Salzer Isabella, Gafar Hend, Gindl Viola, Mahlknecht Peter, Drobny Helmut, Boehm Stefan
Department of Neurophysiology and Neuropharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, 1090, Vienna, Austria.
Pflugers Arch. 2014 Dec;466(12):2289-303. doi: 10.1007/s00424-014-1487-1. Epub 2014 Mar 26.
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.
已知自主神经节中缓慢的胆碱能传递是通过M1毒蕈碱型乙酰胆碱受体对Kv7通道的抑制来介导的。然而，在本实验中，使用大鼠颈上神经节神经元的原代培养物，M1受体激动剂氧化震颤素M引起的去极化程度与同一神经元中Kv7通道的抑制程度并不相关。这一观察结果引发了对其他机制的探索。由于M1受体的激活会导致交感神经元中蛋白激酶C（PKC）活性增强，因此通过不同方式抑制了各种PKC酶。干扰经典PKC亚型会导致氧化震颤素M引起的去极化和去甲肾上腺素释放减少，但毒蕈碱激动剂对Kv7通道的抑制作用不变。当细胞外或细胞内Cl(-)浓度改变时，M1受体诱导的去极化也会改变，对γ-氨基丁酸的去极化反应也是如此。氧化震颤素M引发的去极化和去甲肾上腺素释放被非选择性Cl(-)通道阻滞剂4-乙酰氨基-4'-异硫氰酸基芪-2,2'-二磺酸和氟灭酸降低。氧化震颤素M在负膜电位下诱导缓慢上升的内向电流，该电流被Ca(2+)激活的Cl(-)通道和TMEM16A通道的抑制剂阻断，并被PKC抑制剂减弱。这些通道阻滞剂也减少了氧化震颤素M诱发的去甲肾上腺素释放。总之，这些结果表明，交感神经元的缓慢胆碱能兴奋除了众所周知的对Kv7通道的抑制外，还涉及经典PKC和Ca(2+)激活的Cl(-)通道的激活。
