Rapid Turnover of Calcium in the Endoplasmic Reticulum during Signaling (2000)

HEK293 cells expressing the thyrotropin-releasing hormone (TRH) receptor were transfected with cameleon Ca2+ indicators designed to measure the free Ca2+ concentration in the cytoplasm, [Ca2+]cyt, and the endoplasmic reticulum (ER), [Ca2+]er. Basal [Ca2+]cyt was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca2+]cyt to 1 μm or higher. Basal [Ca2+]er averaged 500 μmand fell to 50–100 μm over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca2+]er to 100 μm, independent of extracellular Ca2+, whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca2+]er to 5–10 μm, indicating that there is substantial store refilling during signaling. TRH increased [Ca2+]cyt and decreased [Ca2+]er if applied after other agonists, whereas other agonists did not alter [Ca2+]cyt or [Ca2+]er if added after TRH. When Ca2+ was added back to cells that had been incubated with TRH in Ca2+-free medium, [Ca2+]cyt and [Ca2+]er increased rapidly. The increase in [Ca2+]er was only partially blocked by thapsigargin but was completely blocked if cells were loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. In conclusion, these new Ca2+ indicators showed that basal [Ca2+]er is ∼500 μm, that [Ca2+]er has to be >100 μm to support an increase in [Ca2+]cyt by agonists, and that during signaling, intracellular Ca2+ stores are continuously refilled with cytoplasmic Ca2+ by the sarcoendoplasmic reticulum Ca2+-ATPase pump. HEK293 cells expressing the thyrotropin-releasing hormone (TRH) receptor were transfected with cameleon Ca2+ indicators designed to measure the free Ca2+ concentration in the cytoplasm, [Ca2+]cyt, and the endoplasmic reticulum (ER), [Ca2+]er. Basal [Ca2+]cyt was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca2+]cyt to 1 μm or higher. Basal [Ca2+]er averaged 500 μmand fell to 50–100 μm over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca2+]er to 100 μm, independent of extracellular Ca2+, whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca2+]er to 5–10 μm, indicating that there is substantial store refilling during signaling. TRH increased [Ca2+]cyt and decreased [Ca2+]er if applied after other agonists, whereas other agonists did not alter [Ca2+]cyt or [Ca2+]er if added after TRH. When Ca2+ was added back to cells that had been incubated with TRH in Ca2+-free medium, [Ca2+]cyt and [Ca2+]er increased rapidly. The increase in [Ca2+]er was only partially blocked by thapsigargin but was completely blocked if cells were loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. In conclusion, these new Ca2+ indicators showed that basal [Ca2+]er is ∼500 μm, that [Ca2+]er has to be >100 μm to support an increase in [Ca2+]cyt by agonists, and that during signaling, intracellular Ca2+ stores are continuously refilled with cytoplasmic Ca2+ by the sarcoendoplasmic reticulum Ca2+-ATPase pump. cytoplasmic free Ca2+concentration 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid ER luminal free Ca2+ concentration Hanks' balanced salt solution inositol 1,4,5-trisphosphate sarcoendoplasmic reticulum Ca2+ ATPase thyrotropin-releasing hormone The cytosolic free Ca2+ concentration ([Ca2+]cyt)1is a dynamic signal regulating a variety of important cellular functions such as secretion, contraction, metabolism, and gene transcription (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar). Ca2+-mobilizing hormones release inositol 1,4,5-trisphosphate (IP3) which acts on the IP3 receptor to release Ca2+ from intracellular sites in the endoplasmic reticulum (ER). It is not certain whether the entire ER or specialized compartments within the ER form the IP3-releasable Ca2+ store (2Bode H.P. Netter K.J. Biochem. Pharmacol. 1996; 51: 993-1001Crossref PubMed Scopus (17) Google Scholar, 3Meldolesi J. Pozzan T. Trends Biochem. Sci. 1998; 23: 10-14Abstract PubMed Scopus Google Scholar, J. J. Pozzan T. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). In IP3 receptors are in a of the ER J. PubMed Scopus Google Scholar). IP3 receptors are Ca2+ that as the IP3 concentration The cytoplasmic and ER Ca2+ the of the IP3 receptor to IP3 Nature. 1998; PubMed Scopus Google Scholar, J. Nature. PubMed Scopus Google Scholar, J. PubMed Google Scholar). The Ca2+ store is refilled by the sarcoendoplasmic reticulum Ca2+-ATPase a Ca2+ pump. Ca2+ in the Ca2+ in intracellular stores is to only free Ca2+ is to in Ca2+ Nature. 1998; PubMed Scopus Google Scholar, Biochem. 23: PubMed Scopus Google Scholar). of Ca2+ stores in by receptors that luminal Ca2+ are for J. PubMed Scopus Google and in the ER J. Pozzan T. Trends Biochem. Sci. 1998; 23: 10-14Abstract PubMed Scopus Google Scholar). of the store by of the to 1996; PubMed Scopus Google the concentration of free Ca2+ in the cytoplasm, [Ca2+]cyt, has been for there been for the free Ca2+ concentration in the ER Trends Pharmacol. Sci. 1998; PubMed Scopus Google Scholar, J. Nature. PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). Ca2+ in the has been but is or to the the ER in cells 1996; PubMed Scopus Google Scholar). has been to but the of to Ca2+ over J. Pozzan T. J. PubMed Scopus Google Scholar). Ca2+ indicators on in a with and a J. Nature. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, Sci. PubMed Scopus Google Scholar). the Ca2+ indicators from the Sci. PubMed Scopus Google been to ER and to [Ca2+]er in cells J. Nature. PubMed Scopus Google Scholar). in the ER is by a Ca2+ to cells loaded with a cytoplasmic Ca2+ ER Ca2+ is The increase in Ca2+ in the an of the of from the the of a expressing receptors for thyrotropin-releasing hormone (TRH) and that the refilling of the intracellular Ca2+ to be the in TRH of [Ca2+]cyt J. PubMed Scopus Google Scholar, 1998; PubMed Google Scholar). In the the to measure the free Ca2+concentration in the ER and the and refilling of the intracellular Ca2+ store in to agonists, and that free Ca2+ in the ER is 500 μm and that during signaling, ER Ca2+ is by the with cytoplasmic Ca2+ that has the from the extracellular the cameleon Ca2+ indicators J. Nature. PubMed Scopus Google to new about the concentration of free Ca2+ in the and endoplasmic reticulum during in a J. PubMed Scopus Google Scholar, J. 1996; Google Scholar). The cytoplasmic cameleon of μm and 100 is to in cytoplasmic Ca2+ in the and Ca2+ indicators are of The in free Ca2+ over a but the in are and is and is and are to J. Nature. PubMed Scopus Google Scholar, Sci. PubMed Scopus Google Scholar). not been a with which has on cytoplasmic cells are Biochem. J. PubMed Scopus Google but was a with which is a of that the in were to in in the but not the of the in in the Ca2+ were in for the cytoplasmic and the is the and the ER Sci. 1998; PubMed Scopus Google Scholar). did not the of or added to cells with intracellular these been to the in of the cytoplasmic has Sci. PubMed Scopus Google of a of [Ca2+]er in were and μm, 500 μm, in with by J. Nature. PubMed Scopus Google for [Ca2+]er in of to of basal [Ca2+]er 100 μm and 1996; PubMed Scopus Google is to that of the Ca2+ by the and TRH was to for the in were added with In refilling is to as [Ca2+]er the of the is by Ca2+ in the ER J. PubMed Google Scholar, J. 1998; PubMed Scopus Google and the of Ca2+ as [Ca2+]cyt J. J. PubMed Google Scholar, Biochem. J. PubMed Scopus Google Scholar). is [Ca2+]er is 100 μm, which the of Ca2+ release the IP3 receptor and Ca2+ refilling by the It be that [Ca2+]er is not to 100 μm, agonists are to other functions of the from the in not the in [Ca2+]er M.J. J. J. PubMed Scopus Google Scholar). In the HEK293 cells the and Ca2+ to Ca2+ in the ER cells are with a of agonists for endogenous receptors cells are with TRH. It is not if is the of in or the of of the transfected TRH the to a of Ca2+ of Ca2+ In the of of ER Ca2+ to be in [Ca2+]er was of extracellular the of Ca2+ in HEK293 cells are in with of Pozzan T. J. 1998; PubMed Scopus Google to [Ca2+]er in cells and that of Ca2+ the ER during cytoplasmic Ca2+ is from by M.J. J. J. PubMed Scopus Google agonists to release the of ER Ca2+ to a cytoplasmic In the release of ER Ca2+ in to agonists is by the of cytoplasmic Ca2+ and luminal Ca2+ on the IP3 In HEK293 agonists a in [Ca2+]er in the of cytoplasmic and luminal Ca2+ and refilling during signaling. in IP3 and metabolism, IP3 receptor and and all to these that extracellular Ca2+ is not for the of the ER Ca2+ store over 10 min if agonists or are In the presence of the intracellular Ca2+ store is continuously stores were to cytoplasmic Ca2+ in increased ER was for in the Ca2+ increase was for the are J. J. PubMed Google Scholar, Biochem. J. PubMed Scopus Google that an increase in cytoplasmic Ca2+ be to to an increase in the of that refilling was completely with indicating that the Ca2+ for refilling from the are with that and the IP3 receptor are for store Ca2+ stores are with the Pozzan T. J. 1998; PubMed Scopus Google Scholar, J. PubMed Google that the intracellular Ca2+ store is refilled with Ca2+ from the by the and by an [Ca2+]er increased extracellular Ca2+ was to cells that had been with thapsigargin or acid in Ca2+-free medium, the increase in [Ca2+]er was were Ca2+ has been J. PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). in and cells been to increase [Ca2+]er in the by the or in Ca2+ been a the the to in free Ca2+ in the ER and that there is a of of ER Ca2+ during Ca2+ signaling. signaling, the intracellular Ca2+ store is continuously refilled by the Ca2+ from the be the of the sites in the ER in release and of The cytosolic free Ca2+ concentration ([Ca2+]cyt)1is a dynamic signal regulating a variety of important cellular functions such as secretion, contraction, metabolism, and gene transcription (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar). Ca2+-mobilizing hormones release inositol 1,4,5-trisphosphate (IP3) which acts on the IP3 receptor to release Ca2+ from intracellular sites in the endoplasmic reticulum (ER). It is not certain whether the entire ER or specialized compartments within the ER form the IP3-releasable Ca2+ store (2Bode H.P. Netter K.J. Biochem. Pharmacol. 1996; 51: 993-1001Crossref PubMed Scopus (17) Google Scholar, 3Meldolesi J. Pozzan T. Trends Biochem. Sci. 1998; 23: 10-14Abstract PubMed Scopus Google Scholar, J. J. Pozzan T. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). In IP3 receptors are in a of the ER J. PubMed Scopus Google Scholar). IP3 receptors are Ca2+ that as the IP3 concentration The cytoplasmic and ER Ca2+ the of the IP3 receptor to IP3 Nature. 1998; PubMed Scopus Google Scholar, J. Nature. PubMed Scopus Google Scholar, J. PubMed Google Scholar). The Ca2+ store is refilled by the sarcoendoplasmic reticulum Ca2+-ATPase a Ca2+ pump. Ca2+ in the Ca2+ in intracellular stores is to only free Ca2+ is to in Ca2+ Nature. 1998; PubMed Scopus Google Scholar, Biochem. 23: PubMed Scopus Google Scholar). of Ca2+ stores in by receptors that luminal Ca2+ are for J. PubMed Scopus Google and in the ER J. Pozzan T. Trends Biochem. Sci. 1998; 23: 10-14Abstract PubMed Scopus Google Scholar). of the store by of the to 1996; PubMed Scopus Google Scholar). the concentration of free Ca2+ in the cytoplasm, [Ca2+]cyt, has been for there been for the free Ca2+ concentration in the ER Trends Pharmacol. Sci. 1998; PubMed Scopus Google Scholar, J. Nature. PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). Ca2+ in the has been but is or to the the ER in cells 1996; PubMed Scopus Google Scholar). has been to but the of to Ca2+ over J. Pozzan T. J. PubMed Scopus Google Scholar). Ca2+ indicators on in a with and a J. Nature. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, Sci. PubMed Scopus Google Scholar). the Ca2+ indicators from the Sci. PubMed Scopus Google been to ER and to [Ca2+]er in cells J. Nature. PubMed Scopus Google Scholar). in the ER is by a Ca2+ to cells loaded with a cytoplasmic Ca2+ ER Ca2+ is The increase in Ca2+ in the an of the of from the the of a expressing receptors for thyrotropin-releasing hormone (TRH) and that the refilling of the intracellular Ca2+ to be the in TRH of [Ca2+]cyt J. PubMed Scopus Google Scholar, 1998; PubMed Google Scholar). In the the to measure the free Ca2+concentration in the ER and the and refilling of the intracellular Ca2+ store in to agonists, and that free Ca2+ in the ER is 500 μm and that during signaling, ER Ca2+ is by the with cytoplasmic Ca2+ that has the from the extracellular the cameleon Ca2+ indicators J. Nature. PubMed Scopus Google to new about the concentration of free Ca2+ in the and endoplasmic reticulum during in a J. PubMed Scopus Google Scholar, J. 1996; Google Scholar). The cytoplasmic cameleon of μm and 100 is to in cytoplasmic Ca2+ in the and Ca2+ indicators are of The in free Ca2+ over a but the in are and is and is and are to J. Nature. PubMed Scopus Google Scholar, Sci. PubMed Scopus Google Scholar). not been a with which has on cytoplasmic cells are Biochem. J. PubMed Scopus Google but was a with which is a of that the in were to in in the but not the of the in in the Ca2+ were in for the cytoplasmic and the is the and the ER Sci. 1998; PubMed Scopus Google Scholar). did not the of or added to cells with intracellular these been to the in of the cytoplasmic has Sci. PubMed Scopus Google of a of [Ca2+]er in were and μm, 500 μm, in with by J. Nature. PubMed Scopus Google for [Ca2+]er in of to of basal [Ca2+]er 100 μm and 1996; PubMed Scopus Google is to that of the Ca2+ by the and TRH was to for the in were added with In refilling is to as [Ca2+]er the of the is by Ca2+ in the ER J. PubMed Google Scholar, J. 1998; PubMed Scopus Google and the of Ca2+ as [Ca2+]cyt J. J. PubMed Google Scholar, Biochem. J. PubMed Scopus Google Scholar). is [Ca2+]er is 100 μm, which the of Ca2+ release the IP3 receptor and Ca2+ refilling by the It be that [Ca2+]er is not to 100 μm, agonists are to other functions of the from the in not the in [Ca2+]er M.J. J. J. PubMed Scopus Google Scholar). In the HEK293 cells the and Ca2+ to Ca2+ in the ER cells are with a of agonists for endogenous receptors cells are with TRH. It is not if is the of in or the of of the transfected TRH the to a of Ca2+ of Ca2+ In the of of ER Ca2+ to be in [Ca2+]er was of extracellular the of Ca2+ in HEK293 cells are in with of Pozzan T. J. 1998; PubMed Scopus Google to [Ca2+]er in cells and that of Ca2+ the ER during cytoplasmic Ca2+ is from by M.J. J. J. PubMed Scopus Google agonists to release the of ER Ca2+ to a cytoplasmic In the release of ER Ca2+ in to agonists is by the of cytoplasmic Ca2+ and luminal Ca2+ on the IP3 In HEK293 agonists a in [Ca2+]er in the of cytoplasmic and luminal Ca2+ and refilling during signaling. in IP3 and metabolism, IP3 receptor and and all to these that extracellular Ca2+ is not for the of the ER Ca2+ store over 10 min if agonists or are In the presence of the intracellular Ca2+ store is continuously stores were to cytoplasmic Ca2+ in increased ER was for in the Ca2+ increase was for the are J. J. PubMed Google Scholar, Biochem. J. PubMed Scopus Google that an increase in cytoplasmic Ca2+ be to to an increase in the of that refilling was completely with indicating that the Ca2+ for refilling from the are with that and the IP3 receptor are for store Ca2+ stores are with the Pozzan T. J. 1998; PubMed Scopus Google Scholar, J. PubMed Google that the intracellular Ca2+ store is refilled with Ca2+ from the by the and by an [Ca2+]er increased extracellular Ca2+ was to cells that had been with thapsigargin or acid in Ca2+-free medium, the increase in [Ca2+]er was were Ca2+ has been J. PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). in and cells been to increase [Ca2+]er in the by the or in Ca2+ been a the the to in free Ca2+ in the ER and that there is a of of ER Ca2+ during Ca2+ signaling. signaling, the intracellular Ca2+ store is continuously refilled by the Ca2+ from the be the of the sites in the ER in release and of the cameleon Ca2+ indicators J. Nature. PubMed Scopus Google to new about the concentration of free Ca2+ in the and endoplasmic reticulum during in a J. PubMed Scopus Google Scholar, J. 1996; Google Scholar). The cytoplasmic cameleon of μm and 100 is to in cytoplasmic Ca2+ in the and Ca2+ indicators are of The in free Ca2+ over a but the in are and is and is and are to J. Nature. PubMed Scopus Google Scholar, Sci. PubMed Scopus Google Scholar). not been a with which has on cytoplasmic cells are Biochem. J. PubMed Scopus Google but was a with which is a of that the in were to in in the but not the of the in in the Ca2+ were in for the cytoplasmic and the is the and the ER Sci. 1998; PubMed Scopus Google Scholar). did not the of or added to cells with intracellular these been to the in of the cytoplasmic has Sci. PubMed Scopus Google Scholar). of a of [Ca2+]er in were and μm, 500 μm, in with by J. Nature. PubMed Scopus Google for [Ca2+]er in of to of basal [Ca2+]er 100 μm and 1996; PubMed Scopus Google is to that of the Ca2+ by the and TRH was to for the in were added with In refilling is to as [Ca2+]er the of the is by Ca2+ in the ER J. PubMed Google Scholar, J. 1998; PubMed Scopus Google and the of Ca2+ as [Ca2+]cyt J. J. PubMed Google Scholar, Biochem. J. PubMed Scopus Google Scholar). is [Ca2+]er is 100 μm, which the of Ca2+ release the IP3 receptor and Ca2+ refilling by the It be that [Ca2+]er is not to 100 μm, agonists are to other functions of the from the in not the in [Ca2+]er M.J. J. J. PubMed Scopus Google Scholar). In the HEK293 cells the and Ca2+ to Ca2+ in the ER cells are with a of agonists for endogenous receptors cells are with TRH. It is not if is the of in or the of of the transfected TRH the to a of Ca2+ of Ca2+ In the of of ER Ca2+ to be in [Ca2+]er was of extracellular the of Ca2+ in HEK293 cells are in with of Pozzan T. J. 1998; PubMed Scopus Google to [Ca2+]er in cells and that of Ca2+ the ER during cytoplasmic Ca2+ is from by M.J. J. J. PubMed Scopus Google agonists to release the of ER Ca2+ to a cytoplasmic In the release of ER Ca2+ in to agonists is by the of cytoplasmic Ca2+ and luminal Ca2+ on the IP3 In HEK293 agonists a in [Ca2+]er in the of cytoplasmic and luminal Ca2+ and refilling during signaling. in IP3 and metabolism, IP3 receptor and and all to these that extracellular Ca2+ is not for the of the ER Ca2+ store over 10 min if agonists or are In the presence of the intracellular Ca2+ store is continuously stores were to cytoplasmic Ca2+ in increased ER was for in the Ca2+ increase was for the are J. J. PubMed Google Scholar, Biochem. J. PubMed Scopus Google that an increase in cytoplasmic Ca2+ be to to an increase in the of that refilling was completely with indicating that the Ca2+ for refilling from the are with that and the IP3 receptor are for store Ca2+ stores are with the Pozzan T. J. 1998; PubMed Scopus Google Scholar, J. PubMed Google that the intracellular Ca2+ store is refilled with Ca2+ from the by the and by an [Ca2+]er increased extracellular Ca2+ was to cells that had been with thapsigargin or acid in Ca2+-free medium, the increase in [Ca2+]er was were Ca2+ has been J. PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). in and cells been to increase [Ca2+]er in the by the or in Ca2+ been a the the to in free Ca2+ in the ER and that there is a of of ER Ca2+ during Ca2+ signaling. signaling, the intracellular Ca2+ store is continuously refilled by the Ca2+ from the be the of the sites in the ER in release and of of a of [Ca2+]er in were and μm, 500 μm, in with by J. Nature. PubMed Scopus Google for [Ca2+]er in of to of basal [Ca2+]er 100 μm and 1996; PubMed Scopus Google is to that of the Ca2+ by the and TRH was to for the in were added with In refilling is to as [Ca2+]er the of the is by Ca2+ in the ER J. PubMed Google Scholar, J. 1998; PubMed Scopus Google and the of Ca2+ as [Ca2+]cyt J. J. PubMed Google Scholar, Biochem. J. PubMed Scopus Google Scholar). is [Ca2+]er is 100 μm, which the of Ca2+ release the IP3 receptor and Ca2+ refilling by the It be that [Ca2+]er is not to 100 μm, agonists are to other functions of the from the in not the in [Ca2+]er M.J. J. J. PubMed Scopus Google Scholar). In the HEK293 cells the and Ca2+ to Ca2+ in the ER cells are with a of agonists for endogenous receptors cells are with TRH. It is not if is the of in or the of of the transfected TRH the to a of Ca2+ of Ca2+ In the of of ER Ca2+ to be in [Ca2+]er was of extracellular the of Ca2+ in HEK293 cells are in with of Pozzan T. J. 1998; PubMed Scopus Google to [Ca2+]er in cells and that of Ca2+ the ER during cytoplasmic Ca2+ is from by M.J. J. J. PubMed Scopus Google agonists to release the of ER Ca2+ to a cytoplasmic In the release of ER Ca2+ in to agonists is by the of cytoplasmic Ca2+ and luminal Ca2+ on the IP3 In HEK293 agonists a in [Ca2+]er in the of cytoplasmic and luminal Ca2+ and refilling during signaling. in IP3 and metabolism, IP3 receptor and and all to these that extracellular Ca2+ is not for the of the ER Ca2+ store over 10 min if agonists or are In the presence of the intracellular Ca2+ store is continuously stores were to cytoplasmic Ca2+ in increased ER was for in the Ca2+ increase was for the are J. J. PubMed Google Scholar, Biochem. J. PubMed Scopus Google that an increase in cytoplasmic Ca2+ be to to an increase in the of that refilling was completely with indicating that the Ca2+ for refilling from the are with that and the IP3 receptor are for store Ca2+ stores are with the Pozzan T. J. 1998; PubMed Scopus Google Scholar, J. PubMed Google that the intracellular Ca2+ store is refilled with Ca2+ from the by the and by an [Ca2+]er increased extracellular Ca2+ was to cells that had been with thapsigargin or acid in Ca2+-free medium, the increase in [Ca2+]er was were Ca2+ has been J. PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). in and cells been to increase [Ca2+]er in the by the or in Ca2+ been a the the to in free Ca2+ in the ER and that there is a of of ER Ca2+ during Ca2+ signaling. signaling, the intracellular Ca2+ store is continuously refilled by the Ca2+ from the be the of the sites in the ER in release and of of a of [Ca2+]er in were and μm, 500 μm, in with by J. Nature. PubMed Scopus Google for [Ca2+]er in of to of basal [Ca2+]er 100 μm and 1996; PubMed Scopus Google is to that of the Ca2+ by the and TRH was to for the in were added with In refilling is to as [Ca2+]er the of the is by Ca2+ in the ER J. PubMed Google Scholar, J. 1998; PubMed Scopus Google and the of Ca2+ as [Ca2+]cyt J. J. PubMed Google Scholar, Biochem. J. PubMed Scopus Google Scholar). is [Ca2+]er is 100 μm, which the of Ca2+ release the IP3 receptor and Ca2+ refilling by the It be that [Ca2+]er is not to 100 μm, agonists are to other functions of the from the in not the in [Ca2+]er M.J. J. J. PubMed Scopus Google Scholar). In the HEK293 cells the and Ca2+ to Ca2+ in the ER cells are with a of agonists for endogenous receptors cells are with TRH. It is not if is the of in or the of of the transfected TRH the to a of Ca2+ of Ca2+ In the of of ER Ca2+ to be in [Ca2+]er was of extracellular the of Ca2+ in HEK293 cells are in with of Pozzan T. J. 1998; PubMed Scopus Google to [Ca2+]er in cells and that of Ca2+ the ER during cytoplasmic Ca2+ is from by M.J. J. J. PubMed Scopus Google agonists to release the of ER Ca2+ to a cytoplasmic In the release of ER Ca2+ in to agonists is by the of cytoplasmic Ca2+ and luminal Ca2+ on the IP3 In HEK293 agonists a in [Ca2+]er in the of cytoplasmic and luminal Ca2+ and refilling during signaling. in IP3 and metabolism, IP3 receptor and and all to these a of [Ca2+]er in were and μm, 500 μm, in with by J. Nature. PubMed Scopus Google for [Ca2+]er in of to of basal [Ca2+]er 100 μm and 1996; PubMed Scopus Google Scholar). It is to that of the Ca2+ by the and TRH was to for the in were added with In refilling is to as [Ca2+]er the of the is by Ca2+ in the ER J. PubMed Google Scholar, J. 1998; PubMed Scopus Google and the of Ca2+ as [Ca2+]cyt J. J. PubMed Google Scholar, Biochem. J. PubMed Scopus Google Scholar). is [Ca2+]er is 100 μm, which the of Ca2+ release the IP3 receptor and Ca2+ refilling by the It be that [Ca2+]er is not to 100 μm, agonists are to other functions of the from the in not the in [Ca2+]er M.J. J. J. PubMed Scopus Google Scholar). In the HEK293 cells the and Ca2+ to Ca2+ in the ER cells are with a of agonists for endogenous receptors cells are with TRH. It is not if is the of in or the of of the transfected TRH the to a of Ca2+ of Ca2+ In the of of ER Ca2+ to be in [Ca2+]er was of extracellular the of Ca2+ in HEK293 cells are in with of Pozzan T. J. 1998; PubMed Scopus Google to [Ca2+]er in cells and that of Ca2+ the ER during cytoplasmic Ca2+ is from by M.J. J. J. PubMed Scopus Google agonists to release the of ER Ca2+ to a cytoplasmic In the release of ER Ca2+ in to agonists is by the of cytoplasmic Ca2+ and luminal Ca2+ on the IP3 In HEK293 agonists a in [Ca2+]er in the of cytoplasmic and luminal Ca2+ and refilling during signaling. in IP3 and metabolism, IP3 receptor and and all to these Ca2+ that extracellular Ca2+ is not for the of the ER Ca2+ store over 10 min if agonists or are In the presence of the intracellular Ca2+ store is continuously stores were to cytoplasmic Ca2+ in increased ER was for in the Ca2+ increase was for the are J. J. PubMed Google Scholar, Biochem. J. PubMed Scopus Google that an increase in cytoplasmic Ca2+ be to to an increase in the of that refilling was completely with indicating that the Ca2+ for refilling from the are with that and the IP3 receptor are for store Ca2+ stores are with the Pozzan T. J. 1998; PubMed Scopus Google Scholar, J. PubMed Google that the intracellular Ca2+ store is refilled with Ca2+ from the by the and by an [Ca2+]er increased extracellular Ca2+ was to cells that had been with thapsigargin or acid in Ca2+-free medium, the increase in [Ca2+]er was were Ca2+ has been J. PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). in and cells been to increase [Ca2+]er in the by the or in Ca2+ been a the the to in free Ca2+ in the ER and that there is a of of ER Ca2+ during Ca2+ signaling. signaling, the intracellular Ca2+ store is continuously refilled by the Ca2+ from the be the of the sites in the ER in release and of that extracellular Ca2+ is not for the of the ER Ca2+ store over 10 min if agonists or are In the presence of the intracellular Ca2+ store is continuously stores were to cytoplasmic Ca2+ in increased ER was for in the Ca2+ increase was for the are J. J. PubMed Google Scholar, Biochem. J. PubMed Scopus Google that an increase in cytoplasmic Ca2+ be to to an increase in the of that refilling was completely with indicating that the Ca2+ for refilling from the are with that and the IP3 receptor are for store Ca2+ stores are with the Pozzan T. J. 1998; PubMed Scopus Google Scholar, J. PubMed Google Scholar). that the intracellular Ca2+ store is refilled with Ca2+ from the by the and by an [Ca2+]er increased extracellular Ca2+ was to cells that had been with thapsigargin or acid in Ca2+-free medium, the increase in [Ca2+]er was were Ca2+ has been J. PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). in and cells been to increase [Ca2+]er in the by the or in Ca2+ been a the the to in free Ca2+ in the ER and that there is a of of ER Ca2+ during Ca2+ signaling. signaling, the intracellular Ca2+ store is continuously refilled by the Ca2+ from the be the of the sites in the ER in release and of