G-protein involvement in central-nervous-system muscarinic-receptor-coupled polyphosphoinositide hydrolysis

Abstract

Potentiation of muscarinic-agonist-stimulated polyphosphoinositide (PPI) hydrolysis was demonstrated in a rat cerebral-cortical membrane preparation prelabelled with myo-[3H]inositol. Accumulation of myo-[3H]inositol 1,4-bisphosphate ([3H]IP2) was used to assess brain [3H]phosphatidylinositol 4,5-bisphosphate hydrolysis as its immediate metabolite, myo-[3H]inositol 1,4,5-trisphosphate, was rapidly hydrolysed to [3H]IP2. Inclusion of ATP (100 microM) and Mg2+ (5 mM) in the assay medium was necessary to demonstrate the effect of GTP analogues on carbachol-stimulated brain [3H]PPI turnover. Carbachol (100 microM) induced only a small increment in [3H]IP2 accumulation (142% of control) in 1 min. However, its effect was markedly enhanced, to 800% and 300% of control, by 100 microM-guanosine 5′-[gamma-thio]triphosphate (GTP[S]) and guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) respectively. GTP[S] and p[NH]ppG also stimulated [3H]IP2 accumulation by over 500% and 200% of control, respectively. The GTP-analogue-potentiated carbachol effect was antagonized by 10 microM-atropine, whereas the GTP-analogue stimulation was unaffected. This report confirms the involvement of a G (GTP-binding) protein(s) in brain PPI metabolism and provides new evidence for the role of G protein(s) in the coupling of stimulated muscarinic receptors to PPI hydrolysis in the central nervous system.