Acute and long-term inhibition of agonist-stimulated phosphoinositide hydrolysis by pulse treatment of cerebellar granule cells with TPA.

Acute pretreatment (30 min) of primary cultures of cerebellar granule cells with TPA (10 nM) resulted in a decrease in carbachol-and glutamate-stimulated phosphoinositide hydrolysis, but not in basal levels of PI hydrolysis. To investigate the mechanism of TPA action, phospholipase C was assayed in membranes prepared from cerebellar granule cells acutely treated with TPA. TPA had no effect on basal, GTP gamma S-, NaF-, and calcium-stimulated phospholipase C when compared with membranes prepared from vehicle-treated cells. The effects of pulsing with TPA (30-min pulse, 10 nM) on agonist-stimulated PI hydrolysis were studied 1, 3, and 5 or 6 d after TPA treatment. TPA treatment results in a statistically significant decrease in glutamate-stimulated PI hydrolysis, and a slight reduction of carbachol-stimulated PI hydrolysis when compared to temporally matched controls. Measurements in membranes prepared from TPA-treated vs control cells 1, 3, and 5 d after treatment showed that calcium- and NaF-stimulated phospholipase C activity was significantly decreased at all days tested, whereas GTP gamma S-stimulated phospholipase C activity was significantly decreased only at d 3. These data demonstrate differences in the acute vs long-term effects of TPA treatment on agonist-stimulated PH hydrolysis, and suggest that the acute effects may be mediated at the level of the receptor, whereas long-term effects of TPA on PI hydrolysis may be mediated by deficits in effector function.