Carbachol in the presence of guanosine 5'-O-(3-thiotriphosphate) stimulates the breakdown of exogenous phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol by rat brain membranes

The breakdown of exogenously added [³H]inositol-labeled phosphoinositides by rat brain cortical membranes was stimulated by the muscarinic cholinergic agonist carbachol. The stimulation required the presence of guanine nucleotide. Optimal conditions were similar to those described for guanosine 5'-O-(3-thiotriphosphate) (GTPγS) + carbachol stimulation of phosphoinositide breakdown in [³H]inositol-prelabeled brain membranes (Claro, E., Garcia, A., and Picatoste, F. (1989) Biochem J. 261, 29-35). Carbachol stimulated [³H]phosphatidylinositol 4,5-bisphosphate (PIP₂) breakdown was inhibited by atropine and guanosine 5'-O-(2-thiobisphosphate). The magnitude of the stimulation of exogenous PIP₂ breakdown by carbachol and GTPγS (2- to 3-fold) was little affected over a PIP₂ concentration range of 0.03-100 μM. Phosphatidylinositol 4-phosphate (PIP) was as good a substrate at all concentrations as PIP₂ for carbachol stimulation of phospholipase C activity. There was appreciable phosphomonoesterase degradation of PIP to phosphatidylinositol (PI) over 10 min. There was also some conversion of added PIP to PIP₂ in the presence of added ATP. The effect of calcium on PIP breakdown was similar to that on PIP₂ breakdown, with an apparent EC₅₀ for Ca²⁺ stimulation of 0.74 and 0.72 μM, respectively, under basal conditions. The stimulation of PIP₂ and PIP breakdown by carbachol in the presence of GTPγS was greatest on a percentage basis at the lowest free Ca²⁺ concentration. Above 1 μM free Ca²⁺, the stimulatory effect was lost, whereas 10 μM free Ca²⁺ gave a maximal stimulation of basal phospholipase C activity. Degradation of added PI was also stimulated by carbachol in the absence of ATP. PI breakdown had an EC₅₀ for Ca²⁺ stimulation of 1.07 μM. The best stimulation of PI breakdown due to carbachol plus GTPγS was seen with 0.3 μM free Ca²⁺ and 100 μM PI. Maximal activation of PI breakdown was seen at 1 mM deoxycholate as was true for PIP₂ and PIP breakdown. There was little effect, even of 30 μM GTPγS alone or of carbachol alone, on PI breakdown. Half-maximal activation of the carbachol response required only 0.2 μM GTPγS. These results indicate that the phospholipase C enzyme(s) activated by carbachol in the presence of GTPγS in rat brain cortical membranes can degrade PIP₂, PIP, and PI to inositol phosphates and diacylglycerol.