be represented by compact rigid particles, scaled particle theory (10) where ri is the characteristic dimension of the ith species (for example, the radius of a quasispherical molecule), and the Aj coefficients are positive-definite functions of the concentrations and characteristic dimensions of all of the species present in the fluid.

Jennings and Schulz (1991. J. Gen. Physiol. 96:799-817) that activation/deactivation of volume-sensitive transport involves phosphorylation/ dephosphorylation of a regulatory protein is supported by these observations.

It is not known whether the activation of Na/H exchange by shrinkage in dog red cells is due to the packing of cell contents or a change in cell configuration. To make this distinction we prepared resealed ghosts that resembled intact cells in hemoglobin concentration and surface area, but had one-third their volume. A shrinkage-induced, amiloride-sensitive Na flux in the ghosts was activated at a much smaller volume in the ghosts than in the intact cells, but at the same concentration (by weight) of dry solids in both preparations. Na/H exchange in ghosts containing a mixture of 40% albumin and 60% hemoglobin (weight/weight) was activated by osmotic shrinkage at a dry solid concentration similar to that of intact cells or of ghosts containing only hemoglobin. We conclude that the process of Na/H exchange activation by cell shrinkage originates with an increase in the concentration of intracellular protein and not with a change in membrane configuration or tension. The macromolecular crowding that accompanies the reduction in cell volume probably alters the activities of key enzymes that in turn modulate the Na/H exchanger.

We previously isolated a family of bone-resorbing proteins from human cancer ascites fluid and established that the three purified bone-resorbing proteins were chemically and immunochemically related to each other and to alpha-2HS glycoprotein (alpha 2HS). After this finding we purified the normal human serum counterpart of these ascites proteins and studied its effects on bone resorption. The bone-resorbing properties of normal human serum alpha 2HS were examined in vitro over a wide dose range. This normal human serum glycoprotein had a biphasic effect on 45Ca2+ release from bone. More specifically, this protein stimulated bone resorption at the lower concentrations tested, with a maximum effect [treated over control ratio of 2.5 +/- 0.30 (+/- SE); P less than 0.01] at 40 micrograms/mL. In contrast, at doses above 40 micrograms/mL, a sharp decline in calcium mobilization occurred, with a return to baseline occurring above 80 micrograms/mL. These results suggest that serum alpha 2HS may participate in the regulation of bone metabolism in vivo.