Agonist-induced, GTP-dependent phosphoinositide hydrolysis in postmortem human brain membranes.

Abstract

Membranes prepared from postmortem human brain were used to measure the activities of three components of the phosphoinositide second messenger system. [3H]Phosphatidylinositol ([3H]PI) hydrolysis was stimulated by directly activating phospholipase C with calcium, by activating guanine nucleotide-binding proteins (G proteins) with guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) or with AIF4, and by receptors 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 acid (a selective excitatory amino acid metabotropic receptor agonist), serotonin, and ATP. Gq/11 was identified as the G protein most likely to mediate [3H]PI hydrolysis in human brain membranes based on the findings that this process was not impaired by pretreatment with pertussis toxin and it was inhibited by antibodies specific for the alpha-subunit of Gq/11 but not by antibodies for G0 or Gi1. The effects of postmortem delay on [3H]PI hydrolysis were examined by studying tissues obtained 6-21 h postmortem. A slight increase in basal [3H]PI hydrolysis was associated with increased postmortem time, suggesting a slow loss of the normal inhibitory control of phospholipase C. GTP gamma S-stimulated [3H]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 that the entire phosphoinositide complex remains functional and experimentally detectable in postmortem human brain membranes. This method provides a means to study the function, regulation, effects of diseases, and responses to drugs of the phosphoinositide system in human brain.