Wiener H, van Os C H
Department of Pharmacology, University of Vienna, Austria.
J Membr Biol. 1989 Sep;110(2):163-74. doi: 10.1007/BF01869471.
Loop diuretic-sensitive (Na+,K+,Cl-)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of gradients of all three ions was essential for optimal transport activity. (Na+,K+) gradient-driven 36Cl fluxes were half-maximally inhibited by 0.14 microM bumetanide and 44 microM furosemide. While 86Rb uptake rates showed hyperbolic dependencies on Na+ and K+ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl concentration, Hill coefficient 1.8, indicating a 1 Na+:1 K+:2 Cl stoichiometry of ion transport. The interaction of putative (Na+,K+,Cl-)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [3H]-bumetanide. The requirement for the simultaneous presence of Na+,K+, and Cl-, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na+,K+,Cl-)-cotransporter. [3H]-bumetanide recognizes a minimum of two classes of diuretic receptor sites, high-affinity (KD1 = 0.13 microM; Bmax1 = 6.4 pmol/mg of protein) and low-affinity (KD2 = 34 microM; Bmax2 = 153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl- (Ki = 60 mM), whereas low-affinity sites seem to be unaffected by Cl-. We have shown that only high-affinity [3H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.
发现兔远端结肠上皮表面细胞和隐窝细胞的基底外侧膜囊泡中存在对袢利尿剂敏感的（Na⁺，K⁺，Cl⁻）协同转运活性。所有三种离子梯度的存在对于最佳转运活性至关重要。（Na⁺，K⁺）梯度驱动的³⁶Cl通量受到0.14微摩尔布美他尼和44微摩尔呋塞米的半数最大抑制。虽然⁸⁶Rb摄取率对Na⁺和K⁺浓度呈双曲线依赖性，希尔系数分别为0.8和0.9，但摄取与Cl⁻浓度呈S形相关，希尔系数为1.8，表明离子转运的化学计量比为1 Na⁺:1 K⁺:2 Cl⁻。使用[³H] - 布美他尼通过平衡结合实验研究了假定的（Na⁺，K⁺，Cl⁻）协同转运蛋白与袢利尿剂的相互作用。对Na⁺、K⁺和Cl⁻同时存在的需求、饱和性、可逆性以及对利尿剂的特异性表明与（Na⁺，K⁺，Cl⁻）协同转运体存在特异性结合。[³H] - 布美他尼识别至少两类利尿剂受体位点，即高亲和力（KD1 = 0.13微摩尔；Bmax1 = 6.4皮摩尔/毫克蛋白质）和低亲和力（KD2 = 34微摩尔；Bmax2 = 153皮摩尔/毫克蛋白质）位点。发现与高亲和力受体的特异性结合与Cl⁻呈线性竞争（Ki = 60毫摩尔），而低亲和力位点似乎不受Cl⁻影响。我们已经表明，只有高亲和力的[³H] - 布美他尼结合与转运抑制相关，这引发了关于在兔远端结肠上皮中观察到的利尿剂受体位点异质性的生理意义的问题。
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