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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− and ionophore substitution on depolarisations by oxotremorine M. Membrane potential in SCG neurons was recorded in current-clamp mode using the amphotericin B- or gramicidin D-perforated patch technique. a Extent of depolarisations elicited by 10 μM oxotremorine M in amphotericin B-perforated patch recordings with pipette solutions containing 75 mM K2SO4 plus 55 mM KCl, 55 mM potassium gluconate (KGluc) or 55 mM CsCl (n = 5–7). *p < 0.05; **p < 0.01 (vs. potassium gluconate, Kruskal–Wallis test). b Original current-clamp traces using either amphotericin B (upper traces) or gramicidin D (lower traces). 10 μM oxotremorine M (OxoM) or 10 μM GABA was present, as indicated by the bars. c Extent of depolarisations elicited by 10 μM oxotremorine M or 10 μM GABA in either amphotericin B- or gramicidin D-perforated patch recordings (n = 5–7)
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Fig5: Effects of Cl− and ionophore substitution on depolarisations by oxotremorine M. Membrane potential in SCG neurons was recorded in current-clamp mode using the amphotericin B- or gramicidin D-perforated patch technique. a Extent of depolarisations elicited by 10 μM oxotremorine M in amphotericin B-perforated patch recordings with pipette solutions containing 75 mM K2SO4 plus 55 mM KCl, 55 mM potassium gluconate (KGluc) or 55 mM CsCl (n = 5–7). *p < 0.05; **p < 0.01 (vs. potassium gluconate, Kruskal–Wallis test). b Original current-clamp traces using either amphotericin B (upper traces) or gramicidin D (lower traces). 10 μM oxotremorine M (OxoM) or 10 μM GABA was present, as indicated by the bars. c Extent of depolarisations elicited by 10 μM oxotremorine M or 10 μM GABA in either amphotericin B- or gramicidin D-perforated patch recordings (n = 5–7)

Mentions: To elucidate the ionic basis of oxotremorine M-induced depolarisations, the composition of the pipette solution was changed by replacing 55 mM KCl by equimolar concentrations of either CsCl or potassium gluconate. The resting membrane potentials determined with these three different pipette solutions were −65.2 + 2.4 mV (KCl, n = 7), −77.7 + 3.3 mV (K-gluconate, n = 5) and −62.8 + 1.9 mV (CsCl, n = 5). Whilst changes in membrane voltage caused by 10 μM oxotremorine M were not affected by alterations in K+, the reduction of intracellular Cl- clearly reduced the depolarising response (Fig. 5a).Fig. 5


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− and ionophore substitution on depolarisations by oxotremorine M. Membrane potential in SCG neurons was recorded in current-clamp mode using the amphotericin B- or gramicidin D-perforated patch technique. a Extent of depolarisations elicited by 10 μM oxotremorine M in amphotericin B-perforated patch recordings with pipette solutions containing 75 mM K2SO4 plus 55 mM KCl, 55 mM potassium gluconate (KGluc) or 55 mM CsCl (n = 5–7). *p < 0.05; **p < 0.01 (vs. potassium gluconate, Kruskal–Wallis test). b Original current-clamp traces using either amphotericin B (upper traces) or gramicidin D (lower traces). 10 μM oxotremorine M (OxoM) or 10 μM GABA was present, as indicated by the bars. c Extent of depolarisations elicited by 10 μM oxotremorine M or 10 μM GABA in either amphotericin B- or gramicidin D-perforated patch recordings (n = 5–7)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4233321&req=5

Fig5: Effects of Cl− and ionophore substitution on depolarisations by oxotremorine M. Membrane potential in SCG neurons was recorded in current-clamp mode using the amphotericin B- or gramicidin D-perforated patch technique. a Extent of depolarisations elicited by 10 μM oxotremorine M in amphotericin B-perforated patch recordings with pipette solutions containing 75 mM K2SO4 plus 55 mM KCl, 55 mM potassium gluconate (KGluc) or 55 mM CsCl (n = 5–7). *p < 0.05; **p < 0.01 (vs. potassium gluconate, Kruskal–Wallis test). b Original current-clamp traces using either amphotericin B (upper traces) or gramicidin D (lower traces). 10 μM oxotremorine M (OxoM) or 10 μM GABA was present, as indicated by the bars. c Extent of depolarisations elicited by 10 μM oxotremorine M or 10 μM GABA in either amphotericin B- or gramicidin D-perforated patch recordings (n = 5–7)
Mentions: To elucidate the ionic basis of oxotremorine M-induced depolarisations, the composition of the pipette solution was changed by replacing 55 mM KCl by equimolar concentrations of either CsCl or potassium gluconate. The resting membrane potentials determined with these three different pipette solutions were −65.2 + 2.4 mV (KCl, n = 7), −77.7 + 3.3 mV (K-gluconate, n = 5) and −62.8 + 1.9 mV (CsCl, n = 5). Whilst changes in membrane voltage caused by 10 μM oxotremorine M were not affected by alterations in K+, the reduction of intracellular Cl- clearly reduced the depolarising response (Fig. 5a).Fig. 5

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