Protein kinase C modulates exogenous acetylcholine current in Xenopus oocytes

A. M. Mileo, E. Palma, L. Polenzani, C. Limatola, F. Grassi, F. Eusebi

Research output: Contribution to journalArticle

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Abstract

The modulation of acetylcholine-activated current (I(ACh)) by protein kinase C (PKC) was studied in opus laevis oocytes microinjected with either mRNA extracted from C2C12 myotubes (C2C12 mRNA) or RNAs encoding murine αβγδ subunits of the nicotinic ACh receptor (nAChR). Voltage-clamped oocytes were treated for 90 sec with 12-O-tetradecanoylphorbol-13-acetate (TPA, 300 nM), a potent PKC activator. Transient increase in the amplitude and acceleration in the decay of I(ACh) were invariably observed within minutes of TPA application, and were independent of extracellular Ca2+ concentration. Both parameters recovered to control within 20-30 min; then a slight depression of I(ACh) developed. By this time, an initial PKC down regulation was observed. At the peak of TPA-induced potentiation, dose- response relations suggested an increased binding affinity of nAChR for the neurotransmitter. 4α-phorbol 12,13-didecanoate (300 nM), a biologically inactive analogue of TPA, did not affect I(ACh), while staurosporine (5-10 μM), a potent inhibitor of PKC activity, suppressed the action of TPA on I(ACh). In oocytes co-injected with C2C12 mRNA and with rat brain mRNA, I(ACh) was potentiated by 5-hydroxytryptamine (10 μM), whose receptors are coupled to phosphoinositide hydrolysis. The nAChR-channel activity in cell- attached patches increased when TPA was applied to the oocytes. In 50% of the oocytes examined, a sustained depression of the single channel activity followed. We conclude that in Xenopus oocytes an endogenous PKC system regulates the function of embryonic-type muscle nAChRs.

Original languageEnglish
Pages (from-to)443-451
Number of pages9
JournalJournal of Neuroscience Research
Volume41
Issue number4
DOIs
Publication statusPublished - 1995

Fingerprint

Xenopus
Protein Kinase C
Acetylcholine
Oocytes
Nicotinic Receptors
Cholinergic Receptors
Messenger RNA
Staurosporine
Skeletal Muscle Fibers
Tetradecanoylphorbol Acetate
Phosphatidylinositols
Neurotransmitter Agents
Serotonin
Hydrolysis
Down-Regulation
RNA
Muscles
Brain

Keywords

  • 12-O-tetradecanoyl-phorbol-13-acetate
  • C2C12 myotube
  • embryonic-type muscle nicotinic acetylcholine receptor
  • receptor modulation

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Protein kinase C modulates exogenous acetylcholine current in Xenopus oocytes. / Mileo, A. M.; Palma, E.; Polenzani, L.; Limatola, C.; Grassi, F.; Eusebi, F.

In: Journal of Neuroscience Research, Vol. 41, No. 4, 1995, p. 443-451.

Research output: Contribution to journalArticle

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abstract = "The modulation of acetylcholine-activated current (I(ACh)) by protein kinase C (PKC) was studied in opus laevis oocytes microinjected with either mRNA extracted from C2C12 myotubes (C2C12 mRNA) or RNAs encoding murine αβγδ subunits of the nicotinic ACh receptor (nAChR). Voltage-clamped oocytes were treated for 90 sec with 12-O-tetradecanoylphorbol-13-acetate (TPA, 300 nM), a potent PKC activator. Transient increase in the amplitude and acceleration in the decay of I(ACh) were invariably observed within minutes of TPA application, and were independent of extracellular Ca2+ concentration. Both parameters recovered to control within 20-30 min; then a slight depression of I(ACh) developed. By this time, an initial PKC down regulation was observed. At the peak of TPA-induced potentiation, dose- response relations suggested an increased binding affinity of nAChR for the neurotransmitter. 4α-phorbol 12,13-didecanoate (300 nM), a biologically inactive analogue of TPA, did not affect I(ACh), while staurosporine (5-10 μM), a potent inhibitor of PKC activity, suppressed the action of TPA on I(ACh). In oocytes co-injected with C2C12 mRNA and with rat brain mRNA, I(ACh) was potentiated by 5-hydroxytryptamine (10 μM), whose receptors are coupled to phosphoinositide hydrolysis. The nAChR-channel activity in cell- attached patches increased when TPA was applied to the oocytes. In 50{\%} of the oocytes examined, a sustained depression of the single channel activity followed. We conclude that in Xenopus oocytes an endogenous PKC system regulates the function of embryonic-type muscle nAChRs.",
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AU - Mileo, A. M.

AU - Palma, E.

AU - Polenzani, L.

AU - Limatola, C.

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AU - Eusebi, F.

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