Calcium uptake in mastocytoma P-815 cells

Kinetic data of 45Ca2+ exchange in exponentially growing mastocytoma P-815 cells at 37 degree C indicated the presence of at least two calcium compartments, fast and slowly exchangeable. The fast phase of calcium exchange at the cell surface had a rate constant of 36.1 min-1 and a compartment size of 8.27 nmol/10(6) cells, while the slow phase, the intracellular calcium exchange, showed a rate constant of 2.4 min-1 and a compartment size of 0.53 nmolS/10(6) cells. The fast phase comprised two calcium binding sites of high and low affinities with dissociation constants of 0.59 and 4.88 mM, respectively, and binding maxima of 5.15 and 34.4 nmol/10(6) cells, respectively. The calcium uptake at both binding sites was inhibited by 80% on the addition of ruthenium red (0.4 mM) or lanthanum chloride (0.5 mM), but not by tetracaine (5 mM). The high-affinity site had two negative reaction enthalpy changes, delta H 0 = -1.55 (from 0 to 22 degree C) and -4.27 (from 22 degree C to 37 degree C) kcal . mol-1, while the low-affinity site showed two positive reaction enthalpy changes, delta H0 = +6.53 (below 22 degree C) and +2.05 (above 22 degree C) kcal . mol-1, suggesting the effect of membrane fluidity on calcium binding. The slow phase of calcium exchange was also inhibited by ruthenium red and lanthanum chloride resulting in a decrease of the Hill's coefficient from 1.70 to 1.28 and 1.13, respectively, but was not affected by tetracaine. Calcium efflux from 45 Ca-loaded cells was also inhibited by ruthenium red and lanthanum chloride and was also temperature sensitive. The efflux rate constant was reduced by 80% and 100% at 20 degree C and 0 degree C, respectively. As calcium uptake fluctuated during the cell cycle of synchronous mastocytoma P-815 cells with a peak at the mid S phase and the lowest level during the late M phase, the compartment size of fast phase also fluctuated, being highest at the S phase and lowest at the M phase. In contrast, the compartment size of the slow phase was highest at the G1 phase and lowest at the M phase. In parallel with these changes, the binding maximum of the high-affinity site was highest at the S phase and lowest at the M phase, while that of the low-affinity site fluctuated little throughout the cell cycle.