Martial S, Neau P, Degeilh F, Lamotte H, Rousseau B, Ripoche P
Département de Biologie Cellulaire et Moléculaire, Centre d'Etudes de Saclay, Gif-sur-Yvette, France.
Pflugers Arch. 1993 Apr;423(1-2):51-8. doi: 10.1007/BF00374960.
The effects of urea structural analogues on the urea-facilitated diffusion system were examined in human red cell membranes (pink ghosts) and in antidiuretic hormone(ADH)-stimulated frog urinary bladder epithelia. In both tissues, urea permeability (P(urea)) was dramatically but reversibly inhibited by a number of urea analogues, such as 1-(3,4-dichlorophenyl)-2-thiourea (DCPTU). This urea derivative reduced the urea flux in a dose-dependent manner (90% inhibition of P(urea) at 0.5 mM concentration of DCPTU). With the aim of obtaining irreversible markers of red cell and urinary bladder urea transport systems, urea derivatives were modified by addition of an azido residue (N3) and preliminary experiments of photoaffinity labelling were carried out. Two synthetic urea derivatives: 1-(3-azido-4-chlorophenyl)-2-thiourea (ACPTU) and 1-(3-azido-4-chlorophenyl)-3-methyl-2-thiourea (Me-ACPTU) were shown to be very potent inhibitors of P(urea) when used in the absence of light, with IC50 values 60.3 microM and 31.6 microM respectively, as measured in frog urinary bladder. Both these molecules appeared to bind covalently to the urea carrier in both frog urinary bladder and human pink red cell ghosts, when illuminated in the presence of the tissue: the urea flux, which fell to 30-70% of the value obtained in the presence of ADH after inhibitor addition, remained low after the preparation had been illuminated for 30 min and the inhibitor removed. These results provide an interesting approach to the urea carrier analysis, particularly to the urea or urea analogue binding site on the transport protein.
在人红细胞膜（血影）和抗利尿激素（ADH）刺激的蛙膀胱上皮中研究了尿素结构类似物对尿素易化扩散系统的影响。在这两种组织中，许多尿素类似物，如1-（3,4-二氯苯基）-2-硫脲（DCPTU），可显著但可逆地抑制尿素通透性（P(urea)）。这种尿素衍生物以剂量依赖的方式降低尿素通量（在0.5 mM DCPTU浓度下对P(urea)的抑制率达90%）。为了获得红细胞和膀胱尿素转运系统的不可逆标记物，通过添加叠氮基（N3）对尿素衍生物进行修饰，并开展了光亲和标记的初步实验。两种合成尿素衍生物：1-（3-叠氮基-4-氯苯基）-2-硫脲（ACPTU）和1-（3-叠氮基-4-氯苯基）-3-甲基-2-硫脲（Me-ACPTU），在无光条件下使用时，显示出对P(urea)非常有效的抑制作用，在蛙膀胱中测得的IC50值分别为60.3 microM和31.6 microM。当在组织存在的情况下光照时，这两种分子似乎都与蛙膀胱和人血影中的尿素载体共价结合：在添加抑制剂后，尿素通量降至ADH存在时所获值的30 - 70%，在制剂光照30分钟并去除抑制剂后，尿素通量仍保持较低水平。这些结果为尿素载体分析，特别是对转运蛋白上的尿素或尿素类似物结合位点，提供了一种有趣的方法。
