Single cell photometry and whole-cell patch clamp recording were used to study caffeine-induced intracellular Ca2+ signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta. 2. Caffeine (5 mM) evoked a transient increase in [Ca2+]i in fura-2-loaded endothelial cells. Pretreatment of cells with 10 microM ryanodine did not alter resting [Ca2+]i but irreversibly inhibited the caffeine-induced rise in [Ca2+]i. The caffeine-induced increase in [Ca2+]i was not attenuated by the removal of extracellular Ca2+ and did not stimulate the rate of Mn2+ quench of fura-2 fluorescence. 3. Bath application of caffeine evoked a dose- and voltage-dependent outward current. The rate of onset and amplitude of the caffeine-evoked outward current increased with higher caffeine concentrations and membrane depolarization. The relationship between caffeine-evoked current amplitude and membrane potential was non linear, suggesting that the channels underlying the current are voltage-sensitive. 4. In the absence of extracellular Ca2+, the amplitude of the caffeine-evoked outward current was reduced by approximately 50% but the duration of the current was prolonged compared to that observed in the presence of external Ca2+. Ca(2+)-free external solutions produced an unexpected increase in both the frequency and amplitude of spontaneous transient outward currents (STOCs). 5. Inclusion of heparin (10 micrograms ml-1) in the patch pipette abolished the acetylcholine (ACh)-induced outward current but failed to inhibit either STOCs or the caffeine-evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, heparin did not affect either STOCs or the caffeine-induced outward current. 6. Externally applied tetraethylammonium ions (TEA, 3-10mM) reversibly inhibited unitary Ca2+-activated K+ currents and STOCs in endothelial cells but failed to inhibit completely the outward current evoked by 20 mM caffeine.7. Bath application of 0.1 mM zinc ion (Zn2+), a chloride channel blocker, did not affect unitary currents or STOCs but reduced the amplitude of the caffeine-evoked current by >75% compared to control. Replacement of extracellular NaCl with Na gluconate also reduced the amplitude of the caffeine-induced outward current. Bath application of 0.1 mM Zn2+ and 10 mM TEA completely blocked the caffeine-evoked outward current in endothelial cells.8. Caffeine-induced Ca2+ release from intracellular stores evokes a transient rise in [Ca2+1, which is correlated with a large, transient outward current. The ionic dependence and inhibition of the caffeine sensitive current by TEA and Zn2+ suggests that Ca2+-activated K+ and Cl- conductances contribute to the caffeine response in rabbit aortic endothelial cells.
摘要
采用单细胞光度测定法和全细胞膜片钳记录法，分别研究从兔主动脉新鲜分离的内皮细胞中咖啡因诱导的细胞内Ca2+信号和膜电流。2. 咖啡因（5 mM）可引起用fura-2负载的内皮细胞中[Ca2+]i的瞬时增加。用10 microM 兰尼碱预处理细胞不会改变静息[Ca2+]i，但会不可逆地抑制咖啡因诱导的[Ca2+]i升高。去除细胞外Ca2+不会减弱咖啡因诱导的[Ca2+]i增加，且不会刺激fura-2荧光的Mn2+淬灭速率。3. 浴槽施加咖啡因可引起剂量和电压依赖性外向电流。咖啡因诱发的外向电流的起始速率和幅度随咖啡因浓度升高和膜去极化而增加。咖啡因诱发电流幅度与膜电位之间的关系是非线性的，表明该电流所依赖的通道对电压敏感。4. 在无细胞外Ca2+的情况下，咖啡因诱发的外向电流幅度降低约50%，但与存在细胞外Ca2+时相比，电流持续时间延长。无Ca2+的细胞外溶液使自发性瞬时外向电流（STOCs）的频率和幅度意外增加。5. 膜片钳微管中加入肝素（10微克/毫升）可消除乙酰胆碱（ACh）诱导的外向电流，但不能抑制天然内皮细胞中的STOCs或咖啡因诱发的外向电流。在无细胞外Ca2+的情况下，肝素对STOCs或咖啡因诱导的外向电流均无影响。6. 外部施加四乙铵离子（TEA，3 - 10 mM）可可逆性抑制内皮细胞中的单通道Ca2+激活K+电流和STOCs，但不能完全抑制20 mM咖啡因诱发的外向电流。7. 浴槽施加0.1 mM锌离子（Zn2+），一种氯离子通道阻滞剂，不影响单通道电流或STOCs，但与对照相比，可使咖啡因诱发电流的幅度降低>75%。用葡萄糖酸钠替代细胞外NaCl也会降低咖啡因诱导的外向电流幅度。浴槽施加0.1 mM Zn2+和10 mM TEA可完全阻断内皮细胞中咖啡因诱发的外向电流。8. 咖啡因诱导细胞内储存库释放Ca2+会引起[Ca2+]i的瞬时升高，这与一个大的、瞬时外向电流相关。TEA和Zn2+对咖啡因敏感电流的离子依赖性和抑制作用表明，Ca2+激活的K+和Cl-电导参与了兔主动脉内皮细胞对咖啡因的反应。
