Effect of extracellular Ca²⁺ on plasma membrane Ca²⁺ inflow and cytoplasmic free Ca²⁺ in isolated hepatocytes

An initial rapid phase and a subsequent slow phase of ⁴⁵Ca²⁺ uptake were observed following the addition of ⁴⁵Ca²⁺ to Ca²⁺-deprived hepatocytes. The magnitude of the rapid phase increased 15-fold over the range 0.1-11 mM extracellular Ca²⁺ (Ca²⁺_o) and was a linear function of [Ca²⁺]_o. The increases in the rate of ⁴⁵Ca²⁺ uptake were accompanied by only small increases in the intracellular free Ca²⁺ concentration. In cells made permeable to Ca²⁺ by treatment with saponin, the rate of ⁴⁵Ca²⁺ uptake (measured at free Ca²⁺ concentrations equal to those in the cytoplasm of intact cells) increased as the concentration of saponin increased from 1.4 to 2.5 μg per mg wet weight cells. Rates of ⁴⁵Ca²⁺ uptake by cells permeabilized with an optimal concentration of saponin were comparable with those of intact cells incubated at physiological [Ca²⁺_o], but were substantially lower than those for intact cells incubated at high [Ca²⁺_o]. It is concluded that (i) Ca²⁺ which enters the hepatocyte across the plasma membrane is rapidly removed by binding and transport to intracellular sites and by the plasma membrane Ca²⁺ + Mg²⁺-ATPase and (ii) the plasma membrane Ca²⁺ inflow transporter is not readily saturated with Ca²⁺_o.