Fluoride-induced depletion of polyphosphoinositides in rat brain cortical slices: A rationale for the inhibitory effects on phospholipase C

Fluoride, which is used commonly as a pharmacological tool to activate phos phoinositide-phospholipase C coupled to the heterotrymeric G(q/11) proteins , inhibited the phosphorylation of phosphatidylinositol (PtdIns) to polypho sphoinositides (PtdIns4P and PtdIns4,5P(2)) in membranes from rat brain cor tex. Fluoride enhanced basal production of H-3-inositol phosphates in membr anes prepared from brain cortical slices that had been prelabeled with [H-3 ]inositol, but inhibited the stimulation elicited by carbachol in the prese nce of GTP gamma S. However in both cases fluoride depleted [H-3]PtdIns4P c ontent by 95%. The inhibitory effects of fluoride on the release of H-3-ino sitol phosphates in slices were nor apparent in a pulse [H-3]inositol-label ing strategy, but became dramatic in a continuous labeling protocol, partic ularly at long incubation times. Prelabeling slices with [H-3]inositol in t he presence of fluoride precluded polyphosphoinositide labeling, and elimin ated phospholipase C responsiveness to carbachol under normal or depolarizi ng conditions, and to the calcium ionophore ionomycin. The lack of response of H-3-polyphosphoinositide-depleted slices to phospholipase C stimuli was not due to fluoride poisoning, unaccesibility of the [H-3]inositol label t o phospholipase C or desensitization of G(q/11), as the effect of carbachol and GTP gamma S was restored, in the presence of ATP, in membranes prepare d from slices that had been labeled in the presence of fluoride. In conclus ion, our data show that fluoride, at a concentration similar to that used t o stimulate directly G(q/11)-coupled phospholipase C, effectively blocks th e synthesis of phospholipase C substrates from PtdIns. (C) 1999 ISDN. Publi shed by Elsevier Science Ltd. All rights reserved.