ABSTRACT
In the estrogen-treated rat myometrium, carbachol increased the generation of inositol phosphates by stimulating the muscarinic receptor-Gq/G11-phospholipase C-beta3 (PLC-beta3) cascade. Exposure to carbachol resulted in a rapid and specific (homologous) attenuation of the subsequent muscarinic responses in terms of inositol phosphate production, PLC-beta3 translocation to membrane, and contraction. Refractoriness was accompanied by a reduction of membrane muscarinic binding sites and an uncoupled state of residual receptors. Protein kinase C (PKC) altered the functionality of muscarinic receptors and contributed to the initial period of desensitization. A delayed phase of the muscarinic refractoriness was PKC independent and was associated with a downregulation of Gqalpha/G11alpha. Atropine failed to induce desensitization as well as Gqalpha/G11alpha downregulation, indicating that both events involve active occupancy of the receptor. Prolonged exposure to AlF-4 reduced subsequent AlF-4 as well as carbachol-mediated inositol phosphate responses and similarly induced downregulation of Gqalpha/G11alpha. Data suggest that a decrease in the level of Gqalpha/G11alpha is subsequent to its activation and may account for heterologous desensitization.
Subject(s)
Carbachol/pharmacology , GTP-Binding Proteins/physiology , Isoenzymes/metabolism , Myometrium/physiology , Receptors, Muscarinic/physiology , Signal Transduction/drug effects , Type C Phospholipases/metabolism , Aluminum Compounds/pharmacology , Animals , Atropine/pharmacology , Enzyme Inhibitors/pharmacology , Female , Fluorides/pharmacology , GTP-Binding Proteins/biosynthesis , In Vitro Techniques , Indoles/pharmacology , Inositol/metabolism , Inositol Phosphates/metabolism , Kinetics , Membrane Potentials/drug effects , Myometrium/drug effects , N-Methylscopolamine/metabolism , Phorbol 12,13-Dibutyrate/pharmacology , Phosphatidylinositols/metabolism , Phospholipase C beta , Radioligand Assay , Rats , Rats, Wistar , Signal Transduction/physiologyABSTRACT
The regulation of the receptor-G protein-phospholipase C (PLC) cascade was investigated in rat myometrium at midgestation (day 12) and at term (day 21) comparatively to the estrogen-treated tissue (day 0). Carbachol-mediated generation of [3H]inositol phosphates was insensitive to pertussis toxin and was enhanced at days 12 and 21 two- and threefold, respectively, with no alteration of muscarinic sites (M3 subtype). A similar increase could be detected in the production of inositol 1,4,5-trisphosphate, indicating the stimulation of a PLC degrading phosphatidylinositol 4,5-bisphosphate. AlF4- also enhanced PLC activation during gestation, suggesting pregnancy-related regulations that bypass receptor activation. Immunoreactive G proteins, Gq alpha and G11 alpha, and PLC-beta 3 were detected in all myometrial preparations. The amount of PLC-beta 3 was similar in day 0 and day 21 myometrium, although decreasing by 75% at midgestation. Of significance was the increased amount of Gq alpha in day 12 and day 21 myometrium (3- and 2-fold, respectively) which coincided with the enhanced phosphoinositide breakdown. The upregulation of Gq alpha may contribute to the enhanced PLC activity during pregnancy and at term.
