Role of intracellular Ca 2+ stores in the regulation of electrogenic plasma membrane Ca 2+ uptake in a B‐lymphocytic cell line

Experiments were undertaken to investigate the role of intracellular Ca 2+ stores in the regulation of Ca 2+ uptake in the cultured B‐lymphocytic cell line CH12.LX.C4.5F5. Release of intracellular Ca 2+ stores by addition of thapsigargin was accompanied by a biphasic increase in intracellular calcium concentration ([Ca 2+ ] i ). The initial rise in [Ca 2+ ] i was due to release of Ca 2+ from intracellular stores as determined by its maintenance in the absence of extracellular Ca 2+ . The secondary phase was (1) dependent on the presence of extracellular Ca 2+ , (2) inhibited by 5 mM extracellular Ni 2+ , and (3) inhibited by high K + , consistent with electrogenic Ca 2+ uptake from the extracellular medium. In order to more accurate y investigate the electrogenic nature of this pathway we measured the membrane potential changes accompanying Ca 2+ influx stimulated by release of Ca 2+ from intracellular stores using bis (1,3‐diethylthiobarbituric acid trimethine) oxonol in Bapta‐loaded cells. Addition of 5 mM Ca 2+ to cells pretreated with doses of thapsigargin or ionomycin shown to release intracellular Ca 2+ stores induced a depolarization which was (1) dependent upon extracellular Ca 2+ , (2) abolished by 5 mM Ni 2+ , (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 [Ca 2+ ] i monitored under identical conditions were monitored fluorimetrically using indo‐1 and were found to correlate with the changes in E m . 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. © 1994 Wiley‐Liss, Inc.