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The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons.

Willets JM, Nelson CP, Nahorski SR, Challiss RA - J. Neurochem. (2007)

Bottom Line: Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin.The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade.In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.

ABSTRACT
To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.

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Translocation of conventional and novel protein kinase C (PKCs) following picrotoxin (PiTx) treatment of hippocampal neuron cultures. eGFP-PKCβII or eGFP-PKCε transfected neurons were challenged with PiTx (100 μmol/L) and PKC translocation to the plasma membrane determined as the decrease in cytosolic fluorescence. In panels (a) and (b), neurons were also loaded with the Ca2+-sensitive dye Fura-Red for 60 min prior to addition of PiTx. Note that downward deflections in the Ca2+ trace indicate increases in [Ca2+]i. (a) representative trace showing that picrotoxin addition alone did not stimulate translocation of eGFP-PKCε. (b) representative trace showing that, in contrast, PiTx addition alone caused transient increases in intracellular [Ca2+] that are mirrored by rapid and transient translocations of eGFP-PKCβII. (c) representative trace showing the effects of sequential methacholine (MCh, 100 μmol/L) and PiTx (100 μmol/L) additions on the translocation of eGFP-PKCβII. (d) representative trace showing the effects of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blocker 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 μmol/L) and tetrodotoxin (TTx, 500 nmol/L) on PiTx-induced eGFP-PKCβII translocations. All representative traces are shown for experiments repeated on 4–10 coverslips from at least three separate hippocampal preparations.
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fig07: Translocation of conventional and novel protein kinase C (PKCs) following picrotoxin (PiTx) treatment of hippocampal neuron cultures. eGFP-PKCβII or eGFP-PKCε transfected neurons were challenged with PiTx (100 μmol/L) and PKC translocation to the plasma membrane determined as the decrease in cytosolic fluorescence. In panels (a) and (b), neurons were also loaded with the Ca2+-sensitive dye Fura-Red for 60 min prior to addition of PiTx. Note that downward deflections in the Ca2+ trace indicate increases in [Ca2+]i. (a) representative trace showing that picrotoxin addition alone did not stimulate translocation of eGFP-PKCε. (b) representative trace showing that, in contrast, PiTx addition alone caused transient increases in intracellular [Ca2+] that are mirrored by rapid and transient translocations of eGFP-PKCβII. (c) representative trace showing the effects of sequential methacholine (MCh, 100 μmol/L) and PiTx (100 μmol/L) additions on the translocation of eGFP-PKCβII. (d) representative trace showing the effects of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blocker 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 μmol/L) and tetrodotoxin (TTx, 500 nmol/L) on PiTx-induced eGFP-PKCβII translocations. All representative traces are shown for experiments repeated on 4–10 coverslips from at least three separate hippocampal preparations.

Mentions: The translocation of eGFP-MARCKS indicates that PKC activity can be stimulated in hippocampal neurons following stimulation of the M1 mACh receptor and also following picrotoxin-mediated enhancement of synaptic activity. To gain a better understanding of PKC isoenzymic recruitment patterns in neurons subject to metabotropic and/or ionotropic stimulation, we transfected neurons with eGFP-tagged constructs of either the Ca2+/diacylglycerol (DAG)-activated PKCβII, or the DAG-activated PKCε. Neurons, recombinantly expressing either eGFP-PKCβII or -ε isoenzymes and loaded with Fura-Red simultaneously to report changes in intracellular Ca2+ concentrations, were subject to picrotoxin and/or M1 mACh receptor agonist additions. Under these conditions it could be shown that picrotoxin treatment caused rapid and transient increases in intracellular Ca2+ (Fig. 7a and b). Perhaps unsurprisingly, picrotoxin addition did not cause the recruitment of the DAG-sensitive PKCε to the plasma membrane (Fig. 7a), however, it did produce rapid, transient recruitments of eGFP-PKCβII (Fig. 7b). Indeed, the translocation of eGFP-PKCβII precisely mirrored the picrotoxin-stimulated changes in intracellular Ca2+ (Fig. 7b). MCh (10 μmol/L) stimulation consistently failed to cause eGFP-PKCβII translocation, while addition of picrotoxin (100 μmol/L) induced rapid, transient translocations of eGFP-PKCβII (Fig. 7c). Further investigation showed that picrotoxin-induced eGFP-PKCβII translocations were inhibited completely following the addition of the AMPA receptor antagonist DNQX (10 μmol/L), and following blockade of synaptic activity by TTx (500 nmol/L) (Fig. 7d). These data suggest that eGFP-PKCβII translocations are driven by Ca2+ entry following AMPA receptor activation and voltage-operated Ca2+ channel opening.


The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons.

Willets JM, Nelson CP, Nahorski SR, Challiss RA - J. Neurochem. (2007)

Translocation of conventional and novel protein kinase C (PKCs) following picrotoxin (PiTx) treatment of hippocampal neuron cultures. eGFP-PKCβII or eGFP-PKCε transfected neurons were challenged with PiTx (100 μmol/L) and PKC translocation to the plasma membrane determined as the decrease in cytosolic fluorescence. In panels (a) and (b), neurons were also loaded with the Ca2+-sensitive dye Fura-Red for 60 min prior to addition of PiTx. Note that downward deflections in the Ca2+ trace indicate increases in [Ca2+]i. (a) representative trace showing that picrotoxin addition alone did not stimulate translocation of eGFP-PKCε. (b) representative trace showing that, in contrast, PiTx addition alone caused transient increases in intracellular [Ca2+] that are mirrored by rapid and transient translocations of eGFP-PKCβII. (c) representative trace showing the effects of sequential methacholine (MCh, 100 μmol/L) and PiTx (100 μmol/L) additions on the translocation of eGFP-PKCβII. (d) representative trace showing the effects of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blocker 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 μmol/L) and tetrodotoxin (TTx, 500 nmol/L) on PiTx-induced eGFP-PKCβII translocations. All representative traces are shown for experiments repeated on 4–10 coverslips from at least three separate hippocampal preparations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2658029&req=5

fig07: Translocation of conventional and novel protein kinase C (PKCs) following picrotoxin (PiTx) treatment of hippocampal neuron cultures. eGFP-PKCβII or eGFP-PKCε transfected neurons were challenged with PiTx (100 μmol/L) and PKC translocation to the plasma membrane determined as the decrease in cytosolic fluorescence. In panels (a) and (b), neurons were also loaded with the Ca2+-sensitive dye Fura-Red for 60 min prior to addition of PiTx. Note that downward deflections in the Ca2+ trace indicate increases in [Ca2+]i. (a) representative trace showing that picrotoxin addition alone did not stimulate translocation of eGFP-PKCε. (b) representative trace showing that, in contrast, PiTx addition alone caused transient increases in intracellular [Ca2+] that are mirrored by rapid and transient translocations of eGFP-PKCβII. (c) representative trace showing the effects of sequential methacholine (MCh, 100 μmol/L) and PiTx (100 μmol/L) additions on the translocation of eGFP-PKCβII. (d) representative trace showing the effects of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blocker 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 μmol/L) and tetrodotoxin (TTx, 500 nmol/L) on PiTx-induced eGFP-PKCβII translocations. All representative traces are shown for experiments repeated on 4–10 coverslips from at least three separate hippocampal preparations.
Mentions: The translocation of eGFP-MARCKS indicates that PKC activity can be stimulated in hippocampal neurons following stimulation of the M1 mACh receptor and also following picrotoxin-mediated enhancement of synaptic activity. To gain a better understanding of PKC isoenzymic recruitment patterns in neurons subject to metabotropic and/or ionotropic stimulation, we transfected neurons with eGFP-tagged constructs of either the Ca2+/diacylglycerol (DAG)-activated PKCβII, or the DAG-activated PKCε. Neurons, recombinantly expressing either eGFP-PKCβII or -ε isoenzymes and loaded with Fura-Red simultaneously to report changes in intracellular Ca2+ concentrations, were subject to picrotoxin and/or M1 mACh receptor agonist additions. Under these conditions it could be shown that picrotoxin treatment caused rapid and transient increases in intracellular Ca2+ (Fig. 7a and b). Perhaps unsurprisingly, picrotoxin addition did not cause the recruitment of the DAG-sensitive PKCε to the plasma membrane (Fig. 7a), however, it did produce rapid, transient recruitments of eGFP-PKCβII (Fig. 7b). Indeed, the translocation of eGFP-PKCβII precisely mirrored the picrotoxin-stimulated changes in intracellular Ca2+ (Fig. 7b). MCh (10 μmol/L) stimulation consistently failed to cause eGFP-PKCβII translocation, while addition of picrotoxin (100 μmol/L) induced rapid, transient translocations of eGFP-PKCβII (Fig. 7c). Further investigation showed that picrotoxin-induced eGFP-PKCβII translocations were inhibited completely following the addition of the AMPA receptor antagonist DNQX (10 μmol/L), and following blockade of synaptic activity by TTx (500 nmol/L) (Fig. 7d). These data suggest that eGFP-PKCβII translocations are driven by Ca2+ entry following AMPA receptor activation and voltage-operated Ca2+ channel opening.

Bottom Line: Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin.The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade.In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.

ABSTRACT
To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.

Show MeSH
Related in: MedlinePlus