Muscarinic receptor activation modulates Ca2+ channels in rat intracardiac neurons via a PTX- and voltage-sensitive pathway
Muscarinic receptor activation modulates Ca2+ channels in rat intracardiac neurons via a PTX- and voltage-sensitive pathway
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DOI：
10.1152/jn.1997.78.3.1476
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发表时间：
1997-09-01
影响因子：
2.5
通讯作者：
Wurster, RD
中科院分区：
文献类型：
--
作者：
Jeong, SW;Wurster, RD
采用全细胞膜片钳技术，研究了毒蕈碱激动剂甲基碘化氧震颤碱（oxotremorine methiodide，oxo-M）对急性分离的成年大鼠心内神经元电压激活钙通道电流的影响。在所有测试的神经元oxo-M可逆地抑制峰值Ba 2+电流。oxo-M对Ba峰电流的抑制与激活动力学的减慢有关，并且具有浓度依赖性。产生电流的半最大抑制和最大抑制所需的oxo-M浓度分别为40.8 nM和75.9%。阿托品可完全阻断oxo-M的抑制作用。在不同的毒蕈碱受体拮抗剂，甲氧四明（100和300纳米）显着拮抗电流抑制oxo-M，在成年大鼠心内神经元的解离常数为8.3的负对数。用鸟苷5 '-（硫代）三磷酸（GTP γ S，0.5 mM）内透析神经元可模拟毒蕈碱对Ba ~（2+）电流峰值的抑制，并显著阻断oxo-M的抑制作用。此外，鸟苷-5 '-O-（2-硫代二磷酸）（GDP β S，2 mM）的内透析也显著降低了oxo-M对峰Ba 2+电流的毒蕈碱抑制。百日咳毒素（PTX，200和400 ng/ml），但不是由霍乱毒素（400 ng/ml）oxo-M的抑制作用显着取消。此外。N-乙基马来酰亚胺（50 μ M）的浴施加显著降低了oxo-M对峰值Ba 2+电流的抑制。oxo-M使从尾电流测量得出的激活曲线向更正的电位移动。一个强的条件预脉冲，以+100 mV显着缓解毒蕈碱抑制峰Ba 2+电流的oxo-M和GTP γ S诱导的电流抑制。在一系列实验中，双-（邻氨基苯氧基）-N，N，N '，N'-四乙酸的细胞内浓度和蛋白激酶活性的变化未能模拟或闭塞oxo-M的电流抑制。二氢吡啶拮抗剂硝苯地平（10 μ M）不能阻断oxo-M的任何抑制作用，oxo-M（3 μ M）不能降低由L型激动剂甲基2，5-二甲基-4-[2-（苯甲基）苯甲酰基]-1H-吡咯-3-羧酸酯（FPL 64176; 2 μ M）诱导的慢尾电流。然而，ω-芋螺毒素（ω-CgTX）GVIA（1 μ M）显着闭塞的毒蕈碱抑制的Ba 2+电流。在ω-CgTX GVIA（1 μ M）和硝苯地平（10 μ M）存在下，oxo-M可进一步抑制。相当于总Ca 2+电流的20%。在使用ω-CgTX GVIA、ω-蛇毒素IVA和硝苯地平完全去除N-、Q-和L-型电流后，70%的R-型电流（类似于总电流的6-7%）被oxo-M（3 μ M）抑制。总之，M-2毒蕈碱受体激活选择性地抑制N-，Q-和R-型钙通道电流，保留L-型钙通道电流主要通过PTX和电压敏感的途径在成年大鼠心内神经元。
With use of the whole cell patch-clamp technique, effects of the potent muscarinic agonist oxotremorine methiodide (oxo-M) on voltage-activated Ca2+ channel currents were investigated in acutely dissociated adult rat intracardiac neurons. In all tested neurons oxo-M reversibly inhibited the peak Ba2+ current. Inhibition of the peak Ba current by oxo-M was associated with slowing of activation kinetics and was concentration dependent. The concentration of oxo-M necessary to produce a half-maximal inhibition of current and the maximal inhibition were 40.8 nM and 75.9%, respectively. Inhibitory effect of oxo-M was completely abolished by atropine. Among different muscarinic receptor antagonists, methoctramine (100 and 300 nM) significantly antagonized the current inhibition by oxo-M, with a negative logarithm of dissociation constant of 8.3 in adult rat intracardiac neurons. Internal dialysis of neurons with guanosine 5'-(thio) triphosphate (GTP gamma S, 0.5 mM) could mimic the muscarinic inhibition of the peak Ba2+ current and significantly occlude inhibitory effects of oxo-M. In addition, the internal dialysis of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S, 2 mM) also significantly reduced the muscarinic inhibition of the peak Ba2+ current by oxo-M. Inhibitory effects of oxo-M were significantly abolished by pertussis toxin (PTX, 200 and 400 ng/ml) but not by cholera toxin (400 ng/ml). Furthermore. the bath application of N-ethylmaleimide (50 mu M) significantly reduced the inhibition of the peak Ba2+ current by oxo-M. The oxo-M shifted the activation curve derived from measurments of tail currents toward more positive potentials. A strong conditioning prepulse to +100 mV significantly relieved the muscarinic inhibition of peak Ba2+ currents by oxo-M and the GTP gamma S-induced current inhibition. In a series of experiments, changes in intracellular concentration of bis-(o-aminophenoxy) -N,N,N',N'-tetraacetic acid and protein kinase activities failed to mimic or occlude the current inhibition by oxo-M. The dihydropyridine antagonist nifedipine (10 mu M) was not able to occlude any of the inhibitory effects of oxo-M, and oxo-M (3 mu M) failed to reduce the slow tail currents induced by the L-type agonist methyl 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate (FPL 64176; 2 mu M). However, omega-conotoxin (omega-CgTX) GVIA (1 mu M) significantly occluded the muscarinic inhibition of the Ba2+ currents. In the presence of omega-CgTX GVIA (1 mu M) and nifedipine (10 mu M), oxo-M could further inhibit. similar to 20% of the total Ca2+ current. After complete removal of N-, Q-, and L-type currents with use of omega-CgTX GVIA, omega-agatoxin IVA, and nifedipine, 70% of the R-type current (similar to 6-7% of the total current) was inhibited by oxo-M (3 mu M). In conclusion, the M-2 muscarinic receptor activation selectively inhibits N-, Q-, and R-type Ca2+ channel currents, sparing L-type Ca2+ channel currents mainly via a PTX-and voltage-sensitive pathway in adult rat intracardiac neurons.