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Suppression of phospholipase C blocks Gi-mediated inhibition of adenylyl cyclase activity
1Shanghai Institute of Cell Biology, Chinese Academy of Sciences, PR China.
Abstract:
The potential effect of inhibition of phospholipase C on the response of Gi-coupled receptors was investigated in neuroblastoma x glioma hybrid (NG108-15) cells. The phospholipase C specific inhibitor 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H -pyrrole-2,5-dione (U73122), which did not affect basal and forskolin-stimulated adenylyl cyclase activities, time- and dose-dependently blocked delta-opioid receptor-mediated inhibition of adenylyl cyclase activity, the EC50 (0.5 microM) of which was consistent with that for inhibition of bradykinin-dependent phospholipase C activation (EC50 = 1 microM). U73122 treatment also blocked functional responses of m4 muscarinic receptor and alpha2-adrenoceptor in NG108-15 cells and three opioid receptors (mu, delta and opioid receptor-like receptor (ORL1)) in human neuroblastoma SK-N-SH cells. 1-[6-((17Beta-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2, 5-pyrrolidinedione (U73343), an inactive analog of U73122, did not show any effect, which suggests that the blockade by U73122 of Gi-coupled receptor-mediated signaling is probably mediated through inhibition of phospholipase C, although a possible direct modification of G proteins can not be excluded. Furthermore, treatment with U73122 but not U73343 blocked the GTP-induced inhibition of adenylyl cyclase, indicating blockade at the level of Gi proteins.
Insights
Inhibition of phospholipase C by U73122 blocks Gi-coupled receptor signaling in neuronal cells. This suggests a role for phospholipase C in mediating responses from opioid, muscarinic, and adrenergic receptors.
Area of Science:
- Neuropharmacology
- Cell Signaling
Background:
- Gi-coupled receptors mediate diverse cellular responses.
- Phospholipase C (PLC) is a key enzyme in signal transduction pathways.
- The precise role of PLC in Gi-coupled receptor signaling remains to be fully elucidated.
Purpose of the Study:
- To investigate the effect of phospholipase C inhibition on Gi-coupled receptor signaling.
- To determine if PLC is involved in the inhibitory effects of Gi-coupled receptors on adenylyl cyclase.
Main Methods:
- Utilized neuroblastoma x glioma hybrid (NG108-15) cells and human neuroblastoma SK-N-SH cells.
- Employed the specific phospholipase C inhibitor U73122 and its inactive analog U73343.
- Assessed adenylyl cyclase activity and functional receptor responses.
Main Results:
- U73122 dose-dependently blocked delta-opioid receptor-mediated inhibition of adenylyl cyclase.
- The effective concentration for U73122 inhibition of PLC activity correlated with its effect on Gi-coupled receptors.
- U73122 also inhibited functional responses of m4 muscarinic and alpha2-adrenoceptors, as well as other opioid receptors.
- U73122 blocked GTP-induced inhibition of adenylyl cyclase, indicating an effect at the level of Gi proteins.
Conclusions:
- Phospholipase C inhibition by U73122 effectively blocks signaling pathways mediated by Gi-coupled receptors.
- These findings suggest a significant role for phospholipase C in the transduction of signals from various Gi-coupled receptors.
- The data indicate that U73122 acts at the level of Gi proteins, potentially through PLC inhibition.