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 phosphoinositide‐phospholipase C coupled to the heterotrymeric G q/11 proteins, inhibited the phosphorylation of phosphatidylinositol (PtdIns) to polyphosphoinositides (PtdIns4P and PtdIns4,5P 2 ) in membranes from rat brain cortex. Fluoride enhanced basal production of 3 H‐inositol phosphates in membranes prepared from brain cortical slices that had been prelabeled with [ 3 H]inositol, but inhibited the stimulation elicited by carbachol in the presence of GTPγS. However in both cases fluoride depleted [ 3 H]PtdIns4P content by 95%. The inhibitory effects of fluoride on the release of 3 H‐inositol phosphates in slices were not apparent in a pulse [ 3 H]inositol‐labeling strategy, but became dramatic in a continuous labeling protocol, particularly at long incubation times. Prelabeling slices with [ 3 H]inositol in the presence of fluoride precluded polyphosphoinositide labeling, and eliminated phospholipase C responsiveness to carbachol under normal or depolarizing conditions, and to the calcium ionophore ionomycin. The lack of response of 3 H‐polyphosphoinositide‐depleted slices to phospholipase C stimuli was not due to fluoride poisoning, unaccesibility of the [ 3 H]inositol label to phospholipase C or desensitization of G q/11 , as the effect of carbachol and GTP γ S was restored, in the presence of ATP, in membranes prepared from slices that had been labeled in the presence of fluoride. In conclusion, our data show that fluoride, at a concentration similar to that used to stimulate directly G q/11 ‐coupled phospholipase C, effectively blocks the synthesis of phospholipase C substrates from PtdIns.