Functional asymmetry of the urea transporter UT-B in human red blood cells.

We studied urea, thiourea, and methylurea transport and interaction in human red blood cells (RBCs) under conditions of self-exchange (SE), net efflux (NE), and net influx (NI) at pH 7.2. We combined four methods, a four-centrifuge technique, the Millipore-Swinnex filtering technique, the continuous flow tube method, and a continuous pump method to measure the transport of the 14C-labeled compounds. Under SE conditions, both urea and thiourea show perfect Michaelis-Menten kinetics with half-saturation constants, K½,SE (mM), of ≈300 (urea) and ≈20 (thiourea). The solutes show no concentration-dependent saturation under NE conditions. Under NI conditions, transport displays saturation or self-inhibition kinetics with a K½,NI (mM) of ≈210 (urea) and ≈20 (thiourea). Urea, thiourea, and methylurea are competitive inhibitors of the transport of analog solutes. This study supports the hypothesis that the three compounds share the same urea transport system (UT-B). UT-B functions asymmetrically as it saturates from the outside only under SE and NI conditions, whereas it functions as a high-capacity channel-like transporter under NE conditions. When the red blood cell enters the urea-rich kidney tissue, self-inhibition reduces the urea uptake in the cell. When the cell leaves the kidney, the channel-like function of UT-B implies that intracellular urea rapidly equilibrates with external urea. The net result is that the cell during the passage in the kidney capillaries carries urea to the kidney to be excreted while the urea transfer from the kidney via the bloodstream is minimized.NEW & NOTEWORTHY The kinetics of urea transport in red blood cells was determined by means of a combination of four methods that ensures a high time resolution. In the present study, we disclose that the urea transporter UT-B functions highly asymmetric being channel-like with no saturation under conditions of net efflux and saturable under conditions of net influx and self-exchange in the concentration range 1-1,000 mM (pH 7.2 and 25-38 °C).

Urea transport in human red blood cells: Donor variation compared to chloride, glucose, and water transport.

We determined the permeability (P, cm/s) of unmodified human red blood cells (HRBC) to urea (Pu), chloride (PCl), glucose (Pglu), and water diffusion (Pd) under conditions of self-exchange (SE) with the continuous flow tube method at pH 7.2, 25°C. Among 24 donors, Pu at 1 mM varied >100%. Two of the donors were also tested in 1983. Their Pu had decreased by 77 and 90%. High age in males and Kidd genotype Jk(a+,b+), but not blood types AB0, appear related to low Pu. For one of the two donors, PCl (150 mM, 38°C, pH 7.2), Pglu (1 mM, 38°C, pH 7.2), and Pd (55.5 M, 25°C, pH 7.2) were determined then and now and showed no significant changes with age. The results from six more donors show donor PCl, Pglu, and Pd in the range of ≈1%. PCl and Pglu are vital for the metabolism of cells and tissues, and we see but little donor variation, and so far, no phenotypes without glucose (GLUT1) and anion (AE1) transporters in HRBC. Phenotypes with no urea transporter (UT-B) or no water transporters (aquaporin, AQP1) are registered and are compatible with life. Our results are in line with the concept that the solutes do not share pathways in common. The great donor variation in Pu must be considered in comparative transport physiological studies.

Urea and water are transported through different pathways in the red blood cell membrane.

Several studies of the urea transporter UT-B expressed in Xenopus oocytes and in genetically modified red blood cells (RBC) have concluded that UT-B also transports water. In the present study, we use unmodified RBC to test that conclusion. We find that the permeability of urea, Pu (cm/s), has a 10-fold donor variation, while the diffusional water permeability, Pd (cm/s), remains unchanged. Additionally, we observe that phloretin inhibits Pu but not Pd, and that the time course of maximum p-chloromercuribenzosulfonate inhibition of Pu and Pd differs-Pu inhibition takes <2 min, whereas Pd inhibition requires ≥1 h of incubation. The findings in the present study are in line with a previous comparative study using unmodified RBC from four animals and a solvent drag study using human RBC, and they lead us to reject the conclusion that the UT-B transporter represents a common pathway for both solutes.