Functional expression of Ca2+ signaling pathways in mouse embryonic stem cells

Mouse embryonic stem (mES) cells have the potential to differentiate into all types of cells, but the physiological properties of undifferentiated mES cells, including Ca2+ signaling systems, are not fully understood. In this study, we investigated Ca2+ signaling pathways in mES cells by using conforcal Ca2+ imaging systems, patch clamp techniques and RT-PCR. The stimulations with ATP and histamine (His) induced a transient increase of intracellular Ca2+ concentration ([Ca2+](i)), which were prevented by the pretreatment of 2-amino-ethoxydiphenyl borate (2-APB), a blocker for inositol-1,4,5-triphosphate receptors (InSP(3)Rs). The application of caffeine (Caff) or ryanodine (Ry) did not change [Ca2+](i). When stores were depleted with Ca2+-ATPase blocker, thapsigargin (TG), or histamine, the capacitative Ca2+ entry (CCE) was observed. In whole cell patch clamp mode, store-operated Ca2+ currents could be recorded in cells treated with histamine and thapsigargin. On the other hand, voltage-operated Ca2+ channels (VOCCs) could not be elicited. The application of blockers for plasma membrane Ca2+ pump (PMCAs) (carboxeosin or caloxin2A1) induced a large increase of [Ca2+](i). When the Na+/Ca2+ exchangers (NCXs) were blocked by Na+ free solution or KBR7943, [Ca2+], was also elevated. Using RT-PCR, mRNAs for InSP(3)Rs type-1, -2, and -3, PMCA-1 and -4, NCX-1, -2, and -3 could be detected. From these results, we conclude that Ca2+ release from ER is mediated by InSP(3)Rs in mES cells before differentiation and Ca2+ entry through plasma membrane is mainly mediated by the store-operated Ca2+ channels (SOCs). For the Ca2+ extrusion systems, both NCXs and PMCAs play important roles for maintaining the low level of [Ca2+](i). (C) 2004 Elsevier Ltd. All rights reserved.