Quantitative study on La~(3+) influx mediated by sodium-calcium exchanger in human lymphocytes

Whether La3+ can enter human peripheral blood lymphocytes by the Na+/Ca2+ exchanger or not and the effect of La3+on the Na+/Ca2+ exchanger activity are examined by fura-2 technique. And that whether La3+ is sequestered by intracellular organelles (mainly endoplasmic reticulum and mitochondria) is studied by this method. La3+uptake is obviously stimulated by pre-treating the cells with ouabain and by removing extracellular Na+, and intracellular La3+concentration (La3+]i) is directly proportional to its extracellular concentration (La3+]o). But when La3+]o exceeds 0.4 mmol/L, the 340/380 nm ratio of fluorescence is no longer varied and the maximum La3+], is 1.5×10-12 mol · L-1. The higher concentration of La3+ (0.1 mmol/L) increases Na+/Ca2+ exchange-mediated calcium influx, but lower concentration (10 μmol/L) appears to block calcium influx. The results also suggest that cytosolic La3+ is transported by the ATP-dependent Ca2+ pump. Intracellular Ca2+ stores are depleted by ionomycin, and then ion

Quantitative study on La3+ influx mediated by sodium-calcium exchanger in human lymphocytes

Whether La3+ can enter human peripheral blood lymphocytes by the Na+/Ca2+ exchanger or not and the effect of La3+ on the Na+/Ca2+ exchanger activity are examined by fura-2 technique. And that whether La3+ is sequestered by intracellular organelles (mainly endoplasmic reticulum and mitochondria) is studied by this method. La3+ uptake is obviously stimulated by pretreating the cells with ouabain and by removing extracellular Na+, and intracellular La3+ concentration (La3+]i) is directly proportional to its extracellular concentration (La3+]o). But when La3+]o exceeds 0.4 mmol/L, the 340/380 nm ratio of fluorescence is no longer varied and the maximum La3+]i is 1.5×10-12 mol@L-1. The higher concentration of La3+ (0.1 mmol/L) increases Na+/Ca2+ exchange-mediated calcium influx, but lower concentration (10 mmol/L) appears to block calcium influx. The results also suggest that cytosolic La3+ is transported by the ATP-dependent Ca2+ pump. Intracellular Ca2+ stores are depleted by ionomycin, and then ionomycin is added again during the period of La3+ uptake, the 340/380 nm ratio of fluorescence is also increased, these results indicate that La3+ is sequestered by intracellular organelles. A characterization of fura-2-La3+ interaction in solution simulating intracellular ionic composition (pH 7.05) shows that La3+ forms a 1:1 fura-2-La3+complex, and the apparent dissociation constant of La3+ for fura-2 (Kd) is 1.7×10-12 mol@L-1. In addition, the limit of detection of fura-2 for La3+ and Ca2+ is 10?12 and 10?8 mol@L-1 respectively.