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

MEMBRANES from human brain cortex (8-12 h post mortem) were labelled with [H-3]inositol, in the presence of CMP, through the back reaction catalysed by PtdIns synthase. The enzyme incorporated [H-3]inositol into 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 headgroup 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 inositol and CMP, respectively. In the presence of ATP, [H-3]polyphosphoinositides 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 (350% of basal) was increased by the muscarinic agonists carbachol and oxotremorine-M (600% of basal) and by serotonin (485% of basal). The relative potencies of carbachol and oxotremorine-M were consistent with an action at muscarinic receptors. These results show that coupling between muscarinic and serotonin receptors and phospholipase C is preserved 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 receptors in human brain samples.