Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of manipulating protein kinase C activity in hippocampal neurons on M1 muscarinic acetylcholine receptor responsiveness. Neurons were co-transfected with PH domain of PLCδ tagged to enhanced green fluorescent protein and empty vector (pcDNA3) or D110A,K220RG protein-coupled receptor kinase (GRK) 2 and changes in receptor responsiveness determined using the standard R1/Rmax/R2 protocol (see Experimental Procedures). Neurons were pre-incubated with either vehicle (Control) or staurosporine (1 μmol/L; Stauro) for 15 min prior to, and throughout the experiment. Representative traces are shown for neurons treated with picrotoxin (PiTx, 100 μmol/L) in the presence of (a) pcDNA3 and vehicle; (b) D110A,K220RGRK2 and vehicle; (c) pcDNA3 + staurosporine (1 μmol/L); (d) D110A,K220RGRK2 and staurosporine (1 μmol/L). (e) cumulative data are shown for either spontaneously active or picrotoxin-treated neurons and are expressed as means ± SEM for the percentage change in R2 relative to the R1 response, for ≥ 5 neurons per treatment taken from at least three separate hippocampal preparations. Significant differences in the R2/R1 ratio caused by either D110A,K220RGRK2 expression are indicated as *p< 0.05; **p< 0.01, while a significant effect of staurosporine pre-treatment is shown as +p< 0.05.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2658029&req=5

fig04: Effects of manipulating protein kinase C activity in hippocampal neurons on M1 muscarinic acetylcholine receptor responsiveness. Neurons were co-transfected with PH domain of PLCδ tagged to enhanced green fluorescent protein and empty vector (pcDNA3) or D110A,K220RG protein-coupled receptor kinase (GRK) 2 and changes in receptor responsiveness determined using the standard R1/Rmax/R2 protocol (see Experimental Procedures). Neurons were pre-incubated with either vehicle (Control) or staurosporine (1 μmol/L; Stauro) for 15 min prior to, and throughout the experiment. Representative traces are shown for neurons treated with picrotoxin (PiTx, 100 μmol/L) in the presence of (a) pcDNA3 and vehicle; (b) D110A,K220RGRK2 and vehicle; (c) pcDNA3 + staurosporine (1 μmol/L); (d) D110A,K220RGRK2 and staurosporine (1 μmol/L). (e) cumulative data are shown for either spontaneously active or picrotoxin-treated neurons and are expressed as means ± SEM for the percentage change in R2 relative to the R1 response, for ≥ 5 neurons per treatment taken from at least three separate hippocampal preparations. Significant differences in the R2/R1 ratio caused by either D110A,K220RGRK2 expression are indicated as *p< 0.05; **p< 0.01, while a significant effect of staurosporine pre-treatment is shown as +p< 0.05.

Mentions: To assess whether PKC plays a role in M1 mACh receptor desensitization we pre-incubated neurons with vehicle (0.01% dimethylsulphoxide, hereafter DMSO) or the PKC inhibitor staurosporine (1 μmol/L) for 15 min. Next neurons were subjected to the standard desensitization R1/Rmax/R2 protocol, in the presence of vehicle or staurosporine throughout the experiment. In spontaneously synaptically-active, pcDNA3-transfected cultures PKC inhibition had no effect on the R2/R1 ratio, implying that PKC does not play a role in agonist-stimulated M1 mACh receptor desensitization (Fig. 4a and e). In addition, staurosporine did not alter the R2/R1 ratio in the presence of D110A,K220RGRK2 in spontaneously synaptically-active neurons. In contrast, staurosporine pre-treatment had a significant effect on the R2/R1 ratio when synaptic activity was enhanced in the presence of picrotoxin (Fig. 4b and e). Furthermore, staurosporine (1 μmol/L) pre-treatment was also able to enhance the effect of D110A,K220RGRK2 expression, resulting in an almost complete blockade of M1 mACh receptor desensitization in picrotoxin-treated neurons (see Fig. 4e). In agreement with the staurosporine data, down-regulation of PKC isoenzymes by a 24 h pre-treatment with phorbol 12,13-dibutyrate (1 μmol/L) significantly attenuated the decrease in R2/R1 ratio caused by agonist challenge in picrotoxin-treated neurons, but was without apparent effect in TTx-treated, or spontaneously-active neurons (data not shown). While neither staurosporine nor chronic phorbol ester treatments act specifically on PKCs in neurons, the fact that these two interventions produce essentially identical data strongly implicate PKCs as the kinases that may additionally regulate M1 mACh receptor responsiveness under conditions of enhanced synaptic activity. Thus, these data are strongly suggestive that under conditions of picrotoxin-enhanced synaptic activity, GRK2 and PKCs act together to enhance M1 mACh receptor desensitization.


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)

Effects of manipulating protein kinase C activity in hippocampal neurons on M1 muscarinic acetylcholine receptor responsiveness. Neurons were co-transfected with PH domain of PLCδ tagged to enhanced green fluorescent protein and empty vector (pcDNA3) or D110A,K220RG protein-coupled receptor kinase (GRK) 2 and changes in receptor responsiveness determined using the standard R1/Rmax/R2 protocol (see Experimental Procedures). Neurons were pre-incubated with either vehicle (Control) or staurosporine (1 μmol/L; Stauro) for 15 min prior to, and throughout the experiment. Representative traces are shown for neurons treated with picrotoxin (PiTx, 100 μmol/L) in the presence of (a) pcDNA3 and vehicle; (b) D110A,K220RGRK2 and vehicle; (c) pcDNA3 + staurosporine (1 μmol/L); (d) D110A,K220RGRK2 and staurosporine (1 μmol/L). (e) cumulative data are shown for either spontaneously active or picrotoxin-treated neurons and are expressed as means ± SEM for the percentage change in R2 relative to the R1 response, for ≥ 5 neurons per treatment taken from at least three separate hippocampal preparations. Significant differences in the R2/R1 ratio caused by either D110A,K220RGRK2 expression are indicated as *p< 0.05; **p< 0.01, while a significant effect of staurosporine pre-treatment is shown as +p< 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

fig04: Effects of manipulating protein kinase C activity in hippocampal neurons on M1 muscarinic acetylcholine receptor responsiveness. Neurons were co-transfected with PH domain of PLCδ tagged to enhanced green fluorescent protein and empty vector (pcDNA3) or D110A,K220RG protein-coupled receptor kinase (GRK) 2 and changes in receptor responsiveness determined using the standard R1/Rmax/R2 protocol (see Experimental Procedures). Neurons were pre-incubated with either vehicle (Control) or staurosporine (1 μmol/L; Stauro) for 15 min prior to, and throughout the experiment. Representative traces are shown for neurons treated with picrotoxin (PiTx, 100 μmol/L) in the presence of (a) pcDNA3 and vehicle; (b) D110A,K220RGRK2 and vehicle; (c) pcDNA3 + staurosporine (1 μmol/L); (d) D110A,K220RGRK2 and staurosporine (1 μmol/L). (e) cumulative data are shown for either spontaneously active or picrotoxin-treated neurons and are expressed as means ± SEM for the percentage change in R2 relative to the R1 response, for ≥ 5 neurons per treatment taken from at least three separate hippocampal preparations. Significant differences in the R2/R1 ratio caused by either D110A,K220RGRK2 expression are indicated as *p< 0.05; **p< 0.01, while a significant effect of staurosporine pre-treatment is shown as +p< 0.05.
Mentions: To assess whether PKC plays a role in M1 mACh receptor desensitization we pre-incubated neurons with vehicle (0.01% dimethylsulphoxide, hereafter DMSO) or the PKC inhibitor staurosporine (1 μmol/L) for 15 min. Next neurons were subjected to the standard desensitization R1/Rmax/R2 protocol, in the presence of vehicle or staurosporine throughout the experiment. In spontaneously synaptically-active, pcDNA3-transfected cultures PKC inhibition had no effect on the R2/R1 ratio, implying that PKC does not play a role in agonist-stimulated M1 mACh receptor desensitization (Fig. 4a and e). In addition, staurosporine did not alter the R2/R1 ratio in the presence of D110A,K220RGRK2 in spontaneously synaptically-active neurons. In contrast, staurosporine pre-treatment had a significant effect on the R2/R1 ratio when synaptic activity was enhanced in the presence of picrotoxin (Fig. 4b and e). Furthermore, staurosporine (1 μmol/L) pre-treatment was also able to enhance the effect of D110A,K220RGRK2 expression, resulting in an almost complete blockade of M1 mACh receptor desensitization in picrotoxin-treated neurons (see Fig. 4e). In agreement with the staurosporine data, down-regulation of PKC isoenzymes by a 24 h pre-treatment with phorbol 12,13-dibutyrate (1 μmol/L) significantly attenuated the decrease in R2/R1 ratio caused by agonist challenge in picrotoxin-treated neurons, but was without apparent effect in TTx-treated, or spontaneously-active neurons (data not shown). While neither staurosporine nor chronic phorbol ester treatments act specifically on PKCs in neurons, the fact that these two interventions produce essentially identical data strongly implicate PKCs as the kinases that may additionally regulate M1 mACh receptor responsiveness under conditions of enhanced synaptic activity. Thus, these data are strongly suggestive that under conditions of picrotoxin-enhanced synaptic activity, GRK2 and PKCs act together to enhance M1 mACh receptor desensitization.

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