Role of intracellular Ca2+ stores in the regulation of electrogenic plasma membrane Ca2+ uptake in a B‐lymphocytic cell line

Experiments were undertaken to investigate the role of intracellular Ca2+ stores in the regulation of Ca2+ uptake in the cultured B-lymphocytic cell line CH12.LX.C4.5F5. Release of intracellular Ca2+ stores by addition of thapsigargin was accompanied by a biphasic increase in intracellular calcium concentration [Ca2+]i). The initial rise in [Ca2+]i was due to release of Ca2+ from intracellular stores as determined by its maintenance in the absence of extracellular Ca2+. The secondary phase was 1) dependent on the presence of extracellular Ca2+, 2) inhibited by 5 mM extracellular Ni2+, and 3) inhibited by high K+, consistent with electrogenic Ca2+ uptake from the extracellular medium. In order to more accurately investigate the electrogenic nature of this pathway we measured the membrane potential changes accompanying Ca2+ influx stimulated by release of Ca2+ from intracellular stores using bis(1,3-diethylthiobarbituric acid trimethine) oxonol in Bapta-loaded cells. Addition of 5 mM Ca2+ to cells pretreated with doses of thapsigargin or ionomycin shown to release intracellular Ca2+ stores induced a depolarization which was 1) dependent upon extracellular Ca2+, 2) abolished by 5 mM Ni2+, 3) independent of extracellular Na+, and 4) dependent upon Bapta loading. This depolarization was followed by a charybdotoxin-sensitive repolarization consistent with secondary activation of K+ channels. Changes in [Ca2+]i monitored under identical conditions were monitored fluorimetrically using indo-1 and were found to correlate with the changes in Em. On the basis of these data we conclude that an electrogenic Ca(2+)-permeable pathway exists in this B-lymphocytic cell line which is regulated by the degree of filling of an internal Ca(2+)-store.