On the mechanism and measurement of shape transformations of constant volume of human red blood cells.

A method is described for measuring the rate of disc-sphere transformations of constant volume by recording changes in the intensity and noise envelope of light transmitted through a stirred suspension of human red blood cells. We used this method to determine the rates and characteristics of disc-sphere transitions induced by various sphering agents (uranyl nitrate, rose bengal, Na glycocholate). It was found that, when sphered by any of these agents, cells spontaneously reverted to discs upon continued incubation in the presence of the agent. The rate of sphering was directly proportional and the rate of spontaneous reversal inversely related to the concentration of sphering agent used. Plasma added before an agent is added prevents subsequent sphering; plasma added after sphering had occurred accelerated reversal. During spontaneous reversal of the spheres to discs, sphering could again be induced prior to subsequent return to discs either by the addition of more agent or, in certain circumstances, by the addition of plasma. Spontaneous reversal of discs to spheres qualitatively correlates with the uptake rate of agent (rose bengal), the cells becoming discs as uptake is completed. Furthermore, establishment of temporary proton gradients across the membrane promotes sphering with return to discs as pH equilibrium takes place. Evidently, the spherical shape is a transient form representing the unequal distribution of agent across the membrane. The spherical form could thus be associated with a change in the relative surface energies between the outer and inner lipid leaflets, such as a difference in their relative surface areas, as hypothesized in the "bilayer couple" models. The molecular mechanism(s) underlying the action of sphering agents and protons may not be the same.