Held B, Pocock J M, Pearson H A
Department of Pharmacology, University of Leeds, Leeds LS2 9JT, UK.
Pflugers Arch. 1998 Oct;436(5):766-75. doi: 10.1007/s004240050700.
In this study, we have investigated the effect of the vasoconstrictor peptide endothelin-1 (ET-1) on voltage-sensitive Ca2+ channels in rat cerebellar granule neurones using the patch-clamp technique. Using amphotericin B perforated-patch recording of whole-cell currents, the Ca2+ channel current was inhibited by 28.4+/-6.4% by 400 nM ET-1, but was unaffected when experiments were repeated using the whole-cell, ruptured-patch configuration. In cell-attached patches, 400 nM ET-1 inhibited unitary L-type Ca2+ channel currents (IBa) by 85+/-5%. ET-1 decreased the open probability (NPo) and the frequency of channel opening and increased the mean closed time of channels. No effects on the mean open time or the time constants for channel opening or closure were observed. L-type Ca2+ channel inhibition was dose dependent with an IC50 of 19 nM. The effect of ET-1 was prevented by the combined endothelin-A and -B receptor antagonist PD145065 (10 microM), indicating a receptor-mediated effect. The ET-A receptor antagonist BQ-123 (10 microM) prevented Ca2+ channel inhibition by ET-1, while the ET-B receptor agonist sarafotoxin 6c (500 nM) had no effect. The inhibition by ET-1 was not due to a change in the voltage of channel activation. Fura-2 Ca2+ imaging showed that no substantial rise in intracellular Ca2+ levels occurred during ET-1 application excluding a Ca2+-dependent inhibition of the channels. Thus in cultured rat cerebellar granule neurones, ET-1 inhibits L-type Ca2+ channels via activation of the ET-A receptor. Inhibition may be mediated by an as yet unidentified cytoplasmic second messenger.
在本研究中，我们使用膜片钳技术研究了血管收缩肽内皮素 -1（ET -1）对大鼠小脑颗粒神经元中电压敏感性Ca2+通道的影响。采用两性霉素B穿孔膜片全细胞电流记录法，400 nM ET -1可使Ca2+通道电流抑制28.4±6.4%，但当采用全细胞破裂膜片记录法重复实验时，该电流未受影响。在细胞贴附式膜片中，400 nM ET -1可使单一L型Ca2+通道电流（IBa）抑制85±5%。ET -1降低了开放概率（NPo）和通道开放频率，并增加了通道的平均关闭时间。未观察到对平均开放时间或通道开放或关闭时间常数的影响。L型Ca2+通道抑制呈剂量依赖性，IC50为19 nM。内皮素A和B受体联合拮抗剂PD145065（10 μM）可阻止ET -1的作用，表明这是一种受体介导的效应。ET -A受体拮抗剂BQ -123（10 μM）可阻止ET -1对Ca2+通道的抑制作用，而ET -B受体激动剂沙拉毒素6c（500 nM）则无作用。ET -1的抑制作用并非由于通道激活电压的改变。Fura -2 Ca2+成像显示，在应用ET -1期间细胞内Ca2+水平未出现显著升高，排除了Ca2+依赖性通道抑制。因此，在培养的大鼠小脑颗粒神经元中，ET -1通过激活ET -A受体抑制L型Ca2+通道。抑制作用可能由一种尚未明确的胞质第二信使介导。
