ACUTE AND LONG-TERM INHIBITION OF AGONIST-STIMULATED PHOSPHOINOSITIDEHYDROLYSIS 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 glutama te-stimulated phosphoinositide hydrolysis, but not in basal levels of PI hydrolysis. To investigate the mechanism of TPA action, phospholipa se C was assayed in membranes prepared from cerebellar granule cells a cutely 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 st atistically significant decrease in glutamate-stimulated PI hydrolysis , and a slight reduction of carbachol-stimulated PI hydrolysis when co mpared to temporally matched controls. Measurements in membranes prepa red from TPA-treated vs control cells 1, 3, and 5 d after treatment sh owed that calcium- and NaF-stimulated phospholipase C activity was sig nificantly decreased at all days tested, whereas GTP gamma S-stimulate d phospholipase C activity was significantly decreased only at d 3. Th ese data demonstrate differences in the acute vs long-term effects of TPA treatment on agonist-stimulated PH hydrolysis, and suggest that th e 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.