Potentiation of stimulus-induced phosphoinositide breakdown by calmodulin antagonists in C6 glioma cells

To investigate the role of calmodulin (CaM)-dependent pathways in agonist-induced phosphoinositide (PI) turnover, the influence of several CaM antagonists on PI-phospholipase C (PLC) activation in intact and permeabilized C₆ glioma cells was examined. The extent of PI turnover was assessed by measuring the accumulation of inositol phosphates (IPs) in the presence of LiCl in C₆ glioma cells prelabelled with myo-³H]inositol. Trifluoperazine, N-(6-aminohexyl)-5-chloro-l-naphthalenesulphonamide (W7), fendiline and calmidazolium themselves had no effect on basal IP formation, but concentration-dependently (1–30 M) potentiated ATP-, NaF- and A23187-stimulated IP formation. The maximal response to ATP (I mM) was increased by up to 50%, while the concentration for half-maximal effect (EC₅₀, 60 M) as unaffected by trifluoperazine. In digitonin-permeabilized C₆ glioma cells, the concentration-dependent increase of PI-PLC activation elicited by free Ca²⁺ was potentiated by the GTP analogue, guanosine 5--thio]triphosphate (GTPS), with an EC₅₀ of 6 M. Trifluoperazine (1–30 M) enhanced the Ca²⁺-stimulated IP formation concentration dependently and this potentiation was counteracted by the addition of CaM. In the combined presence of each CaM antagonist studied and GTPS, an additive increase in IP formation was observed. The results indicate that CaM antagonists enhance stimulus-induced IP formation in C₆ glioma cells primarily by increasing the Ca²⁺-dependent activation of PI-PLC.