The measurement of Ca2+ inflow across the liver cell plasma membrane by using quin2 and studies of the roles of Na+ and extracellular Ca2+ in the mechanism of Ca2+ inflow

1. Rates of Ca2+ inflow across the hepatocyte plasma membrane in the presence of vasopressin were estimated by using quin2. 2. Plots of the rate of Ca2+ inflow as a function of the intracellular quin2 concentration reached a plateau at about 1.7 mM intracellular quin2. Ca2+ inflow was inhibited by 60% in the presence of 400 microM-verapamil. 3. A plot of the rate of Ca2+ inflow as a function of the concentration of extracellular Ca2+ ([Ca2+]o) was biphasic. The second (slower) phase showed no sign of saturation at values of [Ca2+]o up to 5 mM. It is concluded that, in the presence of vasopressin, Ca2+ flows into the liver cell by two different processes, one of which is not readily saturated by Ca2+o. 4. The effect of the replacement of extracellular NaCl by choline or tetramethylammonium chloride on cellular Ca2+ movement was found to depend on the presence or absence of intracellular quin2. 5. In cells loaded with quin2 and incubated in the presence of choline or tetramethylammonium chloride, a small decrease in the basal intracellular free Ca2+ concentration ([Ca2+]i) was observed, and the increase in [Ca2+]i caused by the addition of vasopressin was considerably diminished when compared with cells incubated in the presence of NaCl. In cells loaded with quin2, replacement of NaCl by choline chloride caused a decrease in Ca2+ inflow in the presence of vasopressin, as measured by using quin2 or 45Ca2+ exchange, whereas no change in Ca2+ inflow was observed in the absence of vasopressin. 6. In cells not loaded with quin2, replacement of NaCl by choline chloride did not alter Ca2+ inflow either in the presence or in the absence of vasopressin. 7. It is concluded that (i) Ca2+ inflow through the basal and receptor-activated Ca2+ inflow systems does not involve the inward movement of Ca2+ in exchange for Na+ or the induction of Ca2+ inflow by intracellular Na+, and (ii) the presence of both intracellular quin2 and extracellular choline or tetramethylammonium chloride (in place of NaCl) inhibits Ca2+ inflow through the receptor-activated Ca2+ inflow system but not through the basal Ca2+ inflow system, and inhibits the release of Ca2+ from intracellular stores.

Effect of Ca2+ ions on liver cell plasma membrane polypeptides

Plasma membrane fractions, microsomes, and mitochondrial membrane were prepared from rat liver. The effects of incubation of these membranes in various ionic strengths (1–12 mM) of NaHCO3 (pH 7.4) on the polypeptide patterns were studied in the absence or the presence of various concentrations of CaCl2 (1–12 mM). Many polypeptides were extracted from each of the membranes in the low ionic strength buffers (1 mM); these polypeptides were essentially similar to those remaining in the membrane after incubation. The extraction of these polypeptides continued when concentration of NaHCO3 increased. However, the presence of CaCl2 at a concentration of 4 mM or higher stabilized the membrane proteins and reduced the continuous loss of the polypeptides, and only low molecular weight polypeptides were affected. These data suggest that the organization of proteins in liver cell membranes may depend on the nature of their interaction with other membrane components (i.e., phospholipids), which could be affected by Ca2+ ions.