Antigen-induced Ca2+ mobilization in RBL-2H3 cells: Role of I(1,4,5)P3 and S1P and necessity of I(1,4,5)P3 production

Hyun Sil Lee, Chang Shin Park, Young Mi Lee, Ho Young Suk, Tameka C M Clemons, Oksoon Hong Choi

Research output: Contribution to journalArticle

Abstract

Inositol 1,4,5-trisphosphate (IP3) has long been recognized as a second messenger for intracellular Ca2+ mobilization. Recently, sphingosine 1-phosphate (S1P) has been shown to be involved in Ca2+ release from the endoplasmic reticulum (ER). Here, we investigated the role of S1P and IP3 in antigen (Ag)-induced intracellular Ca2+ mobilization in RBL-2H3 mast cells. Antigen-induced intracellular Ca2+ mobilization was only partially inhibited by the sphingosine kinase inhibitor DL -threo-dihydrosphingosine (DHS) or the IP3 receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB), whereas preincubation with both inhibitors led to complete inhibition. In contrast, stimulation of A3 adenosine receptors with N5 -ethylcarboxamidoadenosine (NECA) caused intracellular Ca2+ mobilization that was completely abolished by 2-APB but not by DHS, suggesting that NECA required only the IP3 pathway, while antigen used both the IP3 and S1P pathways. Interestingly, however, inhibition of IP3 production with the phospholipase C inhibitor U73122 completely abolished Ca2+ release from the ER induced by either stimulant. This suggested that S1P alone, without concomitant production of IP3, would not cause intracellular Ca2+ mobilization. This was further demonstrated in some clones of RBL-2H3 cells excessively overexpressing a β isoform of Class II phosphatidylinositol 3-kinase (PI3KC2β). In such clones including clone 5A4C, PI3KC2β was overexpressed throughout the cell, although endogenous PI3KC2β was normally expressed only in the ER. Overexpression of PI3KC2β in the cytosol and the PM led to depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5) P2), resulting in a marked reduction in IP3 production. This could explain the abolishment of intracellular Ca2+ mobilization in clone 5A4C. Supporting this hypothesis, the Ca2+ mobilization was reconstituted by the addition of exogenous PI(4,5)P2 in these cells. Our results suggest that both IP3 and S1P contribute to FcεRI-induced Ca2+ release from the ER and production of IP3 is necessary for S1P to cause Ca2+ mobilization from the ER.

Original languageEnglish (US)
Pages (from-to)581-592
Number of pages12
JournalCell Calcium
Volume38
Issue number6
DOIs
StatePublished - Dec 2005

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Endoplasmic Reticulum
Antigens
Clone Cells
Class II Phosphatidylinositol 3-Kinases
Adenosine A3 Receptors
Inositol 1,4,5-Trisphosphate Receptors
Inositol 1,4,5-Trisphosphate
Type C Phospholipases
Second Messenger Systems
Phosphatidylinositols
Mast Cells
Cytosol
sphingosine 1-phosphate
Protein Isoforms
2-aminoethoxydiphenyl borate
safingol

Keywords

  • Calcium mobilization
  • FcεRI
  • IP
  • Lipid mediators
  • Signal transduction
  • Sphingosine 1-phosphate

ASJC Scopus subject areas

  • Cell Biology
  • Endocrinology

Cite this

Antigen-induced Ca2+ mobilization in RBL-2H3 cells : Role of I(1,4,5)P3 and S1P and necessity of I(1,4,5)P3 production. / Lee, Hyun Sil; Park, Chang Shin; Lee, Young Mi; Suk, Ho Young; Clemons, Tameka C M; Choi, Oksoon Hong.

In: Cell Calcium, Vol. 38, No. 6, 12.2005, p. 581-592.

Research output: Contribution to journalArticle

Lee, Hyun Sil ; Park, Chang Shin ; Lee, Young Mi ; Suk, Ho Young ; Clemons, Tameka C M ; Choi, Oksoon Hong. / Antigen-induced Ca2+ mobilization in RBL-2H3 cells : Role of I(1,4,5)P3 and S1P and necessity of I(1,4,5)P3 production. In: Cell Calcium. 2005 ; Vol. 38, No. 6. pp. 581-592.
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abstract = "Inositol 1,4,5-trisphosphate (IP3) has long been recognized as a second messenger for intracellular Ca2+ mobilization. Recently, sphingosine 1-phosphate (S1P) has been shown to be involved in Ca2+ release from the endoplasmic reticulum (ER). Here, we investigated the role of S1P and IP3 in antigen (Ag)-induced intracellular Ca2+ mobilization in RBL-2H3 mast cells. Antigen-induced intracellular Ca2+ mobilization was only partially inhibited by the sphingosine kinase inhibitor DL -threo-dihydrosphingosine (DHS) or the IP3 receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB), whereas preincubation with both inhibitors led to complete inhibition. In contrast, stimulation of A3 adenosine receptors with N5 -ethylcarboxamidoadenosine (NECA) caused intracellular Ca2+ mobilization that was completely abolished by 2-APB but not by DHS, suggesting that NECA required only the IP3 pathway, while antigen used both the IP3 and S1P pathways. Interestingly, however, inhibition of IP3 production with the phospholipase C inhibitor U73122 completely abolished Ca2+ release from the ER induced by either stimulant. This suggested that S1P alone, without concomitant production of IP3, would not cause intracellular Ca2+ mobilization. This was further demonstrated in some clones of RBL-2H3 cells excessively overexpressing a β isoform of Class II phosphatidylinositol 3-kinase (PI3KC2β). In such clones including clone 5A4C, PI3KC2β was overexpressed throughout the cell, although endogenous PI3KC2β was normally expressed only in the ER. Overexpression of PI3KC2β in the cytosol and the PM led to depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5) P2), resulting in a marked reduction in IP3 production. This could explain the abolishment of intracellular Ca2+ mobilization in clone 5A4C. Supporting this hypothesis, the Ca2+ mobilization was reconstituted by the addition of exogenous PI(4,5)P2 in these cells. Our results suggest that both IP3 and S1P contribute to FcεRI-induced Ca2+ release from the ER and production of IP3 is necessary for S1P to cause Ca2+ mobilization from the ER.",
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AU - Lee, Hyun Sil

AU - Park, Chang Shin

AU - Lee, Young Mi

AU - Suk, Ho Young

AU - Clemons, Tameka C M

AU - Choi, Oksoon Hong

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KW - FcεRI

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KW - Lipid mediators

KW - Signal transduction

KW - Sphingosine 1-phosphate

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