Urea derivatives as tools for studying the urea-facilitated transport system

Abstract

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.

Urea transport in kidney brush-border membrane vesicles from an elasmobranch, Raja erinacea

“…In BBMVs from the dorsal section, the urea analogue NPTU (80·µmol·l -1 ) significantly reduced urea uptake. This reduction is comparable to that seen in the frog urinary bladder (IC50=79.4·µmol·l -1 ; Martial et al, 1993), indicating a high specificity for this analogue. In the ventral section, acetamide elevated urea uptake above that of the ventral control and the dorsal BBMV in the presence of acetamide.…”

Ammonia and urea excretion in the tidepool sculpin (Oligocottus maculosus): sites of excretion, effects of reduced salinity and mechanisms of urea transport

“…We chose a somewhat lower concentration of DCPTU (0.25 mmol 1-) as the affinity of DCPTU for the urea transporter is known to be relatively high, for example, DCPTU has an inhibition constant (K I ) of 0.01 mmol -in red cell membranes (Mayrand and Levitt 1983). Although urea analogs, phloretin and DCPTU have not been used in aquatic experiments in previous studies, their effects on urea transport in isolated tissues have been shown to be reversible (Chou and Knepper 1989;Martial et al 1993). Jneturea, °Uturea, and Jnetamm were measured in an initial 2h control flux, a 2h experimental period (urea analogs (5.0 mmol N 1-1) i.e., similar to internal urea levels) or inhibitors (0.25 mmol 1-)), and a final 2h recovery period.…”

“…Phloretin (0.25 mmol 1-) blocks facilitated urea transport in mammalian red blood cells and kidney tubules (e.g., Brahm 1983;Chou and Knepper 1989). The inhibitor, DCPTU (0.5 mmol 1-) has been shown to reversibly inhibit urea flux in frog urinary bladder epithelia (Martial et al 1993). We chose a somewhat lower concentration of DCPTU (0.25 mmol 1-) as the affinity of DCPTU for the urea transporter is known to be relatively high, for example, DCPTU has an inhibition constant (K I ) of 0.01 mmol -in red cell membranes (Mayrand and Levitt 1983).…”

Photoaffinity labeling of the human red‐blood‐cell urea‐transporter polypeptide components

“…The control sample was tested as described in Martial et al [16]. For ureaefflux inhibition experiments, 250 pl of anti-Jk3 serum was added to 5 ml ghost suspension (108vesicles/ml).…”