Phorbol myristate acetate inhibition of phospholipase C activation by carbachol in slices of rat brain cortex is a delayed and indirect effect.
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.
Lee
H M
HM
Department of Biochemistry, University of Tennessee, Memphis.
Fain
J N
JN
D000818:Animals; D001030:Apamin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013110:Sphingosine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D010716:Phosphatidylinositols; D001030:Apamin; D002217:Carbachol; D010738:Type C Phospholipases; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium; D002118:Calcium