Passive urea transport
Figure 1A illustrates the results from an experiment measuring the urea uptake in the absence of αMDG in the bathing medium by control and rbSGLT1-expressing oocytes. Urea uptake in oocytes expressing rbSGLT1 (7.6 ± 0.5 pmol oocyte−1 (30 min)−1) was 4-fold higher than in control oocytes (1.7 ± 0.03 pmol oocyte−1 (30 min)−1). Urea uptake in both control and SGLT1-expressing oocytes was linear with the time of incubation (5–120 min), and the rate of uptake was linear with urea concentration from 10 μm to 100 mm. In one experiment the uptake at 55 μm and 100 mm urea was 3.1 ± 0.2 and 5501 ± 65 pmol oocyte−1 h−1, respectively, in control oocytes (n= 4 oocytes), and 13.3 ± 1.3 and 25 500 ± 1000 pmol oocyte−1 h−1, respectively, in rbSGLT1-expressing oocytes (n= 6). The rate of urea uptake in control oocytes corresponds to a urea permeability, Purea, of 4 × 10−8 cm s−1. This is comparable to the urea permeabilities reported by others (e.g. Zhang & Verkman, 1991) after correcting for the increase in the oocyte plasma membrane area due to folding (Zampighi et al. 1995). Thus, the urea permeability due to rbSGLT1, which was obtained by subtraction of the background urea permeability of control oocytes from the total urea permeability of rbSGLT1-expressing oocytes, was 1.3 × 10−7 cm s−1. The increase in urea permeability was directly proportional to the level of SGLT1 expression. The level of expression was gauged in six oocytes by the magnitude of the maximum Na+-glucose current (Mackenzie et al. 1998) immediately before measuring the radioactive urea tracer uptake, and the urea flux (pmol oocyte−1 (30 min)−1) was directly proportional to the Na+-glucose current (nA) over a 3-fold range (regression coefficient, 0.7).
Figure 1. Passive urea transport by rbSGLT1
A, 50 μm[14C]αMDG and 55 μm[14C]urea uptake in control and rabbit SGLT1 (rbSGLT1)-expressing oocytes. Oocytes obtained from the same frog were either injected with 50 ng of rbSGLT1 cRNA or not injected (Control). Uptake was studied in oocytes after 5 days incubation in Barth's medium. For transport experiments, oocytes were incubated in 100 mm NaCl and transport of sugar or urea was measured as a function of the amount (in pmol) per oocyte per 30 min. Each bar in the graph represents the mean αMDG or urea uptake by 4–5 oocytes. Left, ∼250 pmol of αMDG is transported by rbSGLT1-expressing oocytes, 65-fold higher than by control oocytes. Right, ∼8 pmol of urea is transported by rbSGLT1-expressing oocytes, 4-fold higher than by control oocytes. B, inhibition of rbSGLT1 urea transport by phlorizin and phloretin. Uptake of 55 μm[14C]urea was studied after 6 days incubation in non-injected oocytes and in rbSGLT1-injected oocytes. Urea flux was measured in the presence of 100 mm NaCl buffer and in the absence of sugar. Uptake was calculated as the amount of urea transported (in pmol) per oocyte per 30 min. Each bar represents the mean urea uptake by 4–6 rbSGLT1 oocytes from the same batch either in the absence (Control) or in the presence of phlorizin or phloretin. All values have been corrected for endogenous urea uptake. Addition of 1 or 50 μm phlorizin or 1 mm phloretin to the transport buffer had no effect on non-injected oocytes. In rbSGLT1 oocytes, the presence of 1 μm phlorizin in the transport buffer resulted in a 50% decrease in urea transport. Addition of 50 μm phlorizin further inhibited urea transport to ∼20% of that by control oocytes. Identical experiments performed with 1 mm phloretin also reduced rbSGLT1 urea uptake by ∼35% compared to control.
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The rate of αMDG uptake (246 ± 9 pmol oocyte−1 (30 min)−1) in the same batch of oocytes as shown in Fig. 1A was 65-fold higher than in control oocytes (3.8 ± 0.2 pmol oocyte−1 (30 min)−1), corresponding to values obtained previously (Hediger et al. 1987; Ikeda et al. 1989). Using these fluxes we estimate that for the oocytes used in Fig. 1A, approximately 2 × 1010 copies of rbSGLT1 were located in the plasma membrane. Given this density of cotransporters, we obtained a Purea of 7 × 10−18 cm s−1 per rbSGLT1 cotransporter.
The presence of urea analogues did not affect urea uptake into control and rbSGLT1-expressing oocytes. Uptake of 55 μm urea into control and rbSGLT1-expressing oocytes was insensitive to the addition of excess concentrations (100 mm) of the urea analogues. For example, in one experiment where the rbSGLT1-specific uptake of 55 μm urea was 9.8 ± 1.1 pmol oocyte−1 (30 min)−1 (n= 5), the uptake in the presence of 100 mm thiourea, N-methylurea, 1,3-dimethylurea, acetamide and 1,1-dimethylurea was 8.6 ± 0.9 (n= 5), 9.5 ± 0.9 (n= 5), 8.9 ± 1.0 (n= 5), 9.5 ± 1.9 (n= 5) and 9.0 ± 1.7 pmol oocyte−1 (30 min)−1 (n= 5), respectively. This is consistent with the linearity of urea uptake up to 100 mm urea.
Urea uptake by rbSGLT1 was independent of Na+; for example, in two pairs of experiments the specific uptake was 14.2 ± 0.7 (n= 5) and 10.1 ± 0.5 pmol oocyte−1 (30 min)−1 (n= 6) in Na+, and 15.6 ± 0.6 (n= 6) and 10.3 ± 0.7 pmol oocyte−1 (30 min)−1 (n= 6) in choline. However, urea uptake was inhibited by phlorizin, a specific inhibitor of Na+-dependent glucose transport by rbSGLT1 (Fig. 1B). The addition of 1 μm phlorizin reduced the rbSGLT1 uptake of 55 μm urea by ∼50%. Increasing the concentration of phlorizin to 50 μm reduced urea uptake to 23% of that in the absence of phlorizin (Fig. 1B, Control). The apparent phlorizin inhibition constant, Ki, was 1 μm in the presence of 100 mm external Na+, but was greater than 1 mm in choline. Phloretin also inhibited urea uptake by rbSGLT1. The Ki for phloretin was greater than 1 mm in the absence and presence of Na+. Neither phlorizin nor phloretin inhibited urea uptake into non-injected oocytes.
The specificity of the increase in urea permeability of rbSGLT1-expressing oocytes was examined by measuring the uptake of radiolabelled mannitol, glycerol, sulphate, chloride and l-alanine. With these probes there was no observable increase in permeability. For example, in one experiment where the 55 μm urea uptake increased from 1.7 ± 0.3 pmol oocyte−1 (30 min)−1 (n= 5) in control oocytes to 7.6 ± 0.5 pmol oocyte−1 (30 min)−1 (n= 5) in rbSGLT1-expressing oocytes, the uptake of 55 μm mannitol was 1.4 ± 0.1 pmol oocyte−1 (30 min)−1 (n= 5) in control and 1.8 ± 0.2 pmol oocyte−1 (30 min)−1 (n= 5) in rbSGLT1-expressing oocytes. In the case of 55 μml-alanine the uptake was 24 ± 1 (n= 6) and 22 ± 1 pmol oocyte−1 (30 min)−1 (n= 6), respectively.
The temperature sensitivity of urea uptake was determined over the range 14–30°C and the corresponding Arrhenius plots are shown in Fig. 2. In control oocytes, the activation energy, Ea, for urea transport was 14 ± 3 kcal mol−1, a value comparable to that reported previously for the efflux of urea (10 kcal mol−1, Zhang & Verkman, 1991) and to that of passive water diffusion through the plasma membrane (12 kcal mol−1, Loo et al. 1996; 10 kcal mol−1, Zhang & Verkman, 1991). The Ea for urea transport in rbSGLT1 oocytes was 6 ± 1 kcal mol−1.
Figure 2. Arrhenius plots of urea uptake by rbSGLT1 (•) and control (non-injected, ○) oocytes
Urea uptake was measured in oocytes incubated in 55 μm[14C]urea for 30 min at 14, 22 and 30°C. The experiment was performed on 29 oocytes obtained from the same batch and after 5 days expression. Each data point with error bars indicates the mean urea uptake by 4–6 oocytes (±s.e.m.). The lines were drawn by linear regression. For control oocytes, a slope of −3.0 ± 0.6 corresponds to an activation energy, Ea (Ea=−2.3R X slope, where R is the gas constant), of 14 ± 3 kcal mol−1. For rbSGLT1-expressing oocytes, the slope of −1.3 ± 0.3 corresponds to an Ea of 6 ± 1 kcal mol−1.
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Urea uptake was also higher in oocytes expressing the low affinity Na+-glucose (pSGLT3), the Na+-iodide (rNIS) and the Na+-Cl−-GABA (hGAT1) cotransporters (Fig. 3). The values for urea uptake obtained for rNIS- and hGAT1-expressing oocytes were 3-fold higher (8.5 ± 0.5 pmol oocyte−1 (30 min)−1) than when compared to that of control oocytes (3.2 ± 0.2 pmol oocyte−1 (30 min)−1). Urea uptake by pSGLT3 was also significantly higher than in control oocytes (P≤ 0.05; Fig. 3, right). The increase in urea transport was blocked by specific inhibitors, i.e. 100 μm phlorizin blocked pSGLT3 urea uptake by 82%, and 20 μm SKF 89976A reduced hGAT1 urea uptake by 20% (not shown).
Figure 3. Urea transport by different cotransporters
Uptake of 55 μm[14C]urea was measured for 30 min in oocytes after 5 days of expression of different cotransporters. Oocytes from the same batch were injected with 50 ng cRNA of rabbit Na+-glucose cotransporter (rbSGLT1), rat Na+-iodide cotransporter (rNIS), human Na+-Cl−-GABA transporter (hGAT1) or pig low affinity sugar transporter (pSGLT3), or were not injected (Control). Each bar represents the mean uptake by 4–6 oocytes and was tested for significance (P≤ 0.05).
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Urea transport in the presence of substrates
The addition of substrates to the bathing medium further increased urea transport by cotransporters. To minimize complications due to the voltage dependence of cotransport activity, we used substrate concentrations below the K0.5 for transport. This decreased the depolarization of the membrane potential and the concomitant reduction in the rate of Na+-substrate cotransport. Figure 4 summarizes the results of one set of experiments with rbSGLT1, hGAT1 and pSGLT3. Urea transport by rbSGLT1 increased from 4.0 ± 0.3 to 8 ± 0.5 pmol oocyte−1 h−1 in the presence of 0.1 mmαMDG. Phlorizin (100 μm) inhibited this urea transport by rbSGLT1. For pSGLT3, the urea uptake increased from 1.1 ± 0.3 to 2.2 ± 0.01 pmol oocyte−1 h−1 in the presence of 1 mmαMDG. Addition of 5 mmαMDG increased the urea uptake by pSGLT3 from 4.0 ± 0.4 to 6.2 ± 0.4 pmol oocyte−1 (30 min)−1. Urea transport measured in the presence of 1 μm GABA by hGAT1 also increased from 7 ± 0.5 to 14 ± 1 pmol oocyte−1 h−1.
Figure 4. Urea transport in the presence (+) and absence (-) of substrate by oocytes expressing rbSGLT1, hGAT1 or pSGLT3
Urea transport was measured for 1 h in 100 mm NaCl buffer and ±100 μm aMDG for rbSGLT1, ±1 μm GABA for hGAT1, or ±1 mm aMDG for pSGLT3 oocytes. Each bar represents the mean urea uptake by 4–6 oocytes after 5 days expression and has been corrected for the uptake by non-injected oocytes. In the presence of the respective substrate, urea transport through rbSGLT1, hGAT1 and pSGLT3 increases by ∼50%.
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We determined the stoichiometry between urea and Na+-glucose cotransport by rbSGLT1 by measuring the amount of [14C]urea uptake (55 μm) in the presence and absence of 0.2 mmαMDG. We also determined the amount of αMDG transported by rbSGLT1 by measuring the amount of Na+-dependent [14C]αMDG (0.2 mm) uptake in the same batch of rbSGLT1-expressing oocytes. The substrate-dependent urea uptake by rbSGLT1 was 1.6 ± 0.4 pmol oocyte−1 (30 min)−1 (n= 10) and the Na+-dependent αMDG uptake was 776 ± 28 pmol oocyte−1 (30 min)−1 (n= 10). The results from this experiment show that the urea to αMDG transport ratio of rbSGLT1 is approximately 2.1 × 10−3 urea/αMDG.