The conventional whole-cell recording technique was used to study the effects of the chloride channel inhibitors ethacrynic acid, anthracene-9-carboxylic acid (A-9-C) and indanyloxyacetic acid (IAA) on membrane currents in rabbit portal vein smooth muscle cells at a holding potential of 0 mV. 2. Using a pipette solution that contained 1 x 10(-4) M 1,2-bis (2-aminophenoxy)-ethane-N,N,N,N,-tetraacetic acid (BAPTA) and a normal bathing solution the addition of ethacrynic acid (2 x 10(-4) M to 1 x 10(-3) M) inhibited spontaneous transient outward currents (STOCs) and evoked a concentration-dependent current at a holding potential of 0 mV. A similar current was activated by IAA (5 x 10(-4) M to 1 x 10(-3) M) but not by A-9-C (1-5 x 10(-3) M) at a holding potential of 0 mV. 3. The amplitude of the current evoked by ethacrynic acid and IAA was linearly related to potential between -30 and 0 mV and displayed outward rectification at positive potentials. The current induced by A-9-C was evident only at potentials positive to +20 mV. 4. Glibenclamide (1 x 10(-5) M) abolished the current evoked by ethacrynic acid and IAA at potentials negative to +10 mV and partially inhibited the current positive to +10 mV. The glibenclamide-insensitive current at positive potentials was completely inhibited by 1 x 10(-3) M TEA. The A-9-C-evoked current was insensitive to glibenclamide and abolished by 1 x 10(-3) M TEA. 5. The glibenclamide-sensitive current activated by ethacrynic acid was not sustained and declined to control levels in the continued presence of ethacrynic acid. However, the outwardly rectifying current recorded at +50 mV was well maintained over the same period. 6. Outwardly rectifying currents evoked by ethacrynic acid and A-9-C were observed with a pipette solution containing 1 x 10(-2) M BAPTA in cells bathed in Ca-free extracellular solution containing 5 x 10(-4) M BAPTA and 1 x 10(-5) M cyclopiazonic acid. 7. It is concluded that all three chloride-channel blockers activated an outwardly rectifying, TEA-sensitive current. Moreover, ethacrynic acid and IAA evoked an additional glibenclamide-sensitive current which was present at all potentials between -30 and +50 mV.
摘要
采用传统的全细胞记录技术,在0 mV的钳制电位下,研究氯通道抑制剂依他尼酸、蒽-9-羧酸(A-9-C)和茚满氧基乙酸(IAA)对兔门静脉平滑肌细胞膜电流的影响。2. 使用含有1×10⁻⁴ M 1,2-双(2-氨基苯氧基)乙烷-N,N,N,N-四乙酸(BAPTA)的微电极内液和正常的浴液,加入依他尼酸(2×10⁻⁴ M至1×10⁻³ M)可抑制自发性瞬时外向电流(STOCs),并在0 mV的钳制电位下诱发浓度依赖性电流。在0 mV的钳制电位下,IAA(5×10⁻⁴ M至1×10⁻³ M)可激活类似电流,而A-9-C(1 - 5×10⁻³ M)则不能。3. 依他尼酸和IAA诱发的电流幅度在-30至0 mV之间与电位呈线性关系,在正电位时表现出外向整流。A-9-C诱发的电流仅在正于+20 mV的电位时明显。4. 格列本脲(1×10⁻⁵ M)在+10 mV以下的电位时消除依他尼酸和IAA诱发的电流,并在+10 mV以上的电位时部分抑制电流。正电位时对格列本脲不敏感的电流被1×10⁻³ M四乙铵(TEA)完全抑制。A-9-C诱发的电流对格列本脲不敏感,并被1×10⁻³ M TEA消除。5. 依他尼酸激活的对格列本脲敏感的电流不能持续,在依他尼酸持续存在的情况下下降至对照水平。然而,在+50 mV记录的外向整流电流在同一时期内保持良好。6. 在含有5×10⁻⁴ M BAPTA和1×10⁻⁵ M环匹阿尼酸的无钙细胞外溶液中孵育的细胞,使用含有1×10⁻² M BAPTA的微电极内液时,观察到依他尼酸和A-9-C诱发的外向整流电流。7. 得出结论,所有三种氯通道阻滞剂均激活外向整流、对TEA敏感的电流。此外,依他尼酸和IAA诱发了一种额外的对格列本脲敏感的电流,该电流存在于-30至+50 mV之间的所有电位。