Endothelin-1 inhibits voltage-sensitive Ca²⁺ channels in cultured rat cerebellar granule neurones via the ET-A receptor

Abstract

 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 (I _Ba) by 85±5%. ET-1 decreased the open probability (NP _o) 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 µM), indicating a receptor-mediated effect. The ET-A receptor antagonist BQ-123 (10 µM) 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.