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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 PKC inhibitors on 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. Oxotremorine M (OxoM, 10 μM) was present for six periods of 60 s each; these periods of oxotremorine M application were separated by 3-min intervals. From minute 2 after the first oxotremorine M application onward, PKC inhibitors, XE 991, or solvent (0.1 % DMSO) was present throughout the remaining measurements. a Time course of membrane voltage in two different SCG neurons during the first and the sixth exposure to oxotremorine M (OxoM, 10 μM); the agonist was present, as indicated by the bars. After the first oxotremorine M exposure, either 1 μM staurosporine or 1 μM GF 109203 X was present. b Changes in membrane voltage (Δ mV) caused by these six oxotremorine M applications (O1–O6) in the presence of DMSO, staurosporine, GF 109203 X or XE 991 (3 μM); the values of these six depolarisations were normalized to the value of the first one (n = 6). *Significant difference between the four values at O6 (p < 0.05, Kruskal–Wallis test). c Current responses of one neuron that was clamped at a voltage of −30 mV and hyperpolarised to −55 mV once every 15 s and that has been exposed to 1 μM staurosporine. The traces were obtained before (solvent) and during (OxoM) the first application (O1) of 10 μM oxotremorine M as well as before (stauro) and during (stauro+OxoM) the sixth application (O6) of oxotremorine M. d Changes in Kv7 inhibition (quantified by deactivation current amplitudes) caused by these six oxotremorine M applications (O1–O6) in the presence of either DMSO, staurosporine or GF 109203 X; these six values of Kv7 inhibition were normalized to the value of the first one (n = 6–9)
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Fig2: Effects of PKC inhibitors on 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. Oxotremorine M (OxoM, 10 μM) was present for six periods of 60 s each; these periods of oxotremorine M application were separated by 3-min intervals. From minute 2 after the first oxotremorine M application onward, PKC inhibitors, XE 991, or solvent (0.1 % DMSO) was present throughout the remaining measurements. a Time course of membrane voltage in two different SCG neurons during the first and the sixth exposure to oxotremorine M (OxoM, 10 μM); the agonist was present, as indicated by the bars. After the first oxotremorine M exposure, either 1 μM staurosporine or 1 μM GF 109203 X was present. b Changes in membrane voltage (Δ mV) caused by these six oxotremorine M applications (O1–O6) in the presence of DMSO, staurosporine, GF 109203 X or XE 991 (3 μM); the values of these six depolarisations were normalized to the value of the first one (n = 6). *Significant difference between the four values at O6 (p < 0.05, Kruskal–Wallis test). c Current responses of one neuron that was clamped at a voltage of −30 mV and hyperpolarised to −55 mV once every 15 s and that has been exposed to 1 μM staurosporine. The traces were obtained before (solvent) and during (OxoM) the first application (O1) of 10 μM oxotremorine M as well as before (stauro) and during (stauro+OxoM) the sixth application (O6) of oxotremorine M. d Changes in Kv7 inhibition (quantified by deactivation current amplitudes) caused by these six oxotremorine M applications (O1–O6) in the presence of either DMSO, staurosporine or GF 109203 X; these six values of Kv7 inhibition were normalized to the value of the first one (n = 6–9)

Mentions: The activation of PKC contributes to the depolarisation of SCG neurons through B2 bradykinin receptors [34]. Therefore, various kinase inhibitors were tested for their effect on depolarisations triggered by 10 μM oxotremorine M (which was applied repeatedly once every 4 min; Fig. 2a, b). Staurosporine (1 μM), a broad-spectrum kinase inhibitor, increasingly reduced oxotremorine M-evoked depolarisations over a time period of 20 min (Fig. 2a, b): initial depolarisations amounted to 8.4 ± 1.1 mV; after 20 min of staurosporine exposure, this value had decreased to 4.0 ± 0.75 mV (n = 6, p < 0.01, Kruskal–Wallis test). An analogous effect was observed when the PKC inhibitor GF 109203 X (1 μM) was used instead (Fig. 2a, b). After 20 min of its presence, the extent of depolarisation caused by oxotremorine M had decreased from 6.4 ± 0.7 to 3.0 ± 0.6 mV (n = 6, p < 0.01, Kruskal–Wallis test). However, the solvent (0.1 % DMSO) did not cause significant changes when present as long as the kinase inhibitors, and the depolarisations amounted to 7.0 ± 0.9 mV in the beginning and to 6.9 ± 1.1 mV (n = 6, p > 0.1, Kruskal–Wallis test) 20 min later. For a comparison with PKC inhibitors, the effects of the Kv7 channel blocker XE 991 (3 μM) were investigated in an analogous manner: in its presence, the oxotremorine M-induced depolarisations decreased from 8.5 ± 1.0 to 3.8 ± 0.9 mV (n = 6, p < 0.05, Kruskal–Wallis test). When directly comparing the effects of staurosporine, GF 109203 X, XE 991 and DMSO by normalizing the oxotremorine M-induced depolarisations to the respective first value, the values after 20 min exposure to staurosporine, GF 109203 X or XE 991 were smaller than those after exposure to the solvent (Fig. 2b). Thus, the PKC inhibitors staurosporine and GF 109203 X significantly attenuated the depolarising action of the muscarinic agonist, as did the Kv7 channel blocker XE 991.Fig. 2


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 PKC inhibitors on 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. Oxotremorine M (OxoM, 10 μM) was present for six periods of 60 s each; these periods of oxotremorine M application were separated by 3-min intervals. From minute 2 after the first oxotremorine M application onward, PKC inhibitors, XE 991, or solvent (0.1 % DMSO) was present throughout the remaining measurements. a Time course of membrane voltage in two different SCG neurons during the first and the sixth exposure to oxotremorine M (OxoM, 10 μM); the agonist was present, as indicated by the bars. After the first oxotremorine M exposure, either 1 μM staurosporine or 1 μM GF 109203 X was present. b Changes in membrane voltage (Δ mV) caused by these six oxotremorine M applications (O1–O6) in the presence of DMSO, staurosporine, GF 109203 X or XE 991 (3 μM); the values of these six depolarisations were normalized to the value of the first one (n = 6). *Significant difference between the four values at O6 (p < 0.05, Kruskal–Wallis test). c Current responses of one neuron that was clamped at a voltage of −30 mV and hyperpolarised to −55 mV once every 15 s and that has been exposed to 1 μM staurosporine. The traces were obtained before (solvent) and during (OxoM) the first application (O1) of 10 μM oxotremorine M as well as before (stauro) and during (stauro+OxoM) the sixth application (O6) of oxotremorine M. d Changes in Kv7 inhibition (quantified by deactivation current amplitudes) caused by these six oxotremorine M applications (O1–O6) in the presence of either DMSO, staurosporine or GF 109203 X; these six values of Kv7 inhibition were normalized to the value of the first one (n = 6–9)
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Fig2: Effects of PKC inhibitors on 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. Oxotremorine M (OxoM, 10 μM) was present for six periods of 60 s each; these periods of oxotremorine M application were separated by 3-min intervals. From minute 2 after the first oxotremorine M application onward, PKC inhibitors, XE 991, or solvent (0.1 % DMSO) was present throughout the remaining measurements. a Time course of membrane voltage in two different SCG neurons during the first and the sixth exposure to oxotremorine M (OxoM, 10 μM); the agonist was present, as indicated by the bars. After the first oxotremorine M exposure, either 1 μM staurosporine or 1 μM GF 109203 X was present. b Changes in membrane voltage (Δ mV) caused by these six oxotremorine M applications (O1–O6) in the presence of DMSO, staurosporine, GF 109203 X or XE 991 (3 μM); the values of these six depolarisations were normalized to the value of the first one (n = 6). *Significant difference between the four values at O6 (p < 0.05, Kruskal–Wallis test). c Current responses of one neuron that was clamped at a voltage of −30 mV and hyperpolarised to −55 mV once every 15 s and that has been exposed to 1 μM staurosporine. The traces were obtained before (solvent) and during (OxoM) the first application (O1) of 10 μM oxotremorine M as well as before (stauro) and during (stauro+OxoM) the sixth application (O6) of oxotremorine M. d Changes in Kv7 inhibition (quantified by deactivation current amplitudes) caused by these six oxotremorine M applications (O1–O6) in the presence of either DMSO, staurosporine or GF 109203 X; these six values of Kv7 inhibition were normalized to the value of the first one (n = 6–9)
Mentions: The activation of PKC contributes to the depolarisation of SCG neurons through B2 bradykinin receptors [34]. Therefore, various kinase inhibitors were tested for their effect on depolarisations triggered by 10 μM oxotremorine M (which was applied repeatedly once every 4 min; Fig. 2a, b). Staurosporine (1 μM), a broad-spectrum kinase inhibitor, increasingly reduced oxotremorine M-evoked depolarisations over a time period of 20 min (Fig. 2a, b): initial depolarisations amounted to 8.4 ± 1.1 mV; after 20 min of staurosporine exposure, this value had decreased to 4.0 ± 0.75 mV (n = 6, p < 0.01, Kruskal–Wallis test). An analogous effect was observed when the PKC inhibitor GF 109203 X (1 μM) was used instead (Fig. 2a, b). After 20 min of its presence, the extent of depolarisation caused by oxotremorine M had decreased from 6.4 ± 0.7 to 3.0 ± 0.6 mV (n = 6, p < 0.01, Kruskal–Wallis test). However, the solvent (0.1 % DMSO) did not cause significant changes when present as long as the kinase inhibitors, and the depolarisations amounted to 7.0 ± 0.9 mV in the beginning and to 6.9 ± 1.1 mV (n = 6, p > 0.1, Kruskal–Wallis test) 20 min later. For a comparison with PKC inhibitors, the effects of the Kv7 channel blocker XE 991 (3 μM) were investigated in an analogous manner: in its presence, the oxotremorine M-induced depolarisations decreased from 8.5 ± 1.0 to 3.8 ± 0.9 mV (n = 6, p < 0.05, Kruskal–Wallis test). When directly comparing the effects of staurosporine, GF 109203 X, XE 991 and DMSO by normalizing the oxotremorine M-induced depolarisations to the respective first value, the values after 20 min exposure to staurosporine, GF 109203 X or XE 991 were smaller than those after exposure to the solvent (Fig. 2b). Thus, the PKC inhibitors staurosporine and GF 109203 X significantly attenuated the depolarising action of the muscarinic agonist, as did the Kv7 channel blocker XE 991.Fig. 2

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