title: Functional expression of Ca2+ signaling pathways in mouse embryonic stem cells.
abstract: 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 confocal 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 (InsP3Rs). 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+]i was also elevated. Using RT-PCR, mRNAs for InsP3Rs 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 InsP3Rs 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.
authors.affiliation: Department of Cardiovascular Diseases, Medical Research Institute, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Yanagida
authors.forename: Satoshi
authors.initials: S
authors.lastname: Shoji
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Hirayama
authors.forename: Fumio
authors.initials: F
authors.lastname: Yoshikawa
authors.forename: Keishi
authors.initials: K
authors.lastname: Otsu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Uematsu
authors.forename: Masayasu
authors.initials: M
authors.lastname: Hiraoka
authors.forename: Teiichi
authors.initials: T
authors.lastname: Furuichi
authors.forename: Seiko
authors.initials: S
authors.lastname: Kawano
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D051379:Mice; D018408:Patch-Clamp Techniques; D039904:Pluripotent Stem Cells / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D043562:Receptors, G-Protein-Coupled; D019831:Sodium-Calcium Exchanger; D000252:Calcium-Transporting ATPases