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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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Effect of PKC inhibition on noradrenaline release evoked by electrical field stimulation or oxotremorine M. Cultures of SCG were labelled with [3H]noradrenaline and superfused, and subsequent to a 60-min washout period, 4-min fractions of superfusate were collected. Sixty monophasic rectangular pulses (0.5 ms, 60 mA, 50 V/cm) were applied in minute 73, and oxotremorine M (10 μM) was present in minutes 93 and 94. From minute 50 of superfusion onward, the buffer contained either solvent (0.1 % DMSO) or 1 μM staurosporine. Alternatively, cultures had been treated with either 0.1 % DMSO (untreated) or 1 μM phorbol-12-myristate-13-acetate (PMA-treated) for 24 h. a Time course of [3H] outflow as a percentage of radioactivity in the cells in the presence of either solvent (clear circles) or staurosporine (black circles, n = 3). b Summary of the effect of 1 μM staurosporine on [3H] overflow evoked by electrical field stimulation (EFS) or oxotremorine M (OxoM, n = 8–9). c Time course of [3H] outflow as a percentage of radioactivity in the cells which had either been treated with phorbol-12-myristate-13-acetate (black circles) or had remained untreated (clear circles, n = 3). d Summary of the effect of phorbol-12-myristate-13-acetate treatment on [3H] overflow evoked by electrical field stimulation (EFS) or oxotremorine M (OxoM, n = 12). **p < 0.01 (vs. solvent and untreated). n.s. no significance
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Fig3: Effect of PKC inhibition on noradrenaline release evoked by electrical field stimulation or oxotremorine M. Cultures of SCG were labelled with [3H]noradrenaline and superfused, and subsequent to a 60-min washout period, 4-min fractions of superfusate were collected. Sixty monophasic rectangular pulses (0.5 ms, 60 mA, 50 V/cm) were applied in minute 73, and oxotremorine M (10 μM) was present in minutes 93 and 94. From minute 50 of superfusion onward, the buffer contained either solvent (0.1 % DMSO) or 1 μM staurosporine. Alternatively, cultures had been treated with either 0.1 % DMSO (untreated) or 1 μM phorbol-12-myristate-13-acetate (PMA-treated) for 24 h. a Time course of [3H] outflow as a percentage of radioactivity in the cells in the presence of either solvent (clear circles) or staurosporine (black circles, n = 3). b Summary of the effect of 1 μM staurosporine on [3H] overflow evoked by electrical field stimulation (EFS) or oxotremorine M (OxoM, n = 8–9). c Time course of [3H] outflow as a percentage of radioactivity in the cells which had either been treated with phorbol-12-myristate-13-acetate (black circles) or had remained untreated (clear circles, n = 3). d Summary of the effect of phorbol-12-myristate-13-acetate treatment on [3H] overflow evoked by electrical field stimulation (EFS) or oxotremorine M (OxoM, n = 12). **p < 0.01 (vs. solvent and untreated). n.s. no significance

Mentions: To investigate the signalling cascade of oxotremorine M-induced depolarisations not only in single cells, which are quite heterogeneous in this response, a large population of neurons was investigated simultaneously. This was achieved by loading the neurons with [3H]noradrenaline and by subsequently stimulating the overflow of radioactivity by this muscarinic agonist. In such experiments, oxotremorine M, at concentrations <100 μM, triggers overflow of radioactivity through the selective activation of M1 receptors [23]. To control for effects of PKC inhibitors unrelated to the signalling cascade of M1 receptors, tritium overflow was also elicited by electrical field stimulation. Staurosporine (1 μM) did not alter tritium overflow triggered by electrical fields, but reduced that evoked by oxotremorine M by more than 50 % (Fig. 3a, b). Exposure of SCG cultures to phorbol-12-myristate-13-acetate for 24 h downregulates all but atypical PKC isoforms [34]. In cultures treated in that way, electrically evoked release was the same as in untreated sister cultures. However, oxotremorine M-induced tritium overflow in phorbol-12-myristate-13-acetate-treated cultures amounted to only 10 % of that in untreated cultures (Fig. 3c, d).Fig. 3


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)

Effect of PKC inhibition on noradrenaline release evoked by electrical field stimulation or oxotremorine M. Cultures of SCG were labelled with [3H]noradrenaline and superfused, and subsequent to a 60-min washout period, 4-min fractions of superfusate were collected. Sixty monophasic rectangular pulses (0.5 ms, 60 mA, 50 V/cm) were applied in minute 73, and oxotremorine M (10 μM) was present in minutes 93 and 94. From minute 50 of superfusion onward, the buffer contained either solvent (0.1 % DMSO) or 1 μM staurosporine. Alternatively, cultures had been treated with either 0.1 % DMSO (untreated) or 1 μM phorbol-12-myristate-13-acetate (PMA-treated) for 24 h. a Time course of [3H] outflow as a percentage of radioactivity in the cells in the presence of either solvent (clear circles) or staurosporine (black circles, n = 3). b Summary of the effect of 1 μM staurosporine on [3H] overflow evoked by electrical field stimulation (EFS) or oxotremorine M (OxoM, n = 8–9). c Time course of [3H] outflow as a percentage of radioactivity in the cells which had either been treated with phorbol-12-myristate-13-acetate (black circles) or had remained untreated (clear circles, n = 3). d Summary of the effect of phorbol-12-myristate-13-acetate treatment on [3H] overflow evoked by electrical field stimulation (EFS) or oxotremorine M (OxoM, n = 12). **p < 0.01 (vs. solvent and untreated). n.s. no significance
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Related In: Results  -  Collection

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Fig3: Effect of PKC inhibition on noradrenaline release evoked by electrical field stimulation or oxotremorine M. Cultures of SCG were labelled with [3H]noradrenaline and superfused, and subsequent to a 60-min washout period, 4-min fractions of superfusate were collected. Sixty monophasic rectangular pulses (0.5 ms, 60 mA, 50 V/cm) were applied in minute 73, and oxotremorine M (10 μM) was present in minutes 93 and 94. From minute 50 of superfusion onward, the buffer contained either solvent (0.1 % DMSO) or 1 μM staurosporine. Alternatively, cultures had been treated with either 0.1 % DMSO (untreated) or 1 μM phorbol-12-myristate-13-acetate (PMA-treated) for 24 h. a Time course of [3H] outflow as a percentage of radioactivity in the cells in the presence of either solvent (clear circles) or staurosporine (black circles, n = 3). b Summary of the effect of 1 μM staurosporine on [3H] overflow evoked by electrical field stimulation (EFS) or oxotremorine M (OxoM, n = 8–9). c Time course of [3H] outflow as a percentage of radioactivity in the cells which had either been treated with phorbol-12-myristate-13-acetate (black circles) or had remained untreated (clear circles, n = 3). d Summary of the effect of phorbol-12-myristate-13-acetate treatment on [3H] overflow evoked by electrical field stimulation (EFS) or oxotremorine M (OxoM, n = 12). **p < 0.01 (vs. solvent and untreated). n.s. no significance
Mentions: To investigate the signalling cascade of oxotremorine M-induced depolarisations not only in single cells, which are quite heterogeneous in this response, a large population of neurons was investigated simultaneously. This was achieved by loading the neurons with [3H]noradrenaline and by subsequently stimulating the overflow of radioactivity by this muscarinic agonist. In such experiments, oxotremorine M, at concentrations <100 μM, triggers overflow of radioactivity through the selective activation of M1 receptors [23]. To control for effects of PKC inhibitors unrelated to the signalling cascade of M1 receptors, tritium overflow was also elicited by electrical field stimulation. Staurosporine (1 μM) did not alter tritium overflow triggered by electrical fields, but reduced that evoked by oxotremorine M by more than 50 % (Fig. 3a, b). Exposure of SCG cultures to phorbol-12-myristate-13-acetate for 24 h downregulates all but atypical PKC isoforms [34]. In cultures treated in that way, electrically evoked release was the same as in untreated sister cultures. However, oxotremorine M-induced tritium overflow in phorbol-12-myristate-13-acetate-treated cultures amounted to only 10 % of that in untreated cultures (Fig. 3c, d).Fig. 3

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