title: Agonist-induced desensitization of cholinergically stimulated phosphoinositide breakdown is independent of endogenously activated protein kinase C in HT-29 human colon carcinoma cells.
abstract: Activation of M3 muscarinic receptors in HT-29 cells by carbachol rapidly increases polyphosphoinositide breakdown. Pretreatment of these cells with carbachol (0.1 mM) for 5 h completely inhibits the subsequent ability of carbachol to increase [3H]inositol monophosphate ([3H]InsP) accumulation, paralleled by a total loss of muscarinic binding sites. In contrast, protein kinase C (PK-C)-mediated desensitization by incubation with phorbol esters [PMA (phorbol 12-myristate 13-acetate)], leading to a time- and dose-dependent inhibition of cholinergically stimulated InsP release (95% inhibition after 4 h with 0.1 microM-PMA), is accompanied by only a 40% decrease in muscarinic receptor binding, which suggests an additional mechanism of negative-feedback control. Neither carbachol nor PMA pretreatment had any effect on receptor affinity. Incubation with carbachol for 15 min caused a small increase of membrane-associated PK-C activity (15% increase, P less than 0.05) as compared with the potency of phorbol esters (PMA) (3-4-fold increase, P less than 0.01). Long-term incubation (4-24 h) with PMA resulted in a complete down-regulation of cytosolic and particulate PK-C activity. Stimulation of InsP release by NaF (20 mM) was not affected after a pretreatment with phorbol esters or carbachol, demonstrating an intact function of G-protein and phospholipase-C (PL-C) at the effector side. Determination of PL-C activity in a liposomal system with [3H]PtdInsP2 as substrate, showed no change in PL-C activity after carbachol (13 h) and short-term PMA (2.5 h) pretreatment, whereas long-term preincubation with phorbol esters (13 h) caused a small but significant decrease in PL-C activity (19%, P less than 0.05). Our results indicate that agonist-induced desensitization of phosphoinositide turnover occurs predominantly at the receptor level, with a rapid loss of muscarinic receptors. Exogenous activation of PK-C by phorbol esters seems to dissociate the interaction between receptor and G-protein/PL-C, without major effects on total cellular PL-C activity.
authors.affiliation: Medizinische Klinik II, Klinikum Grosshadern, Munich, Federal Republic of Germany.
authors.forename: R
authors.initials: R
authors.lastname: Kopp
authors.forename: P
authors.initials: P
authors.lastname: Mayer
authors.forename: A
authors.initials: A
authors.lastname: Pfeiffer
mesh_terms: D002217:Carbachol / Q000494:pharmacology*; D003110:Colonic Neoplasms / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007294:Inositol / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D019832:N-Methylscopolamine; D010716:Phosphatidylinositols / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D012602:Scopolamine Derivatives / Q000378:metabolism; D012969:Sodium Fluoride / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D007294:Inositol; D015759:Ionomycin; D002217:Carbachol; D012969:Sodium Fluoride; D011493:Protein Kinase C; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D019832:N-Methylscopolamine