title: Kinetic analysis of A23187-mediated polyphosphoinositide breakdown in rat cortical synaptosomes suggests that inositol bisphosphate does not arise primarily by degradation of inositol trisphosphate.
abstract: The kinetics of polyphosphoinositide breakdown and inositol phosphate formation have been studied in rat cortical synaptosomes labelled in vitro with myo-[2-3H]inositol. Intrasynaptosomal Ca2+ concentrations have been varied by the use of Ca-EGTA buffers or by adding the ionophore A23187 in the presence and absence of 1 mM Ca2+. The former studies have revealed that, at very low (20 nM) intrasynaptosomal free Ca2+ levels, inositol bisphosphate, but not inositol monophosphate levels are reduced. Addition of A23187 in the absence of added Ca2+ gives rise to greatly enhanced inositol bisphosphate accumulation, which is further enhanced if 1 mM Ca2+ 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 mM Ca2+ leads to rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and ATP and slower breakdown of phosphatidylinositol 4-phosphate (PtdInsP). The rates of loss of PtdinsP2 and ATP are very highly correlated, suggesting that polyphosphoinositide resynthesis may be limited by ATP availability at high Ca2+ levels. Analysis of 32P-labelled synaptosomes also reveals that A23187 produces Ca2+-dependent losses of PtdInsP2, PtdInsP, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, Institute of Psychiatry, London, England.
authors.forename: M
authors.initials: M
authors.lastname: Brammer
authors.forename: K
authors.initials: K
authors.lastname: Weaver
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D002540:Cerebral Cortex / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013403:Sugar Phosphates / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism*; D013997:Time Factors
chemical_list: D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D013403:Sugar Phosphates; D000001:Calcimycin; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D002118:Calcium