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.
The tritiated urea analogue 1-(3-azido-4-chlorophenyl)-3methyl-2thiourea (rH]MeACPTU) was used as a probe to photolabel the human red-blood-cell membrane facilitated urea transporter. On irradiation, [3H]MeACPTU incorporated irreversibly into white ghost membranes. SDS/gel electrophoresis of membranes revealed radioactive incorporation in five major bands of 200, 110, 60, 40 and 14 kDa. The labeling of the 40-kDa and 60-kDa bands was partly prevented by the presence of a high concentration of other urea analogues such as thiourea and 1-(3,4-dichlorophenyl) 2-thiourea (DCPTU). The photolabeling pattern obtained with white ghosts of the Kidd blood-group type Jk(a-,b-) showed no labeling of the 40-kDa polypeptide. Protecting experiments carried out with anti-Jka, anti-Jkb and anti-Jk3 sera prevented radioactive incorporation in the 60-kDa band and in the 110-kDa band. Urea permeability of pink ghosts of blood type Jk(a+,b+) measured in the presence of Jk3 antibodies was 19% lower than the control values. However, urea permeability of frog urinary bladder epithelial cells was not affected by the presence of Jk-reactive antibodies.These results support the hypothesis that the Kidd antigen and the facilitated urea transporter are the same protein. Our estimation of the number of copies in each cell is close to that of the previously published value of 14000.Urea movements in mammalian kidney play an important role in facilitating hypertonic urine formation and Rich eta]. [4] showed that swelling rates of red blood cells in urea solutions exhibited saturation kinetics, supporting the idea that urea was not transported by simple diffusion. It is now well established that urea crosses the plasma membrane of cells such as mammalian red blood cells, mammalian kidney cells and amphibian urinary bladder epithelial cells by a facilitated diffusion mechanism [5-71 involving a membrane protein which acts as a channel or carrier. Since we do not definitely know whether urea transport is camed out by a channel or by a carrier, we decided for homogeneity in this study to call the protein facilitating the urea movement across the membrane, a channel. This protein has not yet been identified although its heterologous expression has been obtained in Xenopus oocytes [8][9]. However, Gargus et al. [lo] suggested that the Kidd antigen and the urea channel could be one and the same protein in view of the co-segregation of the two pheno- Abbreviations.Me-ACPTU, 1-(3-azido-4-chlorophenyl)-?methyl-2-thiourea; DCPTU, 1-(3,4-dichlorophenyl) 2-thiourea; GSH, glutathione; IC,,,, inhibition constant 50% ; J,,,%, unidirectional mucosal to serosal flux; P,,,,, urea permeability; Rh, Rhesus.types: absence of human erythrocyte Kidd antigen and deficiency in urea transport. To gain insight into the molecular characterization of the urea channel, we developed an approach which consists of photolabeling the molecule. This method, described by Bayley and Knowles [ l l ] has permited the characterization of numerous proteins e.g. receptors, transporte...
Summary.Starting from commercial 4-chloro-3-nitroaniline, through a 5 step synthesis, was prepared 3-azido-4-chlorophenylisothiocyanate 5 which was reacted with [3H]-methylamine. The latter was obtained by three methods : 29 CUmmol). Compound 7 at the highest specific activity had a self radiolysis rate precluding its practical use in biological studies whilst 29 Cifmmol "H1-7 was satisfactory.
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