Effects of pertussis toxin and G alpha-protein-specific antibodies on phosphoinositide hydrolysis in rat brain membranes after cholinergic denervation and hippocampal sympathetic ingrowth

Cholinergic denervation of the hippocampal formation, via medial septal les ions, induces peripheral noradrenergic fibers, originating from the superio r cervical ganglion, to grow into the hippocampus. We have previously repor ted that cholinergic denervation and hippocampal sympathetic ingrowth diffe rentially affect guanosine-5'-O-(3-thiotriphosphate)- as well as guanosine- 5'-O-(3-thiotriphosphate) + carbachol-stimulated polyphosphoinositide hydro lysis, suggesting an alteration in G proteins and/or the entire receptor co mplex, To examine the type of G protein which may be involved in these effe cts, rat dorsal hippocampal membranes were preincubated with pertussis toxi n in the presence of guanosine-5'-O-(3 -thiotriphosphate) and guanosine-5'- O-(3-thiotriphosphate) + carbachol. Pertussis toxin reduced guanosine-5'-O- (3-thiotriphosphate) in all groups, while guanosine-5'-O-(3-thiotriphosphat e) + carbachol-stimulated phosphoinositide hydrolysis was reduced in contro ls and animals without sympathetic ingrowth but not in animals with hippoca mpal sympathetic ingrowth. This suggests that pertussis toxin-sensitive G:p roteins may be involved in the mediation of phosphoinositide hydrolysis. To confirm this hypothesis, membranes were preincubated with antibodies to G alpha o and Gq/11. The Go antibody significantly decreased guanosine-5'-O-( 3-thiotriphosphate) in all groups, while guanosine-5'-O-(3-thiotriphosphate ) + carbachol-stimulated phosphoinositide hydrolysis was reduced only in hi ppocampal sympathetic ingrowth, Impairment of guanosine-5'-O-(3-thiotriphos phate) and carbachol-stimulated phosphoinositide hydrolysis was also decrea sed in all groups when preincubated with Gq/11 antibody. To determine wheth er hippocampal sympathetic ingrowth or cholinergic denervation altered the concentration of various G proteins, immunoblotting methodology was utilize d. Gq/11 concentrations were found to be equivalent among groups. The densi ty of Gel, Go2, and Go3 isoforms was significantly increased in the choline rgic denervation, while in the hippocampal sympathetic ingrowth only group Go3 was significantly increased. When assessed as total Go protein, density was increased significantly only in the cholinergic denervation group. Ove rall, these results suggest that hippocampal sympathetic ingrowth and choli nergic denervation induce alterations in phosphoinositide hydrolysis throug h both the Gq/11 and the Go proteins and that the coupling between muscarin ic receptor and G protein is the possible site which affects changes in pho sphoinositide turnover. Our results also suggest that cholinergic denervati on and hippocampal sympathetic ingrowth may mediate phosphoinositide hydrol ysis through an effect on different isoforms of the Same G protein. (C) 200 0 Academic Press.