| Abstract: | The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of 3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of 3H]phosphatidylinositol, 3H]phosphatidylinositol 4-phosphate and 3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in 3H]inositol 1,4-bisphosphate and 3H]inositol 1,4,5-trisphosphate, but not in 3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. 3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol. |
