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Excitation of rat sympathetic neurons via M1 muscarinic receptors independently of Kv7 channels.

Salzer I, Gafar H, Gindl V, Mahlknecht P, Drobny H, Boehm S - Pflugers Arch. (2014)

Bottom Line: However, in the present experiments using primary cultures of rat superior cervical ganglion neurons, the extent of depolarisation caused by the M1 receptor agonist oxotremorine M did not correlate with the extent of Kv7 channel inhibition in the very same neuron.These channel blockers also reduced oxotremorine M-evoked noradrenaline release.Together, these results reveal that slow cholinergic excitation of sympathetic neurons involves the activation of classical PKCs and of Ca(2+)-activated Cl(-) channels in addition to the well-known inhibition of Kv7 channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurophysiology and Neuropharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.

ABSTRACT
The slow cholinergic transmission in autonomic ganglia is known to be mediated by an inhibition of Kv7 channels via M1 muscarinic acetylcholine receptors. However, in the present experiments using primary cultures of rat superior cervical ganglion neurons, the extent of depolarisation caused by the M1 receptor agonist oxotremorine M did not correlate with the extent of Kv7 channel inhibition in the very same neuron. This observation triggered a search for additional mechanisms. As the activation of M1 receptors leads to a boost in protein kinase C (PKC) activity in sympathetic neurons, various PKC enzymes were inhibited by different means. Interference with classical PKC isoforms led to reductions in depolarisations and in noradrenaline release elicited by oxotremorine M, but left the Kv7 channel inhibition by the muscarinic agonist unchanged. M1 receptor-induced depolarisations were also altered when extra- or intracellular Cl(-) concentrations were changed, as were depolarising responses to γ-aminobutyric acid. Depolarisations and noradrenaline release triggered by oxotremorine M were reduced by the non-selective Cl(-) channel blockers 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid and niflumic acid. Oxotremorine M induced slowly rising inward currents at negative membrane potentials that were blocked by inhibitors of Ca(2+)-activated Cl(-) and TMEM16A channels and attenuated by PKC inhibitors. These channel blockers also reduced oxotremorine M-evoked noradrenaline release. Together, these results reveal that slow cholinergic excitation of sympathetic neurons involves the activation of classical PKCs and of Ca(2+)-activated Cl(-) channels in addition to the well-known inhibition of Kv7 channels.

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Comparison of depolarisation and Kv7 channel inhibition by oxotremorine M. Membrane potential and currents through Kv7 channels in SCG neurons were recorded in current-clamp and voltage-clamp mode, respectively, using the amphotericin B-perforated patch technique. a Time course of membrane voltage in two different SCG neurons; oxotremorine M (OxoM, 10 μM) was present, as indicated by the bars. b Frequency distribution of resting membrane potentials (voltage in bins of 2 mV) as determined in 42 SCG neurons. c Frequency distribution of changes in membrane potentials (Δ mV in bins of 2 mV) caused by 10 μM oxotremorine M as determined in these 42 SCG neurons. d Correlation between resting membrane potential (voltage) and membrane potential changes (Δ mV) caused by 10 μM oxotremorine M in the same 42 SCG neurons; experiments were carried out as shown in (a). The Spearman’s coefficient for this correlation is −0.053 (95% confidence interval, −0.3594 to 0.2638). e Subset of 16 neurons categorized according to the extent of depolarisation caused by 10 μM oxotremorine M (<5 mV, n = 8; >5 mV, n = 8); the means of the depolarisation observed in these two groups are shown. f Subsequently, currents through Kv7 channels were recorded by holding these 16 cells at a voltage of −30 mV and by applying hyperpolarisations to −55 mV once every 15 s. The traces show current responses of two neurons, one out of each of these two categories. g Mean values of densities of deactivation currents caused by the steps from −30 to −55 mV in the neurons from both categories (n = 8). h Time course of deactivation current amplitudes caused by the steps from −30 to −55 mV in the neurons from both categories; oxotremorine M (OxoM, 10 μM) was present, as indicated by the bar (n = 8)
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Fig1: Comparison of depolarisation and Kv7 channel inhibition by oxotremorine M. Membrane potential and currents through Kv7 channels in SCG neurons were recorded in current-clamp and voltage-clamp mode, respectively, using the amphotericin B-perforated patch technique. a Time course of membrane voltage in two different SCG neurons; oxotremorine M (OxoM, 10 μM) was present, as indicated by the bars. b Frequency distribution of resting membrane potentials (voltage in bins of 2 mV) as determined in 42 SCG neurons. c Frequency distribution of changes in membrane potentials (Δ mV in bins of 2 mV) caused by 10 μM oxotremorine M as determined in these 42 SCG neurons. d Correlation between resting membrane potential (voltage) and membrane potential changes (Δ mV) caused by 10 μM oxotremorine M in the same 42 SCG neurons; experiments were carried out as shown in (a). The Spearman’s coefficient for this correlation is −0.053 (95% confidence interval, −0.3594 to 0.2638). e Subset of 16 neurons categorized according to the extent of depolarisation caused by 10 μM oxotremorine M (<5 mV, n = 8; >5 mV, n = 8); the means of the depolarisation observed in these two groups are shown. f Subsequently, currents through Kv7 channels were recorded by holding these 16 cells at a voltage of −30 mV and by applying hyperpolarisations to −55 mV once every 15 s. The traces show current responses of two neurons, one out of each of these two categories. g Mean values of densities of deactivation currents caused by the steps from −30 to −55 mV in the neurons from both categories (n = 8). h Time course of deactivation current amplitudes caused by the steps from −30 to −55 mV in the neurons from both categories; oxotremorine M (OxoM, 10 μM) was present, as indicated by the bar (n = 8)

Mentions: In order to evaluate the relation between the depolarisation of SCG neurons and the inhibition of Kv7 channels, both through the activation of M1 receptors by oxotremorine M, an initial set of 42 neurons was investigated. The values of resting membrane potentials in these neurons ranged between −55 and −75 mV. Changes in membrane potential caused by 10 μM oxotremorine M varied between −1 and +13 mV. There was no correlation between these values of resting membrane potential and the changes induced by the muscarinic agonist (Fig. 1a–d). In a subset of neurons with oxotremorine M-induced depolarisations of either less (n = 8) or more (n = 8) than 5 mV, currents through Kv7 channels were determined subsequently to the current-clamp measurements (Fig. 1e, f). The densities of Kv7 deactivation currents (triggered by hyperpolarisations from −30 to −55 mV) were comparable in these two groups of neurons (Fig. 1g). Likewise, the extent as well as the time course of current inhibition by 10 μM oxotremorine M for these two sets of neurons were indiscernible (Fig. 1h). We therefore concluded that mechanisms other than the inhibition of Kv7 channels also contribute to the depolarisation caused by oxotremorine M. As the extent of oxotremorine M-induced depolarisation varies considerably between single neurons (Fig. 1b), the underlying signalling cascade was investigated only in neurons that displayed depolarisations of at least 5 mV.Fig. 1


Excitation of rat sympathetic neurons via M1 muscarinic receptors independently of Kv7 channels.

Salzer I, Gafar H, Gindl V, Mahlknecht P, Drobny H, Boehm S - Pflugers Arch. (2014)

Comparison of depolarisation and Kv7 channel inhibition by oxotremorine M. Membrane potential and currents through Kv7 channels in SCG neurons were recorded in current-clamp and voltage-clamp mode, respectively, using the amphotericin B-perforated patch technique. a Time course of membrane voltage in two different SCG neurons; oxotremorine M (OxoM, 10 μM) was present, as indicated by the bars. b Frequency distribution of resting membrane potentials (voltage in bins of 2 mV) as determined in 42 SCG neurons. c Frequency distribution of changes in membrane potentials (Δ mV in bins of 2 mV) caused by 10 μM oxotremorine M as determined in these 42 SCG neurons. d Correlation between resting membrane potential (voltage) and membrane potential changes (Δ mV) caused by 10 μM oxotremorine M in the same 42 SCG neurons; experiments were carried out as shown in (a). The Spearman’s coefficient for this correlation is −0.053 (95% confidence interval, −0.3594 to 0.2638). e Subset of 16 neurons categorized according to the extent of depolarisation caused by 10 μM oxotremorine M (<5 mV, n = 8; >5 mV, n = 8); the means of the depolarisation observed in these two groups are shown. f Subsequently, currents through Kv7 channels were recorded by holding these 16 cells at a voltage of −30 mV and by applying hyperpolarisations to −55 mV once every 15 s. The traces show current responses of two neurons, one out of each of these two categories. g Mean values of densities of deactivation currents caused by the steps from −30 to −55 mV in the neurons from both categories (n = 8). h Time course of deactivation current amplitudes caused by the steps from −30 to −55 mV in the neurons from both categories; oxotremorine M (OxoM, 10 μM) was present, as indicated by the bar (n = 8)
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Related In: Results  -  Collection

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Fig1: Comparison of depolarisation and Kv7 channel inhibition by oxotremorine M. Membrane potential and currents through Kv7 channels in SCG neurons were recorded in current-clamp and voltage-clamp mode, respectively, using the amphotericin B-perforated patch technique. a Time course of membrane voltage in two different SCG neurons; oxotremorine M (OxoM, 10 μM) was present, as indicated by the bars. b Frequency distribution of resting membrane potentials (voltage in bins of 2 mV) as determined in 42 SCG neurons. c Frequency distribution of changes in membrane potentials (Δ mV in bins of 2 mV) caused by 10 μM oxotremorine M as determined in these 42 SCG neurons. d Correlation between resting membrane potential (voltage) and membrane potential changes (Δ mV) caused by 10 μM oxotremorine M in the same 42 SCG neurons; experiments were carried out as shown in (a). The Spearman’s coefficient for this correlation is −0.053 (95% confidence interval, −0.3594 to 0.2638). e Subset of 16 neurons categorized according to the extent of depolarisation caused by 10 μM oxotremorine M (<5 mV, n = 8; >5 mV, n = 8); the means of the depolarisation observed in these two groups are shown. f Subsequently, currents through Kv7 channels were recorded by holding these 16 cells at a voltage of −30 mV and by applying hyperpolarisations to −55 mV once every 15 s. The traces show current responses of two neurons, one out of each of these two categories. g Mean values of densities of deactivation currents caused by the steps from −30 to −55 mV in the neurons from both categories (n = 8). h Time course of deactivation current amplitudes caused by the steps from −30 to −55 mV in the neurons from both categories; oxotremorine M (OxoM, 10 μM) was present, as indicated by the bar (n = 8)
Mentions: In order to evaluate the relation between the depolarisation of SCG neurons and the inhibition of Kv7 channels, both through the activation of M1 receptors by oxotremorine M, an initial set of 42 neurons was investigated. The values of resting membrane potentials in these neurons ranged between −55 and −75 mV. Changes in membrane potential caused by 10 μM oxotremorine M varied between −1 and +13 mV. There was no correlation between these values of resting membrane potential and the changes induced by the muscarinic agonist (Fig. 1a–d). In a subset of neurons with oxotremorine M-induced depolarisations of either less (n = 8) or more (n = 8) than 5 mV, currents through Kv7 channels were determined subsequently to the current-clamp measurements (Fig. 1e, f). The densities of Kv7 deactivation currents (triggered by hyperpolarisations from −30 to −55 mV) were comparable in these two groups of neurons (Fig. 1g). Likewise, the extent as well as the time course of current inhibition by 10 μM oxotremorine M for these two sets of neurons were indiscernible (Fig. 1h). We therefore concluded that mechanisms other than the inhibition of Kv7 channels also contribute to the depolarisation caused by oxotremorine M. As the extent of oxotremorine M-induced depolarisation varies considerably between single neurons (Fig. 1b), the underlying signalling cascade was investigated only in neurons that displayed depolarisations of at least 5 mV.Fig. 1

Bottom Line: However, in the present experiments using primary cultures of rat superior cervical ganglion neurons, the extent of depolarisation caused by the M1 receptor agonist oxotremorine M did not correlate with the extent of Kv7 channel inhibition in the very same neuron.These channel blockers also reduced oxotremorine M-evoked noradrenaline release.Together, these results reveal that slow cholinergic excitation of sympathetic neurons involves the activation of classical PKCs and of Ca(2+)-activated Cl(-) channels in addition to the well-known inhibition of Kv7 channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurophysiology and Neuropharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.

ABSTRACT
The slow cholinergic transmission in autonomic ganglia is known to be mediated by an inhibition of Kv7 channels via M1 muscarinic acetylcholine receptors. However, in the present experiments using primary cultures of rat superior cervical ganglion neurons, the extent of depolarisation caused by the M1 receptor agonist oxotremorine M did not correlate with the extent of Kv7 channel inhibition in the very same neuron. This observation triggered a search for additional mechanisms. As the activation of M1 receptors leads to a boost in protein kinase C (PKC) activity in sympathetic neurons, various PKC enzymes were inhibited by different means. Interference with classical PKC isoforms led to reductions in depolarisations and in noradrenaline release elicited by oxotremorine M, but left the Kv7 channel inhibition by the muscarinic agonist unchanged. M1 receptor-induced depolarisations were also altered when extra- or intracellular Cl(-) concentrations were changed, as were depolarising responses to γ-aminobutyric acid. Depolarisations and noradrenaline release triggered by oxotremorine M were reduced by the non-selective Cl(-) channel blockers 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid and niflumic acid. Oxotremorine M induced slowly rising inward currents at negative membrane potentials that were blocked by inhibitors of Ca(2+)-activated Cl(-) and TMEM16A channels and attenuated by PKC inhibitors. These channel blockers also reduced oxotremorine M-evoked noradrenaline release. Together, these results reveal that slow cholinergic excitation of sympathetic neurons involves the activation of classical PKCs and of Ca(2+)-activated Cl(-) channels in addition to the well-known inhibition of Kv7 channels.

Show MeSH
Related in: MedlinePlus