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Phorbol ester inhibition of current responses and simultaneous protein phosphorylation in Xenopus oocyte injected
1Department of Pharmacology, Toyama Medical and Pharmaceutical University, Japan.
Abstract:
The role of protein kinase C activation in a coupling of Ca2+-mobilizing receptors/GTP-binding protein/phospholipase C was examined using Xenopus oocytes before and after microinjection of mRNA purified from rat brains. Under voltage-clamp conditions, although the phorbol ester TPA per se never elicited any changes in ionic conductance, chloride current responses of mRNA-injected cells to 5-hydroxytryptamine and acetylcholine (ACh) were suppressed by an 8-min pretreatment of 12-O-tetradecanoyl-4 beta-phorbol-13-acetate (TPA), at nanomolar concentrations. Native ACh response in intact follicular oocytes was also inhibited by the TPA treatment. However, similar current responses triggered by the direct activation of their intracellular signalling pathway with guanosine-5'-O-(3-thio)triphosphate or Ca2+ were not affected by TPA. Biochemical analyses indicated that phosphorylation of 33,000- and 45,000-dalton proteins was markedly enhanced by TPA in vivo, and that stimulation of receptors with agonists as well as TPA treatment increased phosphoproteins in the membrane fraction of mRNA-injected oocytes. These observations suggest that protein kinase C may switch off the signal transduction from receptors to GTP-binding proteins and may participate in the negative feedback modulation of receptor-operated ion channel responses.
Insights
Protein kinase C activation suppresses signaling from receptors to GTP-binding proteins. This suggests a negative feedback role for protein kinase C in modulating ion channel responses.
Area of Science:
- Cellular signaling
- Molecular biology
- Neuroscience
Background:
- Investigating the role of protein kinase C (PKC) in signal transduction pathways.
- Examining the coupling of Ca2+-mobilizing receptors, GTP-binding proteins, and phospholipase C.
- Utilizing Xenopus oocytes as a model system for studying receptor-mediated signaling.
Purpose of the Study:
- To determine the effect of protein kinase C activation on the signaling cascade initiated by Ca2+-mobilizing receptors.
- To elucidate the mechanism by which PKC influences the coupling of receptors to downstream effectors.
- To explore the potential negative feedback role of PKC in modulating ion channel activity.
Main Methods:
- Microinjection of rat brain mRNA into Xenopus oocytes.
- Voltage-clamp electrophysiology to measure ionic currents.
- Treatment with phorbol ester (TPA) to activate PKC.
- Stimulation with neurotransmitters (acetylcholine, 5-hydroxytryptamine) and direct pathway activators (GTPγS, Ca2+).
- Biochemical analysis of protein phosphorylation.
Main Results:
- TPA pretreatment suppressed chloride current responses to acetylcholine and 5-hydroxytryptamine in mRNA-injected oocytes.
- TPA did not affect responses mediated by direct activation of the intracellular signaling pathway.
- TPA enhanced in vivo phosphorylation of 33,000- and 45,000-dalton proteins.
- Receptor stimulation and TPA treatment increased membrane-associated phosphoproteins.
Conclusions:
- Protein kinase C activation appears to inhibit signal transduction between receptors and GTP-binding proteins.
- PKC may play a role in the negative feedback regulation of receptor-operated ion channels.
- These findings contribute to understanding the complex regulation of cellular signaling pathways.