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Potentiation of stimulus-induced phosphoinositide breakdown by calmodulin antagonists in C6 glioma cells
1Department of Pharmacology, National Taiwan University, Taipei.
Abstract:
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-1- naphthalenesulphonamide (W-7), fendiline and calmidazolium themselves had no effect on basal IP formation, but concentration-dependently (1-30 microM) potentiated ATP-, NaF- and A23187-stimulated IP formation. The maximal response to ATP (1 mM) was increased by up to 50%, while the concentration for half-maximal effect (EC50, 60 microM) was 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'-[gamma-thio]triphosphate (GTP gamma S), with an EC50 of 6 microM. Trifluoperazine (1-30 microM) enhanced the Ca(2+)-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 GTP gamma S, 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 Ca(2+)-dependent activation of PI-PLC.
Insights
Calmodulin antagonists boost phosphoinositide turnover in glioma cells by enhancing calcium-dependent activation of PI-PLC. This study reveals a key mechanism in cellular signaling pathways.
Area of Science:
- Cellular Biology
- Biochemistry
- Neuroscience
Background:
- Phosphoinositide (PI) turnover is crucial for cellular signaling.
- Calmodulin (CaM) plays a role in various calcium-dependent pathways.
- PI-phospholipase C (PLC) activation is a key step in PI turnover.
Purpose of the Study:
- To investigate the role of CaM-dependent pathways in agonist-induced PI turnover.
- To examine the influence of CaM antagonists on PI-PLC activation in C6 glioma cells.
Main Methods:
- Assessed PI turnover by measuring inositol phosphate (IP) accumulation in C6 glioma cells.
- Utilized various CaM antagonists (trifluoperazine, W-7, fendiline, calmidazolium).
- Examined intact and digitonin-permeabilized cells, including stimulation with ATP, NaF, A23187, Ca2+, and GTPγS.
Main Results:
- CaM antagonists potentiated stimulated IP formation concentration-dependently without affecting basal levels.
- Trifluoperazine increased maximal ATP-stimulated IP formation by up to 50%.
- CaM antagonists enhanced Ca2+-stimulated PI-PLC activation, an effect counteracted by CaM.
Conclusions:
- CaM antagonists enhance stimulus-induced PI turnover in C6 glioma cells.
- The primary mechanism involves increased Ca2+-dependent activation of PI-PLC.
- This highlights the involvement of CaM-dependent pathways in regulating PI turnover.