Abstract
Recent evidence has revealed that a highly sensitive and specific guanine nucleotide regulatory process controls intracellular Ca2+ release within N1E-115 neuroblastoma cells (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464). The present report documents GTP-induced Ca2+ release within quite distinct cell types, including the DDT1MF-2 smooth muscle cell line. GTP-induced Ca2+ release has similar GTP sensitivity and specificity among cells and rapidly mobilizes up to 70% of Ca2+ specifically accumulated within a nonmitochondrial Ca2+-pumping organelle within permeabilized DDT2MF-2 cells. Maximal GTP-induced release of Ca2+ is observed to be greater than inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (the latter being approximately 30% of total releasable Ca2+). After maximal IP3-induced release, further IP3 addition is ineffective, whereas subsequent addition of GTP further releases Ca2+ to equal exactly the extent of Ca2+ release observed by addition of GTP in the absence of IP3. This suggests that IP3 releases Ca2+ from the same pool as GTP, whereas GTP also releases from an additional pool. The effects of GTP appear to be reversible since simple washing of GTP-treated cells restores their previous Ca2+ uptake properties. Electron microscopic analysis of GTP-treated membrane vesicles reveals their morphology to be unchanged, whereas treatment of vesicles with 3% polyethylene glycol, known to enhance GTP-mediated Ca2+ release, clearly induces close coalescence of membranes. In the presence of 4 mM oxalate, GTP induces a rapid and profound uptake, as opposed to release, of Ca2+. The findings suggest that GTP-activated Ca2+ movement is a widespread phenomenon among cells, which can function on the same Ca2+ pool mobilized by IP3, and although activating Ca2+ movement by a mechanism distinct from IP3, does so via a process that does not appear to involve fusion between membranes.
Highlights
Total releasable Ca2+).After maximal IP3-induced re- release is induced by submicromolar GTP concentrations and lease, further IP3addition is ineffective, whereassub- shows high specificity for guanine nucleotides (4, 5)
The sequent additionof GTP furtherreleases Ca2+to equal release process appears to be dependent on terminal phosexactly the extenotf Ca2+release observed by addition phate hydrolysis of GTP
DDTIMF-2 cells were grown under similar conditions to those we described previously for N1E-115 cells (6), with the followingexceptions: the Dulbecco's modified Eagle's medium was supplemented with 5% as opposed to 10%fetal bovine serum; cells were passaged every 7 days, and medium changes were performed on days 3, 5, and 6 after subculture
Summary
Using microsomes derived (A), 10 pM GTP (V),[5] pM A23187 (V), or buffer control (0)A. t the from guinea pig parotid gland, Henne and Soling (12) have indicated times after these additions, samples were withdrawn from observed very similar effects on release of accumulated Ca2+ vials and Ca2+remaining within cells was determined by rapid La3+
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