NEUROTRANSMITTER-STIMULATED BREAKDOWN OF ENDOGENOUS POLYPHOSPHOINOSITIDES IN POST-MORTEM HUMAN BRAIN

MEMBRANES from human brain cortex (8-12 h post mortem) were labelled w ith [H-3]inositol, in the presence of CMP, through the back reaction c atalysed by PtdIns synthase. The enzyme incorporated [H-3]inositol int o phosphoinositides at a maximal rate of 419 pmol min(-1) mg protein(- 1). In the absence of CMP, the labelling rate due to the PtdIns headgr oup exchanging enzyme was 36 pmol min(-1) mg protein(-1). Human brain PtdIns synthase showed Km(app) values of 0.49 mM and 18 mu M for inosi tol and CMP, respectively. In the presence of ATP, [H-3]polyphosphoino sitides formed after [H-3]PtdIns were hydrolysed by phospholipase C in a GTP gamma S and neurotransmitter receptor agonist-dependent manner. Production of H-3-inositol phosphates as stimulated by GTP gamma S (3 50% of basal) was increased by the muscarinic agonists carbachol and o xotremorine-M (600% of basal) and by serotonin (485% of basal). The re lative potencies of carbachol and oxotremorine-M were consistent with an action at muscarinic receptors. These results show that coupling be tween muscarinic and serotonin receptors and phospholipase C is preser ved in membranes from post mortem human brain cortex and validate the use of a method involving direct [H-3]inositol labelling of a membrane fraction to study the functional state of phospholipase C-coupled rec eptors in human brain samples.