Phorbol Myristate Acetate Inhibition of Phospholipase C Activation by Carbachol in Slices of Rat Brain Cortex Is a Delayed and Indirect Effect

Abstract

We examined the effect of phorbol esters on phospholipase C activation in rat brain cortical slices and membranes. There was little effect of concurrent addition of phorbol 12-myristate 13-acetate (PMA) with carbachol on phosphoinositide breakdown due to carbachol over a 1-h incubation of brain slices. However, if slices were preincubated for 3 h with 1 microM PMA or 200 microM sphingosine before addition of carbachol, there was a 35-50% inhibition of phosphoinositide breakdown. There was also a marked loss of protein kinase C (PKC) activity from both cytosol and membranes after a 3-h exposure to PMA. The loss in responsiveness to the muscarinic agonists in slices was not reflected in carbachol-stimulated phospholipase C activation using isolated membranes. However, the decrease in carbachol-induced phosphoinositide breakdown seen in slices after a 3-h exposure to PMA was abolished if the extracellular K+ concentration was elevated from 5.9 to 55mM. Because elevation of the K+ level induces depolarization and increases Ca2+ entry, we examined the effect of ionomycin, a Ca2+ ionophore. Ionomycin potentiated the effects of carbachol on phosphoinositide breakdown but was unable to reverse the effects of a 3-h incubation with PMA. Because apamin, an inhibitor of Ca2(+)-dependent K+ channels, mimicked the effects of exposure to PMA for 3 h, it is possible that these channels are involved in muscarinic cholinergic regulation of phosphoinositide breakdown in rat brain slices. These results support the hypothesis that prolonged PMA treatment in rat brain cortex has no direct effect on phospholipase C activation by muscarinic cholinergic stimulation.

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

“…Our data demonstrating the lack of a significant effect several days after TPA on carbachol-stimulated PI hydrolysis is consistent with the findings of a previous study using rat brain cortical slices (Lee and Fain, 1991). Their data demonstrated that although acute TPA treatment of slices blocked carbachol-stimulated PI hydrolysis in slices, it could not inhibit carbachol-stimulated phospholipase C activity in membranes prepared from treated slices.…”

“…These data indicate that, for carbachol-stimulated PI hydrolysis, both the receptor and coupling mechanism were unaffected by TPA. The decrease in PI hydrolysis in this study was attributed to a TPA-induced inhibition of calcium-dependent K+ channels (Lee and Fain, 1991). The possibility that the effect of TPA on carbachol-stimulated PI hydrolysis is not mediated at the level of the receptor or the signal transducing proteins may explain why we observe no significant changes in carbachol-stimulated PI hydrolysis several days after TPA.…”

Magnesium‐Dependent Inhibition of Agonist‐Stimulated Phosphoinositide Breakdown in Rat Cortical Slices by Excitatory Amino Acids

“…Previously we found that exposure of rat brain cortical slices for 3 h to 1 pM PMA also reduced the subsequent stimulation of phosphoinositide breakdown by carbachol (Lee and Fain, 1991). Furthermore, this inhibition was mimicked by addition of 100 nM apamin, a specific blocker of Ca'+-dependent K+ channels (Castle et al, 1989).…”

“…We previously demonstrated that muscarinic receptor activation of phosphoinositidase C is coupled to a G protein in membranes prepared from rat cortical slices (Claro et al, 1989) and that prolonged phorbol 12-myristate 13-acetate (PMA) treatment inhibited carbachol-stimulated phosphoinositide hydrolysis in rat cortical slices (Lee and Fain, 1991). In the present study, we tested the effects of excitatory amino acids on phosphoinositidase C activation in membranes; failing to find any, we investigated their effects on phosphoinositide breakdown in brain slices.…”

Arecoline Desensitizes Carbachol‐Stimulated Phosphatidylinositol Breakdown in Rat Brain Cortices

“…Measurements of levels of labeled inositol phosphates and inositol phospholipids were performed as described previously (Lee and Fain, 1991). In brief, slices were labeled with 1~.tCi/ml[3Hlinositol for 1 h. At the end of the labeling period, slices were washed three times with ice-cold Krebs-Henseleit buffer, and 50-gil aliquots of slices were transferred to tubes containing 200~slof Krebs-Henseleit buffer containing 10 mM Lit, 0.25 mM Ca2~,and appropriate agonists.…”