CONTRIBUTION OF G-PROTEIN ACTIVATION TO FLUORIDE STIMULATION OF PHOSPHOINOSITIDE HYDROLYSIS IN HUMAN NEUROBLASTOMA-CELLS

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(p)), we have evaluated the contribution of G(p) activation to NaF-stimulated phosphoinositide 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; EC50 of approximately 8 mM). Inclusion of U-73122, an aminosteroid inhibitor of guanine nucleotide-regulated PIC activity in these cells, resulted in a dose-dependent inhibition of NaF-stimulated inositol lipid hydrolysis (IC50 of approximately 3.5 muM). When added to digitonin-permeabilized cells, NaF or guanosine-5'-O-thiotriphosphate (GTPgammaS) resulted in a three- and sevenfold enhancement, respectively, of inositol phosphate release. In the combined presence of optimal concentrations of NaF and GTPgammaS, inositol phosphate release was less than additive, indicative of a common site of action. Inclusion of 2-5 mM concentrations of guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) fully blocked phosphoinositide hydrolysis elicited by GTPgammaS, whereas that induced by NaF was partially inhibited (65%). However, preincubation of the cells with GDPbetaS resulted in a greater reduction in the ability of NaF to stimulate inositol phosphate release (87% inhibition). Both GTPgammaS and NaF-stimulated inositol phosphate release were inhibited by inclusion of 10 muM U-73122 (54-71%). The presence of either NaF or GTPgammaS also resulted in a marked lowering of the Ca2+ requirement for activation of PIC in permeabilized cells. These results indicate 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 of G(p).