Contribution of G Protein Activation to Fluoride Stimulation of Phosphoinositide Hydrolysis

Abstract: To examine the possibility that NaF enhances phosphoinositide-specific phospholipase C (PIC) activity in neural tissues by a mechanism independent of a guanine nucleotide binding protein (G,). we have evaluated the con-tribution of G, activation to NaF-stimulated phosphoinosi-tide hydrolysis in human SK-N-SH neuroblastoma cells. Addition of NaF to intact cells resulted in an increase in the release of inositol phosphates (450 % of control values: ECso of-8 mM). Inclusion of U-73 122. an aminosteroid inhibi-tor of guanine nucleotide-regulated PIC activity in these cells, resulted in a dose-dependent inhibition of NaF-stimu-lated inositol lipid hydrolysis (ICso of-3.5 p M). When added to digitonin-permeabilized cells, NaF or guanosine-5'-0-thiotriphosphate (GTPyS) resulted in a three- and sev-enfold enhancement. respectively, of inositol phosphate re-lease. In the combined presence of optimal concentrations of NaF and GTP-yS, inositol phosphate release was less than additive. indicative of a common site of action. Inclusion of 2-5 mM concentrations of guanosine-5'-0-(2-thiodiphos-phate) (GDPBS) fully blocked phosphoinositide hy-drolysis elicited by GTPyS. whereas that induced by NaF was partially inhibited (65%,). However, preincubation of the cells with GDPgS resulted in a greater reduction in the ability of NaF to stimulate inositol phosphate release (87% inhibition). Both GTPyS and NaF-stimulated inositol phos-phate release were inhibited by inclusion of 10 piZf U-73 122 (54-719h). The presence of either NaF or GTPyS also re-sulted in a marked lowering of the Ca2 ' requirement for activation of PIC in permeabilized cells. These results indi-cate that in SK-N-SH cells, little evidence exists for direct stimulation of PIC by NaF and that the majority of inositol phosphate release that occurs in the presence of NaF can be attributed to activation ofG,. Key Words: lnositol phospho-lipid-Phosphoinositide-specific phospholipase C-Fluo-ride-Guanine nucleotide-Neuroblastoma-Aminoster