Journal
CELL CALCIUM
Volume 36, Issue 2, Pages 135-146Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.ceca.2004.01.022
Keywords
mouse ES cell; Ca2+; InSP3 receptors; store-operated Ca2+ channels; Na+/Ca2+ exchange; plasma membrane Ca2+ pump; patch clamp
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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.
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