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Kinetic Analysis of A23187‐Mediated Polyphosphoinositide Breakdown in Rat Cortical Synaptosomes Suggests that Inositol Bisphosphate Does Not Arise Primarily by Degradation of Inositol Trisphosphate
Author(s) -
Brammer Michael,
Weaver Keith
Publication year - 1989
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1989.tb07348.x
Subject(s) - inositol , inositol trisphosphate , egta , phosphatidylinositol , chemistry , inositol phosphate , calcium , synaptosome , ionophore , phosphatidylinositol 4,5 bisphosphate , biophysics , biochemistry , biology , in vitro , receptor , signal transduction , organic chemistry
The kinetics of polyphosphoinositide breakdown and inositol phosphate formation have been studied in rat cortical synaptosomes labelled in vitro with myo ‐[2‐ 3 H]inositol. Intrasynaptosomal Ca 2+ concentrations have been varied by the use of Ca‐EGTA buffers or by adding the onophore A23187 in the presence and absence of 1 mM Ca 2+ . The former studies have revealed that, at very low (20 n M ) intrasynaptosomal free Ca 2+ levels, inositol pisphos‐phate, but not inositol monophosphate levels are reduced. Addition of A23187 in the absence of added Ca 2+ gives rise to greatly enhanced inositol bisphosphate accumulation, which is further enhanced if 1 mM Ca 2+ is present in the extrasynaptosomal medium. At all time points examined (down to 2 s after adding ionophore), the ratio of inositol trisphosphate/inositol bisphosphate accumulation does not exceed 0.2, and calculations based on inositol bis‐ and trisphosphate breakdown rates in synaptosomal lysates suggest that only a minority of the inositol bisphosphate arises from degradation of inositol trisphosphate. Addition of ionophore in the presence (but not in the absence) of 1 m M Ca 2+ leads to rapid breakdown of phosphatidylinositol 4,5‐bisphosphate (PtdInsP 2 ) and ATP and slower breakdown of phosphatidylinositol 4‐phosphate (PtdInsP). The rates of loss of PtdinsP 2 and ATP are very highly correlated, suggesting that polyphosphoinositide resynthesis may be limited by ATP availability at high Ca 2+ levels. Analysis of 32 P‐labelled synaptosomes also reveals that A23187 produces Ca 2+ ‐dependent losses of PtdInsP 2 , PtdlnsP, ATP, and GTP radioactivity and a marked increase in the radioactivity of a compound distinct from nucleotides or any of the lipid breakdown products tested. This compound may be a hexose bisphosphate arising from stimulation of glycolysis by A23187.