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

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.