Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line

T. Ueda, Chueh Sheau-Huei Chueh, M. W. Noel, Donald Gill

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Abstract

The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D.L., and Chueh, S.H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 μM IP3 effects rapid release (t( 1/2 ) < 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 μM A23187. Half-maximal Ca2+ release occurs with 0.5 μM IP3, and maximal release with 3 μM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 μM IP3 rapidly releases (t( 1/2 ) < 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 μM GTP, but not guanosine 5'-(β,γ-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t( 1/2 ) value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 μM GTP, with maximal release at 3-5 μM GTP; 20 μM GMPPNP has no effect on release and only slightly inhibits 5 μM GTP; 20 μM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 μM GTP. Using permeabilized N1E-115 cells, 5 μM GTP with 3% PEG releases >50% of releasable Ca2+; without PEG, GTP still mediates ~30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.

Original languageEnglish (US)
Pages (from-to)3184-3192
Number of pages9
JournalJournal of Biological Chemistry
Volume261
Issue number7
StatePublished - 1986

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Inositol 1,4,5-Trisphosphate
Guanine Nucleotides
Guanosine Triphosphate
Cells
Calcium
Cell Line
Polyethylene glycols
Organelles
Saponins
Cell Membrane
Cell membranes
Synaptic Membranes
Adenosine Triphosphate
Neuroblastoma
Pumps
Endoplasmic Reticulum
Membranes

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{ad908833285949f185e0bfea99c571a1,
title = "Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line",
abstract = "The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D.L., and Chueh, S.H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 μM IP3 effects rapid release (t( 1/2 ) < 20 s) of approximately 40{\%} of accumulated Ca2+ releasable with 5 μM A23187. Half-maximal Ca2+ release occurs with 0.5 μM IP3, and maximal release with 3 μM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 μM IP3 rapidly releases (t( 1/2 ) < 30 s) 10-20{\%} of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3{\%} polyethylene glycol (PEG). 10 μM GTP, but not guanosine 5'-(β,γ-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40{\%} of releasable Ca2+) with a t( 1/2 ) value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3{\%} PEG. Half-maximal Ca2+ release occurs with 1 μM GTP, with maximal release at 3-5 μM GTP; 20 μM GMPPNP has no effect on release and only slightly inhibits 5 μM GTP; 20 μM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 μM GTP. Using permeabilized N1E-115 cells, 5 μM GTP with 3{\%} PEG releases >50{\%} of releasable Ca2+; without PEG, GTP still mediates ~30{\%} release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.",
author = "T. Ueda and {Sheau-Huei Chueh}, Chueh and Noel, {M. W.} and Donald Gill",
year = "1986",
language = "English (US)",
volume = "261",
pages = "3184--3192",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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}

Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line. / Ueda, T.; Sheau-Huei Chueh, Chueh; Noel, M. W.; Gill, Donald.

In: Journal of Biological Chemistry, Vol. 261, No. 7, 1986, p. 3184-3192.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line

AU - Ueda, T.

AU - Sheau-Huei Chueh, Chueh

AU - Noel, M. W.

AU - Gill, Donald

PY - 1986

Y1 - 1986

N2 - The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D.L., and Chueh, S.H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 μM IP3 effects rapid release (t( 1/2 ) < 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 μM A23187. Half-maximal Ca2+ release occurs with 0.5 μM IP3, and maximal release with 3 μM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 μM IP3 rapidly releases (t( 1/2 ) < 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 μM GTP, but not guanosine 5'-(β,γ-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t( 1/2 ) value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 μM GTP, with maximal release at 3-5 μM GTP; 20 μM GMPPNP has no effect on release and only slightly inhibits 5 μM GTP; 20 μM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 μM GTP. Using permeabilized N1E-115 cells, 5 μM GTP with 3% PEG releases >50% of releasable Ca2+; without PEG, GTP still mediates ~30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.

AB - The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D.L., and Chueh, S.H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 μM IP3 effects rapid release (t( 1/2 ) < 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 μM A23187. Half-maximal Ca2+ release occurs with 0.5 μM IP3, and maximal release with 3 μM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 μM IP3 rapidly releases (t( 1/2 ) < 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 μM GTP, but not guanosine 5'-(β,γ-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t( 1/2 ) value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 μM GTP, with maximal release at 3-5 μM GTP; 20 μM GMPPNP has no effect on release and only slightly inhibits 5 μM GTP; 20 μM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 μM GTP. Using permeabilized N1E-115 cells, 5 μM GTP with 3% PEG releases >50% of releasable Ca2+; without PEG, GTP still mediates ~30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.

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