Curtis T M, Scholfield C N
Physiology Department, Queens University, Belfast, United Kingdom.
Invest Ophthalmol Vis Sci. 2000 Jul;41(8):2279-85.
To characterize the effects of endothelin (ET)-1 on the Ca2+-activated Cl- conductance of choroidal arteriolar smooth muscle.
Microvascular smooth muscle cells were enzymatically isolated from choroidal arterioles from the eyes of freshly killed rabbits. Cells were voltage-clamped at -60 mV using the whole-cell perforated patch-clamp technique. Internal pipette solutions were K+ based and contained amphotericin B (200 microg/ml). The cells were bathed in a 20 mM tetraethyl-ammonium solution to block outward K+ currents.
Within 2 to 5 seconds of adding ET-1 (10 nM), inward current pulses were generated at a frequency of around 1 Hz. These evoked transient inward currents were blocked by niflumic acid (10 microM) or anthracene-9-carboxylic acid (1 mM). They were increased 2.4+/-0.1-fold when Cl- was replaced by I in the bathing medium and lost within 4 minutes when external Cl- was reduced from 151.6 to 20 mM. The reversal potential was -1+/-2 mV with 135 mM Cl- in the recording pipette and with 54 mM Cl it was -18+/-4 mV. When gramicidin D (100 microg/ml), which maintains [Cl-]i, was used instead of amphotericin B, the reversal potential was -18+/-1 mV. Ca2+ release by caffeine (10 mM) produced a single transient inward current. Endothelin-evoked transient inward currents were slowly reduced and eventually abolished in Ca2+-free solution (approximately 2 to 3 minutes) and were eliminated after approximately 30 seconds by the sarcoplasmic reticulum Ca2+-uptake inhibitor cyclopiazonic acid (5 microM). The ET(A) receptor antagonist BQ123 (1 microM) prevented an effect by endothelin but did not inhibit the current oscillations once they had been triggered.
In choroidal arteriolar smooth muscle ET-1 evokes transient inward Ca2+-activated Cl- currents induced through the cyclical release and re-uptake of Ca2+ from intracellular stores after ET(A) receptor stimulation.
研究内皮素（ET）-1对脉络膜小动脉平滑肌钙激活氯电导的影响。
采用酶解法从刚处死的兔眼脉络膜小动脉中分离微血管平滑肌细胞。使用全细胞穿孔膜片钳技术将细胞钳制在-60 mV电压。内电极液以钾离子为基础，含有两性霉素B（200 μg/ml）。细胞浸浴在20 mM四乙铵溶液中以阻断外向钾电流。
加入ET-1（10 nM）后2至5秒内，以约1 Hz的频率产生内向电流脉冲。这些诱发的瞬时内向电流被尼氟灭酸（10 μM）或蒽-9-羧酸（1 mM）阻断。当浸浴液中的氯离子被碘离子取代时，它们增加2.4±0.1倍，当细胞外氯离子从151.6 mM降至20 mM时，4分钟内消失。记录电极内为135 mM氯离子时，反转电位为-1±2 mV，54 mM氯离子时为-18±4 mV。当使用维持细胞内氯离子浓度的短杆菌肽D（100 μg/ml）代替两性霉素B时，反转电位为-18±1 mV。咖啡因（10 mM）引起的钙释放产生单个瞬时内向电流。内皮素诱发的瞬时内向电流在无钙溶液中缓慢降低并最终在约2至3分钟内消失，在约30秒后被肌浆网钙摄取抑制剂环匹阿尼酸（5 μM）消除。ET(A)受体拮抗剂BQ123（1 μM）可阻止内皮素的作用，但一旦触发电流振荡则不能抑制。
在脉络膜小动脉平滑肌中，ET-1通过ET(A)受体刺激后细胞内钙库中钙的周期性释放和再摄取，诱发瞬时内向钙激活氯电流。
