AGONIST-INDUCED, GTP-DEPENDENT PHOSPHOINOSITIDE HYDROLYSIS IN POSTMORTEM HUMAN BRAIN MEMBRANES

Membranes prepared from postmortem human brain were used to measure th e activities of three components of the phosphoinositide second messen ger system. [H-3]Phosphatidylinositol ([H-3]PI) hydrolysis was stimula ted by directly activating phospholipase C with calcium, by activating guanine nucleotide-binding proteins (G proteins) with guanosine-5'-O- (2-thiotriphosphate) (GTP(gamma)S) or with r with AIF(4), and by recep tors activated with several agonists (in the presence of (GTP(gamma)S) , including (in order of increasing magnitudes of responses) carbachol , pilocarpine, histamine, trans-1-aminocyclopentyl-1,3-dicarboxylic ac id (a selective excitatory amino acid metabotropic receptor agonist), serotonin, and ATP. G(q/11) was identified as the G protein most likel y to mediate [H-3]PI hydrolysis in human brain membranes based on the findings that this process was not impaired by pretreatment with pertu ssis toxin and it was inhibited by antibodies specific for the alpha-s ubunit of G(q/11) but not by antibodies for G(o) or G(i1). The effects of postmortem delay on [H-3]PI hydrolysis were examined by studying t issues obtained 6-21 h postmortem. A slight increase in basal [H-3]PI hydrolysis was associated with increased postmortem time, suggesting a slow loss of the normal inhibitory control of phospholipase C. GTP(ga mma)S-stimulated [H-3]PI hydrolysis was unaffected by postmortem times within this range, but carbachol-induced [3H]PI hydrolysis tended to decrease with increasing postmortem times. These results demonstrate t hat the entire phosphoinositide complex remains functional and experim entally detectable in postmortem human brain membranes. This method pr ovides a means to study the function, regulation, effects of diseases, and responses to drugs of the phosphoinositide system in human brain.