Meyer G, Doppierio S, Vallin P, Daffonchio L
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
Eur Respir J. 1996 Dec;9(12):2461-7. doi: 10.1183/09031936.96.09122461.
Frusemide can be used as an antiasthma drug and appears to inhibit the release (conditioned by activation of Cl- channels) of mast cell proinflammatory mediators. We studied the cause of the effects of frusemide, checking its action on Cl- channels. The patch-clamp technique was used to study single-channel currents, and differences in electrical potential of the cellular membrane of rat peritoneal mast cells were measured. In inside-out configuration, outwardly-rectifying Cl- channels were identified whose conductance was 2.4/1.7 pS at positive and negative voltages. In cell-attached configuration, the open probability (Po) of the channel increased with depolarization or with the presence of cyclic adenosine monophosphate (cAMP) in the incubation medium. Po increased with a rise of cytoplasmic free calcium concentration [Ca2+] and was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and by 4-4'-diisothiocyanatoostilbene-2-2'-disulphonic acid (DIDS). These channels seem to be the main cause of mast cell Cl- conductance. Frusemide (10(-5) and 10(-3) M) did not affect Cl- channel activity when using excised patches. In cell-attached configuration experiments, the presence of frusemide (from 10(-5) to 10(-3) M) in the cell incubation medium, increasingly reduced Po (median inhibitory concentration (IC50) = 4.3 x 10(-7) M). In similar conditions, bumetanide also inhibited Po (IC50 = 5.7 x 10(-3) M). The results of this study suggest that frusemide can inhibit mast cell Cl- channels only via an indirect mechanisms, which probably involves an inhibition of a Na(+)-K(+)-2Cl- symport.
速尿可用作抗哮喘药物，似乎能抑制肥大细胞促炎介质的释放（由氯离子通道激活所调节）。我们研究了速尿产生作用的原因，检测了其对氯离子通道的作用。采用膜片钳技术研究单通道电流，并测量大鼠腹膜肥大细胞膜电位的差异。在内面向外模式下，鉴定出外向整流氯离子通道，其在正电压和负电压下的电导分别为2.4/1.7 pS。在细胞贴附模式下，通道的开放概率（Po）随去极化或孵育介质中存在环磷酸腺苷（cAMP）而增加。Po随细胞质游离钙浓度[Ca2+]升高而增加，并受到5-硝基-2-（3-苯丙基氨基）-苯甲酸酯（NPPB）和4,4'-二异硫氰酸根合芪-2,2'-二磺酸（DIDS）的抑制。这些通道似乎是肥大细胞氯离子电导的主要原因。使用切除的膜片时，速尿（10^(-5)和10^(-3) M）不影响氯离子通道活性。在细胞贴附模式实验中，细胞孵育介质中存在速尿（10^(-5)至10^(-3) M）时，Po逐渐降低（半数抑制浓度（IC50）= 4.3×10^(-7) M）。在类似条件下，布美他尼也抑制Po（IC50 = 5.7×10^(-3) M）。本研究结果表明，速尿仅通过间接机制抑制肥大细胞氯离子通道，这可能涉及对钠-钾-2氯同向转运体的抑制。
