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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 subtype preferring PKC inhibitors 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 0.01 to 1 μM of GF 109203 X (GF), Gö 6976 or Gö 6983. a Time course of [3H] outflow as a percentage of radioactivity in the cells in the presence of either solvent (clear circles) or 1 μM of GF 109203 X (black circles, n = 3). b Summary of the effects of the indicated concentrations of PKC inhibitors on [3H] overflow evoked by electrical field stimulation (EFS). Overflow in the presence of the inhibitors is depicted as a percentage of the overflow in the presence of solvent (% of control). c Summary of the effects of the indicated concentrations of PKC inhibitors on [3H] overflow evoked by oxotremorine M (OxoM). Overflow in the presence of the inhibitors is depicted as a percentage of the overflow in the presence of solvent (% of control). In (b) and (c), n = 6–9. *p < 0.05; **p < 0.01; ***p < 0.001 (vs. solvent)
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Fig4: Effects of subtype preferring PKC inhibitors 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 0.01 to 1 μM of GF 109203 X (GF), Gö 6976 or Gö 6983. a Time course of [3H] outflow as a percentage of radioactivity in the cells in the presence of either solvent (clear circles) or 1 μM of GF 109203 X (black circles, n = 3). b Summary of the effects of the indicated concentrations of PKC inhibitors on [3H] overflow evoked by electrical field stimulation (EFS). Overflow in the presence of the inhibitors is depicted as a percentage of the overflow in the presence of solvent (% of control). c Summary of the effects of the indicated concentrations of PKC inhibitors on [3H] overflow evoked by oxotremorine M (OxoM). Overflow in the presence of the inhibitors is depicted as a percentage of the overflow in the presence of solvent (% of control). In (b) and (c), n = 6–9. *p < 0.05; **p < 0.01; ***p < 0.001 (vs. solvent)

Mentions: Together, the above results indicate that some PKC isoforms, with the exception of atypical ones, are involved in the excitation of SCG neurons via M1 receptors. To further elaborate which PKC subtypes may contribute, GF 109203 X and related PKC inhibitors (GÖ 6976 and GÖ 6983) with divergent subtype preferences [37] were employed. None of these drugs caused significant alterations in electrically induced tritium overflow (Fig. 4b). In contrast, at 0.01 μM, GÖ 6976 and GÖ 6983, but not GF 109203 X, significantly diminished oxotremorine M-evoked overflow, and at higher concentrations, all the PKC inhibitors shared this effect (Fig. 4c). Thus, with respect to the inhibition of noradrenaline release caused by oxotremorine M, GÖ 6976 and GÖ 6983 were more potent than GF 109203 X.Fig. 4


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 subtype preferring PKC inhibitors 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 0.01 to 1 μM of GF 109203 X (GF), Gö 6976 or Gö 6983. a Time course of [3H] outflow as a percentage of radioactivity in the cells in the presence of either solvent (clear circles) or 1 μM of GF 109203 X (black circles, n = 3). b Summary of the effects of the indicated concentrations of PKC inhibitors on [3H] overflow evoked by electrical field stimulation (EFS). Overflow in the presence of the inhibitors is depicted as a percentage of the overflow in the presence of solvent (% of control). c Summary of the effects of the indicated concentrations of PKC inhibitors on [3H] overflow evoked by oxotremorine M (OxoM). Overflow in the presence of the inhibitors is depicted as a percentage of the overflow in the presence of solvent (% of control). In (b) and (c), n = 6–9. *p < 0.05; **p < 0.01; ***p < 0.001 (vs. solvent)
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Fig4: Effects of subtype preferring PKC inhibitors 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 0.01 to 1 μM of GF 109203 X (GF), Gö 6976 or Gö 6983. a Time course of [3H] outflow as a percentage of radioactivity in the cells in the presence of either solvent (clear circles) or 1 μM of GF 109203 X (black circles, n = 3). b Summary of the effects of the indicated concentrations of PKC inhibitors on [3H] overflow evoked by electrical field stimulation (EFS). Overflow in the presence of the inhibitors is depicted as a percentage of the overflow in the presence of solvent (% of control). c Summary of the effects of the indicated concentrations of PKC inhibitors on [3H] overflow evoked by oxotremorine M (OxoM). Overflow in the presence of the inhibitors is depicted as a percentage of the overflow in the presence of solvent (% of control). In (b) and (c), n = 6–9. *p < 0.05; **p < 0.01; ***p < 0.001 (vs. solvent)
Mentions: Together, the above results indicate that some PKC isoforms, with the exception of atypical ones, are involved in the excitation of SCG neurons via M1 receptors. To further elaborate which PKC subtypes may contribute, GF 109203 X and related PKC inhibitors (GÖ 6976 and GÖ 6983) with divergent subtype preferences [37] were employed. None of these drugs caused significant alterations in electrically induced tritium overflow (Fig. 4b). In contrast, at 0.01 μM, GÖ 6976 and GÖ 6983, but not GF 109203 X, significantly diminished oxotremorine M-evoked overflow, and at higher concentrations, all the PKC inhibitors shared this effect (Fig. 4c). Thus, with respect to the inhibition of noradrenaline release caused by oxotremorine M, GÖ 6976 and GÖ 6983 were more potent than GF 109203 X.Fig. 4

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