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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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Effects of Cl− substitution on noradrenaline release evoked by electrical field stimulation, oxotremorine M or GABA. 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) (a, b) or GABA (10 μM) (c, d) was present in minutes 93 and 94. From minute 50 of superfusion onward, the buffer contained 134 or 74 mM Cl− (the lacking Cl− was replaced by gluconate). a, c Time course of [3H] outflow as a percentage of radioactivity in the cells (n = 3). b Summary of the amount of [3H] overflow evoked by electrical field stimulation (EFS) and oxotremorine M (OxoM), respectively (n = 12). d Summary of the amount of [3H] overflow evoked by electrical field stimulation (EFS) and GABA, respectively (n = 10–12). **p < 0.01 (vs. 134 mM Cl−). n.s. no significance
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Fig6: Effects of Cl− substitution on noradrenaline release evoked by electrical field stimulation, oxotremorine M or GABA. 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) (a, b) or GABA (10 μM) (c, d) was present in minutes 93 and 94. From minute 50 of superfusion onward, the buffer contained 134 or 74 mM Cl− (the lacking Cl− was replaced by gluconate). a, c Time course of [3H] outflow as a percentage of radioactivity in the cells (n = 3). b Summary of the amount of [3H] overflow evoked by electrical field stimulation (EFS) and oxotremorine M (OxoM), respectively (n = 12). d Summary of the amount of [3H] overflow evoked by electrical field stimulation (EFS) and GABA, respectively (n = 10–12). **p < 0.01 (vs. 134 mM Cl−). n.s. no significance

Mentions: To explore the role of Cl− conductances in the stimulatory action of oxotremorine M on noradrenaline release, [3H] overflow was triggered by this agonist either in quasi-physiological solution (containing 134 mM Cl−) or in a solution in which 60 mM NaCl had been replaced by 60 mM sodium gluconate. In these two different solutions, tritium overflow triggered by electrical field stimulation was essentially the same (and if anything, reduced in the presence of sodium gluconate rather than enhanced; Fig. 6d). Oxotremorine M-evoked overflow, however, was significantly enhanced when extracellular Cl− had been reduced (Fig. 6a, b). For comparison, cultures were exposed to 10 μM GABA instead of the same concentration of the muscarinic agonist. As expected, GABA-induced overflow was also enhanced by lowering extracellular Cl− (Fig. 6c, d). Thus, the stimulation of noradrenaline release from SCG neurons through the activation of M1 receptors depends on the extracellular Cl− concentration.Fig. 6


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)

Effects of Cl− substitution on noradrenaline release evoked by electrical field stimulation, oxotremorine M or GABA. 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) (a, b) or GABA (10 μM) (c, d) was present in minutes 93 and 94. From minute 50 of superfusion onward, the buffer contained 134 or 74 mM Cl− (the lacking Cl− was replaced by gluconate). a, c Time course of [3H] outflow as a percentage of radioactivity in the cells (n = 3). b Summary of the amount of [3H] overflow evoked by electrical field stimulation (EFS) and oxotremorine M (OxoM), respectively (n = 12). d Summary of the amount of [3H] overflow evoked by electrical field stimulation (EFS) and GABA, respectively (n = 10–12). **p < 0.01 (vs. 134 mM Cl−). n.s. no significance
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Related In: Results  -  Collection

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Fig6: Effects of Cl− substitution on noradrenaline release evoked by electrical field stimulation, oxotremorine M or GABA. 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) (a, b) or GABA (10 μM) (c, d) was present in minutes 93 and 94. From minute 50 of superfusion onward, the buffer contained 134 or 74 mM Cl− (the lacking Cl− was replaced by gluconate). a, c Time course of [3H] outflow as a percentage of radioactivity in the cells (n = 3). b Summary of the amount of [3H] overflow evoked by electrical field stimulation (EFS) and oxotremorine M (OxoM), respectively (n = 12). d Summary of the amount of [3H] overflow evoked by electrical field stimulation (EFS) and GABA, respectively (n = 10–12). **p < 0.01 (vs. 134 mM Cl−). n.s. no significance
Mentions: To explore the role of Cl− conductances in the stimulatory action of oxotremorine M on noradrenaline release, [3H] overflow was triggered by this agonist either in quasi-physiological solution (containing 134 mM Cl−) or in a solution in which 60 mM NaCl had been replaced by 60 mM sodium gluconate. In these two different solutions, tritium overflow triggered by electrical field stimulation was essentially the same (and if anything, reduced in the presence of sodium gluconate rather than enhanced; Fig. 6d). Oxotremorine M-evoked overflow, however, was significantly enhanced when extracellular Cl− had been reduced (Fig. 6a, b). For comparison, cultures were exposed to 10 μM GABA instead of the same concentration of the muscarinic agonist. As expected, GABA-induced overflow was also enhanced by lowering extracellular Cl− (Fig. 6c, d). Thus, the stimulation of noradrenaline release from SCG neurons through the activation of M1 receptors depends on the extracellular Cl− concentration.Fig. 6

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