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 language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - 1986|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology