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ABSTRACT |
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1. In order to study its role in steady state water transport, the Na+-glucose cotransporter (SGLT1) was expressed in Xenopus laevis oocytes; both the human and the rabbit clones were tested. The transport activity was monitored as a clamp current and the flux of water followed optically as the change in oocyte volume.
2. SGLT1 has two modes of water transport. First, it acts as a molecular water pump: for each 2 Na+ and 1 sugar molecule 264 water molecules were cotransported in the human SGLT1 (hSGLT1), 424 for the rabbit SGLT1 (rSGLT1). Second, it acts as a water channel.
3. The cotransport of water was tightly coupled to the sugar-induced clamp current. Instantaneous changes in clamp current induced by changes in clamp voltage were accompanied by instantaneous changes in the rate of water transport.
4. The cotransported solution was predicted to be hypertonic, and an osmotic gradient built up across the oocyte membrane with continued transport; this resulted in an additional osmotic influx of water. After 5-10 min a steady state was achieved in which the total influx was predicted to be isotonic with the intracellular solution.
5. With the given expression levels, the steady state water transport was divided about equally between cotransport, osmosis across the SGLT1 and osmosis across the native oocyte membrane.
6. Coexpression of AQP1 with the SGLT1 increased the water permeability more than 10-fold and steady state isotonic transport was achieved after less than 2 s of sugar activation. One-third of the water was cotransported, and the remainder was osmotically driven through the AQP1.
7. The data suggest that SGLT1 has three roles in isotonic water transport: it cotransports water directly, it supplies a passive pathway for osmotic water transport, and it generates an osmotic driving force that can be employed by other pathways, for example aquaporins.
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INTRODUCTION |
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The apical membrane of leaky epithelia such as the small intestine and the kidney proximal tubule incorporates several cotransporters, among them the Na+-dependent glucose transporter SGLT1. This protein mediates inwardly directed fluxes of Na+, glucose and, as recently suggested, water. The water transport by the SGLT1 can proceed by two mechanisms. The protein cotransports water together with Na+ and glucose in a ratio which is fixed by a mechanism within the protein, and which is independent of external parameters, such as clamp voltage, osmotic gradients and substrate concentrations. The cotransported solution is hypertonic to plasma; between 80 and 150 water molecules are transported for each non-aqueous substrate molecule (Loo et al. 1996; Zeuthen et al. 1997; Meinild et al. 1998a). In addition SGLT1 has a passive water permeability through which water can be driven by external osmotic forces (Loike et al. 1996; Loo et al. 1999). Water cotransport has also been found in a number of other cotransporters of the symport type (Zeuthen, 1991, 1994; Zeuthen et al. 1996; Meinild et al. 2000; MacAulay et al. 2000). For a review see Zeuthen (2000).
The purpose of this study was to separate and quantify the cotransport and osmotic components of water transport in the SGLT1 under conditions of steady state isotonic transport. In addition, we wanted to test how the transmembrane osmotic gradient generated by the cotransport modality could be utilized for additional osmotic transport of water. This would have important implications for a transcellular model of epithelial transport. In the human small intestine, for example, it has been suggested that cotransport by the hSGLT1 could account for about half of the daily uptake of isotonic solution across the brush border membrane, about 4 l (Loo et al. 1996). Here we tested whether the other half of the steady state absorption could be osmotic, driven by a transmembrane osmotic gradient generated by the SGLT1 itself.
We used over-expression of SGLT1 in Xenopus laevis oocytes as a model for isotonic transport across the brush border membrane of the small intestine. Long-term activation of the SGLT1, for about 10 min, resulted in steady state swellings of the oocyte, in which the influx was divided about equally between cotransport and osmosis. In this state the cotransport had built up a sufficiently large osmotic gradient to render the total transportate isotonic with the intracellular solution. In order to model more water-permeable brush border membranes, say from kidney proximal tubule cells, specific water channels (aquaporins) were coexpressed with the SGLT1. In this case the water transport also became isotonic, divided about equally between cotransport and osmotic transport. The presence of aquaporins caused the coupling between the fluxes to be more efficient than in the small intestine model; the osmotic flux was obtained faster and by a comparatively small osmotic gradient.
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METHODS |
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Human or rabbit SGLT1 and human AQP1 were expressed in Xenopus laevis oocytes as described previously (Hediger et al. 1989; Meinild et al. 1998a,b). For coexpression of SGLT1 and AQP1, the respective cRNAs were injected in a ratio of between 75/25 and 90/10. Oocytes were incubated in Kulori medium (mM: 90 NaCl, 1 KCl, 1 CaCl2, 1 MgCl2, 5 Hepes, pH 7.4, 182 mosmol l-1) at 19 °C for 3-7 days before experiments. Osmolarities of the test solutions were determined with an accuracy of less than 1 mosmol l-1. The experimental chamber was perfused by control solution (90 NaCl, 20 mannitol (or urea), 2 KCl, 1 CaCl2, 1 MgCl2, 10 Hepes, pH 7.4, 213 mosmol l-1). Oocytes were transferred to the control medium 30 min or more prior to experiments. Isotonic test solutions were obtained by replacing mannitol (or urea) by methyl-
-D-glucopyranoside (-MDG) (Sigma, catalogue no. M-9376), a non-metabolizable sugar which is transported by the SGLT1 in the same way as glucose (Loo et al. 1993). The majority of the experiments were performed at saturating conditions, clamp potentials of -50 or -70 mV and sugar concentrations higher than 1 mM. The inhibitor phlorizin (200 
M; Sigma) was added directly, without ethanol, to the test solutions. Hyperosmotic test solutions were obtained by addition of mannitol (or urea). The refractive index of the solutions depended on the concentration of mannitol but not on the concentration of urea. As a result, mannitol addition might lead to a transient blurring of the picture. For optical reasons, therefore, urea was used instead of mannitol in the majority of the experiments measuring water permeability (Lp), with no difference in the results. Under conditions of no sugar, the Lp measured by means of urea was 0.97 ± 0.07 times the Lp measured by mannitol (paired experiments in 6 oocytes expressing hSGLT1); in the presence of sugar the ratio was 0.96 ± 0.04 (paired experiments in 5 oocytes expressing hSGLT1). Similar data were obtained for rSGLT1.
When the influence of unstirred layers and electrode artefacts was tested, the cation-selective pore former gramicidin (150-300 nM; Sigma, catalogue no. G-5002) was added to both bathing and test solutions. After about 10 min gramicidin had decreased the input resistance of the oocyte by one or two orders of magnitude and was removed from the solutions (Zeuthen et al. 1997; Meinild et al. 1998a).
The experimental chamber, the voltage clamp, and the optical system for the volume measurements have been described in detail elsewhere (Parent et al. 1992; Zeuthen et al. 1997). In accordance with national guidelines, Xenopus oocytes were collected under anaesthesia (2 g l-1 Tricaine, 3-aminobenzoic acid ethyl ester; Sigma, catalogue no. A 5040). An ovarian lobe was removed from the abdominal cavity through a small (1 cm) incision. The anaesthetized frogs were finally killed by decapitation. The oocytes were impaled by two microelectrodes, one providing the clamp current and the other measuring the membrane potential. The electrodes were filled with KCl (concentrations between 0.3 and 1 M). The clamp current will be carried by equal and opposite fluxes of K+ and Cl-. Consequently, the current electrode does not contribute to the net solute flux into the cell (see Appendix).
The experimental chamber had a volume of 30 l. At maximal flow rates (12 l s-1) solution changes were 90 % complete in 5 s. In experiments which required a number of subsequent measurements (i.e. Table 1) a 3 times slower flow rate was employed in order to increase the number of successful series. The solutions were fed into the chamber via a mechanical valve with a dead space of 5 l. The oocyte was observed from below via a low-magnification objective and a charge coupled device camera. To achieve a high stability of the oocyte image, the upper surface of the bathing solution was determined by the flat end of a Perspex rod, which also provided an illuminated background. Images were captured directly from the camera to the random access memory of a computer. The oocyte was focused at the circumference and assumed to be spherical. The volume was recorded and calculated on-line at a rate of one point per second with an accuracy of 3 in 10 000. Only oocytes with well-defined and clean surfaces were used.
The water permeabilities (Lp) are given per true membrane surface area (Verkman & Ives, 1986). This is about 9 times the apparent area due to membrane foldings (Soreq & Seidman, 1992; Zampighi et al. 1995). Lp is given in units of cm s-1. To obtain the water permeability in units of cm s-1 (osmol l-1)-1, the Lp values were multiplied by the partial molar volume of water (Vw), 18 cm3 mol-1. The diameter of the oocytes was 1.34 ± 0.02 mm (n = 13). The coupling ratio of the SGLT1 is defined as the number of water molecules cotransported per turnover of the protein, in which 2 Na+ and 1 glucose molecule are translocated. Accordingly the coupling ratio equals 2FJH2O(VwIs)-1, where JH2O is the cotransported water flux, Is the sugar-induced current, and F Faraday's constant. Experiments were performed at room temperature (20 °C). All numbers are given as means ± S.E.M., with n equal to the number of oocytes tested unless otherwise stated. Comparisons were done with Student's t test, and P < 0.05 was taken as the criterion of statistical difference.
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RESULTS |
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Initial and steady state transport of water
Human SGLT1. We employed oocytes in which over-expression of hSGLT1 gave currents of up to 700 nA at saturating sugar (-MDG) concentrations (1 mM). This low expression level was chosen in order to conform to the experiments where SGLT1 and AQP1 were coexpressed (see below).
When sugar was added under isosmolar and voltage-clamped conditions the clamp current increased from its baseline value by the amount Is. Simultaneously, the oocyte began to swell at a rate 
V/dt, which defines the influx of water JH2O (Fig. 1 and 4). There was no measurable delay between the onset of water influx and sugar-induced current; JH2O was given by the integrated current to within 2 s (Meinild et al. 1998a). JH2O and Is were correlated with a coupling ratio of 269 ± 12 (n = 9) water molecules per turnover of the protein, in which 2 Na+ and 1 sugar molecule are transported. The coupling ratio was similar to that of previous findings (Meinild et al. 1998a).
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V) in a hSGLT1-expressing oocyte
Sugar (
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If the sugar challenge was maintained, JH2O increased progressively. After about 5-10 min, JH2O stabilized at a steady state value 2.7 ± 0.3 (n = 5) times the initial rate (Fig. 1 and Table 1). This rate of swelling remained constant for another 10 min at least. In the steady state the intracellular osmolarity must be constant and the solution transported into the oocyte isotonic with the intracellular solution (see Appendix).
In order to separate the cotransport and the osmotic component of water transport, a number of transport parameters were recorded in the steady state period 10-25 min after the initial application of sugar. The data were obtained from five oocytes which served as their own controls (Table 1).
(i) Isomolar removals of sugar, lasting about 2 min, reduced JH2O by an average of 38 ± 4 % and abolished Is (see Fig. 2 for an example of a similar experiment in rSGLT1). The reductions in JH2O and Is were equivalent to a coupling ratio 1.05 ± 0.06 times that obtained from the initial values of JH2O and Is; this difference was not significant.
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V) in an rSGLT-expressing oocyte
The initial steady state was obtained by continuous application of sugar (+
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(ii) Application of 200 M of phlorizin, a high affinity blocker of SGLT1, reduced JH2O by an average of 73 ± 6 % (see Fig. 3 for an example obtained in rSGLT1). This was twice the reduction obtained by sugar removal.
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V) and water permeabilities (Lp) in rSGLT-expressing oocytes
A steady state rate of swelling of 42 pl s-1 and a clamp current, Is, of 480 nA had been obtained by continuous application of sugar (+
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(iii) The water permeability (Lp) was recorded as the change in the rate of swelling induced by the addition of 20 mosmol l-1 of urea or mannitol. Lp was recorded during the steady state induced by sugar and after the inhibition of the hSGLT1 by phlorizin, which blocks the water permeability of the hSGLT1 and reduces the Lp of the oocyte to that of the native oocyte membrane (Loo et al. 1999). Lp recorded in the presence of phlorizin was 38 ± 5 % smaller than in the unpoisoned case (Fig. 3). Accordingly, the Lp was shared between 38 ± 5 % for the rSGLT1 and 62 ± 5 % for the native oocyte membrane, i.e. in a ratio of 38/62.
In summary, the isosmotic influx of water at steady state can be estimated to consist of 38 ± 4 % via cotransport, and the remainder (62 %) by osmosis. The osmotic component can be divided between the SGLT1 and the native oocyte membrane. According to the ratio of the Lp values determined above (38/62), the osmotic component in the hSGLT1 will contribute 24 ± 4 % to the total JH2O, and osmosis via the native oocyte membrane 38 ± 3 %. A slightly different estimate was obtained if the division was based on the phlorizin data, which suggested that 27 % of JH2O was due to osmosis via the native oocyte membrane and 36 % via the SGLT1. It seems fair to conclude that the three pathways contribute about one-third each (Fig. 7). The steady state intracellular osmolarity can be estimated from the osmotic contribution to JH2O (= JH2O in steady state minus JH2O reduction at sugar removal) and the Lp (sugar present, in Table 1). It appears that the intracellular osmolarity had increased by 7 ± 1 mosmol l-1 to about 220 mosmol l-1 in order to drive the steady state osmotic component of JH2O.
Rabbit SGLT1. Five oocytes were tested under conditions identical to those of the hSGLT1s above. In another three oocytes mannitol was used instead of urea. There was no apparent difference between the two groups of results and the data from the eight oocytes are pooled in the following. All oocytes were tested under conditions of saturating sugar concentrations and clamp potentials. The average initial coupling ratio was 423 ± 24 water molecules per 2 Na+ and 1 sugar molecule. As the sugar challenge was maintained the steady state value of JH2O after 10 min was 2.3 ± 0.3 times the initial value (Fig. 1, Table 1).
(i) Isomolar removals of sugar reduced JH2O by an average of 37 ± 2 % and abolished Is (Fig. 2). The reductions in JH2O and Is were equivalent to a coupling ratio equal to 1.07 ± 0.08 times that obtained from the initial values of JH2O and Is; this difference is not significant.
(ii) Application of phlorizin reduced the rate of swelling by an average of 61 ± 5 % (Fig. 3), which is almost twice that obtained by sugar removal.
(iii) Lp recorded in the presence of phlorizin was 33 ± 6 % smaller than in the unpoisoned case (Fig. 3). Accordingly, the Lp was shared, with 33 ± 6 % to the rSGLT1 and 67 ± 6 % to the native oocyte membrane.
In summary, the isosmotic influx of water in steady state was divided between 37 ± 2 % via cotransport and the remainder (63 %) by osmosis. According to the ratio of the Lp values determined above (33/67), the osmotic component of the rSGLT1 will contribute 21 ± 4 % to the total JH2O, and osmosis in the native oocyte membrane 42 ± 4 %. From the osmotic contribution to the steady state value of JH2O and the Lp given in Table 1, it can be calculated that the intracellular osmolarity must have increased by 7 ± 1 mosmol l-1 in the steady state.
JH2O during rapid changes of clamp voltage
In order to investigate the tightness of the coupling between the cotransport component of the water transport and Is, we studied the changes in JH2O induced by instantaneous changes in clamp voltage (Fig. 4). To begin the experiment sugar was added isosmotically at a given clamp voltage, JH2O and Is were recorded, and after about 20 s the clamp voltage was changed abruptly (within 10 ms) and new steady state values of JH2O and Is were obtained (Fig. 4A). The results from 18 tests on six oocytes expressing rSGLT1 to the same degree are presented. In most experiments the voltage was changed between a high (-70 to -90 mV) and a low negative value (0 to -10 mV), and both upward and downward jumps were tested. The instantaneous change in clamp voltage was associated with instantaneous changes in JH2O and Is (Fig. 4B). The time of change in JH2O was estimated from the intercept between the regression lines that represent JH2O before and after the jump in voltage. This intercept took place 1.1 ± 0.7 s (n = 18) after the jump in voltage. This delay was not significantly different from zero.
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A, isotonic addition of sugar (
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The relation between sugar-induced current Is and clamp voltage (Fig. 4C) was identical to earlier findings (i.e. Parent et al. 1992). The ratio between JH2O and Is was the same before and after the change in clamp voltage (Fig. 4D). An average of 220 water molecules were transported per unit charge or 440 water molecules per turnover of the protein.
Effects of Na+ fluxes on JH2O in oocytes expressing SGLT1
In order to test the ability of conductive Na+ fluxes per se to generate water transport, the cation-selective ionophore gramicidin was inserted into SGLT1-expressing oocytes. By clamping the oocyte to a potential between -20 and -100 mV, large inward Na+ fluxes were generated via gramicidin, given by inward clamp currents in the range 680-2060 nA. Gramicidin does not affect the Lp of the SGLT1-expressing oocyte (Zeuthen et al. 1997). Rabbit SGLT1 was employed for the experiments.
The gramicidin-mediated Na+ fluxes did not produce any initial influxes of water (Fig. 5A). The rate of volume change was zero (0.15 ± 1.0 pl s-1, 7 tests in 3 oocytes) and remained so for about 35 s (range 20-60 s); this agrees with earlier findings (Zeuthen et al. 1997; Meinild et al. 1998a). After 61 ± 10 s (range 25-75 s), JH2O had reached a steady state of 26.9 ± 1.5 pl s-1. Due to the relatively high currents employed, the steady state swelling was obtained somewhat faster than in the experiments given in Table 1. Given an Lp of 4.8 X 10-4 cm s-1 (Table 1) the transmembrane osmotic gradient in the steady state can be calculated to be about 7 mosmol l-1. When the clamp was switched off, the volume increase receded gradually (Fig. 5A). The cotransport component was kept inactivated by the absence of sugar.
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A, the SGLT1 was kept inactivated by the absence of sugar throughout. Application of the clamp potential (-70 mV, first vertical line) caused an inward Na+ current of 1400 nA (not shown) via the ionophore gramicidin (see Methods). After about 30 s the oocyte began to swell; after about 70 s JH2O (=
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The cotransport capacity of SGLT1 in the presence of gramicidin-mediated Na+ fluxes was tested in paired experiments in five oocytes. First, the gramicidin-mediated Na+ fluxes were initiated by applying a clamp voltage under conditions of no sugar. When the gramicidin-mediated flux had resulted in a steady state volume increase, sugar was applied and the increments in clamp current (Is) and water flow (JH2O) recorded (Fig. 5B). This was compared to the sugar-induced JH2O and Is obtained prior to the insertion of gramicidin. Is obtained in the presence of gramicidin was 1.1 ± 0.12 times larger, which was not significant. The initial value of the sugar-induced water flux JH2O was slightly smaller by a factor 0.82 ± 0.06 in the presence of gramicidin. In the comparison Is has been taken as the average over the first 50 s.
Transport by AQP1 and SGLT1
The coupling between solute and water transport was studied in oocytes coexpressing human AQP1 and human SGLT1 and compared to the coupling observed in oocytes expressing only the SGLT1. The two groups were selected so as to express SGLT1 to the same extent, given by a current of about 700 nA at saturating sugar concentrations. This limit was set by the coexpressing oocytes where it was difficult to achieve higher expressions of the hSGLT1. The Lp of the oocytes expressing both AQP1 and SGLT1 was (43 ± 2) X 10-4 cm s-1 (n = 21 in 9 oocytes); this was more than 10 times larger than the Lp of the SGLT1-expressing oocytes, (3.7 ± 0.3) X 10-4 cm s-1 (n = 9 in 4 oocytes).
The SGLT1 was activated and deactivated by switching on and off the clamp voltage, while the oocytes were bathed in sugar containing solutions (Fig. 6A). This protocol avoids errors that might arise from the high water permeabilities of the coexpressing oocytes combined with submilliosmolar differences in bathing solution osmolarity. Experiments in four oocytes (not shown) showed that the protocol gave the same results as experiments in which the cotransporter was activated by additions of sugar under clamped conditions. It should be noted, however, that the total current (It) induced by turning on the clamp is a sum of the sugar-induced cotransport current (Is) and a smaller leak current through the hSGLT1 and the native oocyte membrane (Parent et al. 1992). The leak current was estimated by measuring It with and without sugar. The leak current was largest, about 20 % of It, at the maximal clamp potential employed, -110 mV. These are probably overestimates; there is evidence that the Na+-dependent component of the leakage current through the SGLT1 is reduced in the presence of sugar (Parent et al. 1992; Mackenzie et al. 1998). Accordingly, it is assumed that It is close to Is for most of the experiments described below.
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A, the oocyte coexpressed hSGLT1 and AQP1, and sugar (
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When the hSGLT1 was activated, a linear increase in oocyte volume commenced instantaneously (time resolution about 2 s). The slope of the increase defines the water flux, JH2O. The increase in volume was associated with an abrupt increase in inwardly directed current, It (Fig. 6A). The relation between JH2O and It was determined from experiments in which the clamp voltage was maintained for 1 min; different currents were obtained by choosing different clamp voltages. The results are summarized in Fig. 6B where the number of water molecules transported for each Na+, nw/Na+, is plotted versus It. For oocytes coexpressing hSGLT1 and AQP1, nw/Na+ was relatively independent of It as indicated by the regression line nw/Na+ = 423 - 0.22It (with S.E.M. values of 31 and 0.09, respectively, n = 55 in 15 oocytes). nw/Na+ averaged 358 ± 12 (Fig. 6B, upper panel). For oocytes expressing only hSGLT1, nw/Na+ was independent of It as indicated by the regression line nw/Na+ = 142 - 0.07It (with S.E.M. values of 38 and 0.1, respectively, n = 17 in 4 oocytes). nw/Na+ was on average 117 ± 11 (Fig. 6B, middle panel). Since the hSGLT1 cotransports 2 Na+ (+ 1 sugar molecule) per turnover it follows that the combination of hSGLT1 and AQP1 maintains a transport of 2 X 358 = 715 water molecules per turnover of the hSGLT1, while the hSGLT itself cotransports 2 X 117 = 234 water molecules per turnover. The difference of 481 water molecules per turnover is expected to arise from osmotic effects generated by the Na+ and glucose fluxes.
Effects of Na+ fluxes on JH2O in oocytes coexpressing SGLT1 and AQP1
The ability of Na+ currents to generate an influx of water in the oocytes coexpressing the human SGLT1 and AQP1 was tested by inserting the ionophore gramicidin while keeping the SGLT1 in its inactivated state by the absence of sugar. Inward currents were induced by clamping the oocyte to between -50 and -110 mV for 1 min. Gramicidin levels and clamp voltages were chosen to produce inward currents between 200 and 700 nA. The currents induced an immediate linear volume increase, the slope of which defines JH2O. The rapid onset of JH2O is a strong indication that unstirred layers drive the water transport. The number of water molecules transported per ion, nw/Na+, was independent of It as indicated by the regression line nw/Na+ = 166 - 0.04It (with S.E.M. values of 22 and 0.05, respectively, n = 36 in 6 oocytes). On average nw/Na+ was 148 ± 7 (Fig. 6B lower panel). This ratio predicts that 444 (3 X 148) water molecules are transported for 3 Na+ ions. This is close to the osmotic component of 481 water molecules per 2 Na+ and 1 sugar estimated above in relation to the coexpressing oocytes (see Fig. 6C and Discussion).
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DISCUSSION |
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The number of water molecules cotransported per solute molecule in the SGLT1 is between 80 and 150 compared to the bathing solution that has 258 water molecules per solute molecule. It follows that activation of the SGLT1 will gradually increase the osmolarity of the oocyte and give rise to a progressive rate of osmotic water influx both via the SGLT1 itself and via other passive pathways in the membrane. This will continue until a steady rate of swelling has been achieved. At this point the intracellular osmolarity is constant and the total flux of water and solutes into the oocyte is isotonic with the intracellular solution (see Appendix). Our data separate this steady state water flux into a cotransport and an osmotic component.
Two components of water flow during steady state
There are several lines of evidence which suggest that the flux of water during steady state can be separated into a cotransport component and an osmotic component. In general the cotransport component will be tightly coupled to the sugar-dependent clamp current while the osmotic component will depend on the slow build up and decay of osmotic gradients.
(1) The removal of sugar (MDG) during the steady state transport reduced the water transport rate (JH2O) and the current (Is) by amounts equivalent to the cotransport component. The return of the sugar re-established the cotransport component (Fig. 2 and Table 1).
The cotransport component was initially determined from the JH2O elicited by isosmolar addition of sugar under voltage clamp conditions (Fig. 1 and 4, and Table 1). For the hSGLT1 the coupling ratio, Na+:MDG:H2O, was 2:1:269, which is slightly higher than a previous estimate of 2:1:210 (Meinild et al. 1998a). For the rSGLT1 the coupling ratio was 2:1:424, slightly higher than a previous estimate of 2:1:390 (Zeuthen et al. 1997). The early estimate of 260 for rSGLT1 (Loo et al. 1996) is probably an underestimate; it was obtained with a lower time and volume resolution than employed in the present study.
(2) A jump in clamp voltage changed JH2O and Is abruptly by amounts equivalent to the cotransport component (Fig. 4). The coupling ratio was constant under all clamp conditions and identical to the ratio determined from removal and addition of sugar to the bathing solution. The rapidity of the response demonstrated the tightness of the coupling between the cotransport component of water transport and the turnover of the protein and was suggestive of there being no unstirred layer effects (see below).
(3) Phlorizin induced a relatively larger change in JH2O (65 %) than in the passive water permeability Lp (35 %) (Table 1 and Fig. 3). If the steady state JH2O was entirely osmotic, the relative reductions in Lp and JH2O should have been identical.
Phlorizin blocks both the cotransport component and the Lp of the SGLT1 and our data were consistent with a model in which the steady state JH2O was divided between cotransport and osmosis. If the phlorizin data were combined with the data obtained from sugar removal (see above), it appeared that the steady state JH2O generated by the hSGLT1 was divided about equally between cotransport, osmosis in the SGLT1, and osmosis through the native oocyte membrane (Fig. 7). The findings were similar for the rSGLT1. Given the Lp of the membranes (Table 1) the transmembrane steady state osmotic gradient can be estimated to be about 7 mosmol l-1 for both clones. Given a bathing solution osmolarity of 213 mosmol l-1 the intracellular osmolarity amounts to 220 mosmol l-1. Since it takes 5-10 min to build up the steady state it is fair to assume that this osmolarity applies to the bulk of the intracellular compartment.
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A, the water transport consists of three components of roughly equal magnitude: for the hSGLT1 it can be estimated that one-third is cotransported via a mechanism within the SGLT1, one-third is osmotically driven via the SGLT1, and one-third is osmotically driven through the native oocyte membrane (ooc). The SGLT1 has a relatively low intrinsic Lp, so the hypertonic cotransport by the SGLT1 has to build up a relatively large transmembrane osmotic gradient (
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(4) The osmotic component could be mimicked by conductive Na+ fluxes. The JH2O resulting from the gramicidin-generated Na+ fluxes was similar to the osmotic component of the SGLT1-generated JH2O on two accounts. First, the Na+ fluxes did not give rise to any immediate JH2O. Second, the steady state JH2O could be explained by an osmotic gradient of 7 mosmol l-1 and the Lp of the oocyte (Fig. 5A). This was identical to the osmotic component determined by removing sugar from oocytes in which the SGLT1 was active (Table 1 and Fig. 2).
(5) The sugar-induced cotransport of water could be superimposed upon the water flux induced by gramicidin. When sugar was added to gramicidin-treated oocytes under voltage-clamp conditions there was an abrupt increase in JH2O and clamp current (Fig. 5B). The ratio of these increases was similar to that of the cotransport component. This confirmed that cotransport can proceed in parallel with, and independent, of an osmotic flux.
(6) The coexpression of AQP1 did not alter the magnitudes of the water cotransport and of the osmotic water flow; the major effect was to enable the osmotic component to be driven by a small osmotic gradient.
In the coexpressing oocytes the steady state value of JH2O was divided between about 1/3 cotransport and 2/3 osmotic transport (Fig. 6), the same fractions as determined in steady state experiments for oocytes expressing only hSGLT1. The high Lp of the AQP1 had two effects. First, a constant value of JH2O was achieved instantaneously (Fig. 6A). This rapid response indicates that the osmotic component is driven by an unstirred layer osmotic gradient (see below). Second, the osmotic component took place almost exclusively via the AQP1, driven by a small osmotic gradient. The osmotic component of JH2O could be estimated to be about 20 pl s-1 (Fig. 6B): given the high Lp this could be driven by an osmotic gradient of only 0.5 mosmol l-1. As the bathing solution osmolarity is 213 mosmol l-1, it follows that the isotonic transport into the oocyte has an osmolarity of about 213.5 mosmol l-1.
In summary, the steady state isotonic water transport can be divided into one-third cotransport and two-thirds osmosis (Fig. 7). This applies both to the case where hSGLT1 is expressed and where hSGLT1 and AQP1 are coexpressed. The rates of Na+ transport and water transport are about the same in the two cases, determined by the turnover of the hSGLT1. In both instances the intracellular hyperosmolarity will attain a steady state value which renders the transported solution isotonic. In the case of hSGLT1 expression, the osmolarity of the isotonic transport is about 220 mosmol l-1 (213 + 7) while it is about 213.5 mosmol l-1 (213 + 0.5) in the case of coexpression. Given similar rates of Na+ transport, it follows that the total rate of water transport (cotransport plus osmosis) at steady state differs by only about 3 % between the two cases. This similarity is also reflected in the experimental estimates of the number of water molecules transported per turnover of the hSGLT1 during the steady states, 726 in the case of hSGLT1 expression (Table 1) and 715 in the case of coexpression (Fig. 6C). The major difference between the two cases is the magnitude of the osmotic gradient required to produce the osmotic component of the steady state water transport: about 7 mosmol l-1 in the case of hSGLT1 expression, 0.5 mosmol l-1 in the case of coexpression.
Unstirred layers and electrode artefacts
The high Lp of the oocytes coexpressing hSGLT1 and AQP1 allowed the immediate utilization of unstirred layers for osmotic transport. Given the Lp, the gradient could be estimated to be about 0.5 mosmol l-1, which is close to the value predicted from unstirred layer theory (Dainty & House, 1966; Loo et al. 1996). The situation was different in oocytes expressing only SGLT1. Here the Na+ fluxes did not give rise to any immediate inflow of water as investigated directly in the gramicidin experiments (Fig. 5). Apparently, unstirred layer gradients were too small to generate any initial influx of water in these oocytes given their relatively low Lp. This agrees with earlier findings (Zeuthen et al. 1997; Meinild et al. 1998a).
Unstirred layer effects cannot be the basis for the cotransport component of the water transport. Abrupt changes in clamp voltage (and Is) induced changes in JH2O that were complete in times not significantly different from zero (see Fig. 4B). This also applies to experiments where the clamp voltage was switched off (see Fig. 5B and 6A). In contrast to this, in experiments in which the water transport was driven entirely by osmosis, i.e. the gramicidin experiments (Fig. 5A), changes in clamp voltage induced changes in JH2O characterized by time constants of about 1 min.
It might be argued that the sugar, due to its larger size and slower diffusion coefficient than Na+, could provide larger unstirred layer osmotic gradients. This is unlikely for two reasons. First, transport of 1 sugar molecule was associated with the transport of 464 water molecules in the rabbit SGLT1 but only 269 in the human SGLT1. If unstirred layers determined the cotransport component, the two clones would exhibit the same coupling ratio. Second, in the oocytes coexpressing hSGLT1 and AQP1, the unstirred layer determined from the diffusion of 2 Na+ and 1 sugar molecule was not different from that determined from the diffusion of 3 Na+ ions. The combination of cotransport and osmosis resulted in the transport of 715 water molecules per 2 Na+ ions and 1 sugar molecule (Fig. 6B), out of which the cotransport component accounted for 233 water molecules. Accordingly, the difference of 482 water molecules should be driven osmotically by the transmembrane differences in Na+ and glucose concentrations. The same unstirred layer effect could be estimated from experiments using gramicidin. Here, the water flow was given by a coupling of 148 water molecules per Na+ ion or 444 per 3 Na+ ions. This estimate based entirely on Na+ ions is not significantly different from the estimate of 482 water molecules based on the diffusion of 2 Na+ and 1 sugar molecule (Fig. 6C). In summary, there is no basis for assuming that glucose contributes more to unstirred layer osmotic gradients than Na+ ions.
In order to minimize net solute flow from the microelectrodes, KCl was used as the filling solution. This ensures that currents are carried by equal and opposite fluxes of K+ and Cl-. During steady state isosmotic transport, it can be estimated from the Lp that about 250 water molecules enter the oocyte per solute molecule via the membrane and the electrode. The membrane component can be estimated from the steady state influx of water and solute maintained by the SGLT1 (Table 1), which indicates that 225 water molecules enter per solute molecule by this route. The small difference between the two estimates confirms that only a little water transport originates from net ion fluxes from the electrodes (see also Appendix).
Physiological relevance
Our data suggest that SGLT1 has three functions in regard to water transport: it cotransports water, it provides the driving force for parallel osmotic transport, and it provides passive water permeability. This has relevance for models of transcellular isotonic transport in leaky epithelia. If surface folding and experimental temperatures are taken into account, the data obtained with SGLT1 expression are compatible with those obtained with brush border membranes from small intestine while those obtained with coexpression of SGLT1 and AQP1 are compatible with those obtained with brush borders from kidney proximal tubule (references below).
For oocytes expressing hSGLT1, steady state JH2O was 1 nl cm-2 s-1, the Lp of the SGLT1 was 1.2 X 10-3 cm s-1, and the Lp of the native membrane was 1.9 X 10-3 cm s-1. For comparison, the values are expressed per square centimetre of the oocyte membrane, which is folded by a factor of about 9 (Zampighi et al. 1995). The Arrhenius activation energy for JH2O is high, 26 kcal mol-1 (109 kJ mol-1) for the hSGLT1 and 23 kcal mol-1 (97 kJ mol-1) for the rSGLT1 (Loo et al. 1996; Meinild et al. 1998a). This suggests that oocyte experiments performed at 37 °C would give JH2O values 20 times larger, around 20 nl cm-2 s-1, which is similar to the values obtained in the intact epithelia (see below). The activation energy for the Lp is small, about 4 kcal mol-1 (17 kJ mol-1) (Loo et al. 1996, 1999; Meinild et al. 1998a) and an increase to 37 °C would only increase the Lp by a factor of 5, to about 1 X 10-2 cm s-1. The relatively larger increase in JH2O than in Lp with temperature shows that it would require a 10 times larger osmotic gradient (about 70 mosmol l-1) to drive the osmotic influx of water into the oocyte at 37 °C than at 20 °C.
The apical membrane of the small intestine epithelium contains no known aquaporins and the water transport properties are probably determined by the cotransport proteins and the lipid component. The most complete set of data is from rat, where JH2O under sugar transporting conditions is about 25 nl cm-2 s-1 and Lp about 5 X 10-2 cm s-1, values per epithelial square centimetre (Worman & Field, 1985; Heeswijk & Van Os, 1986; Pappenheimer & Reis, 1987; Dempster et al. 1991; for a review see Zeuthen, 1996). The properties of the rat SGLT1 are similar to those of the rabbit and human clones (Wright et al. 1998), and a comparison with the oocyte data shows that the JH2O values are similar while the Lp of the brush border is larger by a factor of five. This would suggest that an osmotic gradient of about 15 mosmol l-1 is required to ensure steady state isotonic transport across the brush border of the small intestine. This is likely to be a minimum estimate. Activation of other cotransporters with properties similar to the SGLT1 and transport by proteins with no capacity for water cotransport would impose a larger osmotic gradient.
For oocytes coexpressing hSGLT1 and AQP1, JH2O was found to be about 1 nl cm-2 s-1 and Lp about 4 X 10-2 cm s-1 at 20 °C (per cm2 of folded oocyte membrane). If activation energies are taken into account (see above), JH2O would be about 20 nl cm-2 s-1 and Lp about 0.2 cm s-1 at 37 °C. At this temperature it would require an osmotic gradient of about 4 mosmol l-1 to drive the osmotic component.
The brush border of the kidney proximal tubule is rich in cotransporters. We have expressed two of these, the SGLT1 and the Na+-dicarboxylate cotransporter, NaDC1 (Meinild et al. 2000), in Xenopus oocytes and have found them to function as molecular water pumps; the rabbit NaDC1 cotransports 175 water molecules per turnover together with 3 Na+ and 1 dicarboxylate. Furthermore, the epithelial cells express AQP1 in the apical (and basolateral) membrane (for a recent review see Borgnia et al. 1999). Most kidney data are from rat, where JH2O lies between 10 and 30 nl cm-2 s-1 and the Lp of the apical membrane is about 0.4 cm s-1, values per epithelial square centimetre (Green & Giebish, 1984; Gonzáles et al. 1984; Pratz et al. 1986; Heswijk & Van Os, 1986; Carpi-Medina et al. 1988; Dempster et al. 1991). A comparison between the oocyte and epithelial data suggests that the expression of SGLT1 in the oocyte is similar to that of the brush border, and that the expression of AQP1 is about 2 times lower in the oocyte. This suggests that the osmotic gradient required to produce steady state isotonic transport in the epithelial cell should be of the order of 2 mosmol l-1. By analogy to the arguments put forward in relation to the small intestine, the presence of other transporters could increase this estimate.
In conclusion, the mammalian small intestine and kidney proximal tubule have roughly the same transepithelial rates of Na+ and water transport. Their major difference is their passive water permeabilities; the apical membrane of the proximal tubule is 10 times more permeable than the small intestine. By analogy, the oocytes expressing SGLT1 and the ones coexpressing SGLT1 and AQP1 have also the same steady state transport rates for Na+ and water and exhibit the same 10-fold difference between their passive water permeabilities. A comparison between the two types of oocytes will therefore give insights into the mechanisms of salt and water fluxes into the respective epithelial cells.
Our results also have relevance for the interpretation of data obtained in transgenic mice lacking AQP1. Here the rate of water transport in the proximal tubule was reduced by a factor of 2, the water permeability reduced by almost 80 %, and the luminal hypotonicity increased by up to 30 mosmol l-1 as compared to wild-type mice (Schnermann et al. 1998; Verkmann, 1999; Vallon et al. 2000). A comparison with the oocyte models suggests that the mode of water transport in the transgenic mice resembles the case where only SGLT1 is expressed. The lack of aquaporins leads to a large reduction in the water permeability, to the build up of a larger osmotic gradient, and to a more hypertonic absorbate.
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APPENDIX |
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TopAbstractIntroductionMethodsResultsDiscussionAppendixReferences |
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Intracellular osmolarity and solute fluxes under steady state swelling
The oocyte is bathed in a solution of osmolarity 213 mosmol l-1 (O (o), eqn (A1)). When the SGLT1 is activated, the initial rate of swelling equals the rate of water cotransport, JH2O. As the cotransported solution is hypertonic to the bathing solution, the transmembrane osmotic gradient increases until a steady state has been achieved. At this point the influx is isotonic with the intracellular solution, the osmolarity of which, O (
), will be constant and determined by (Zeuthen, 1996):
|
JS /[JH2O + (O () - O (o))LpA] = O (),
| (A1) |
where JS is the solute flux into the oocyte: the sum of the Na+ and the glucose influxes via the SGLT1 (JNa + JGlu) plus the net flux of ions into or out of the current clamp microelectrode, JE; Lp is the osmotic water permeability in units of cm s-1 (osmol l-1)-1 equal to LpVw, where Vw is the partial molar volume of water, 18 cm3 mol-1; and A is the surface area of the oocyte equal to 9
d 2, where d is the oocyte diameter and 9 is the folding factor of the oocyte (see Methods). From eqn (A1):
|
O () = O (o)/2 - JH2O/2LpA + [(O (o)/2 - JH2O/2LpA)2 + JS/LpA]1/2.
| (A2) |
Consider a typical numerical example (Table 1): a sugar-induced current, Is, of 600 nA, which equals 2FJNa (F = 105 mol coulomb-1), a water cotransport component, JH2O, of 20 pl s-1, an oocyte diameter of 1.3 mm, and a Lp of 3.3 X 10-4 cm s-1. If JE is zero, i.e. the current in the microelectode consists of equal and opposite fluxes of K+ and Cl-, JS = 900 X 10-14 mol s-1 (= JNa + JGlu). In this case O () was calculated to be 220 mosmol l-1, 7 mosmol l-1 above the bathing solution osmolarity, in agreement with the experimental estimate. In the case where the clamp current was carried entirely by Cl- flowing from the electrode into the oocyte, a maximal estimate of JS of 1500 X 10-14 mol s-1 was obtained. In this case case O () was calculated to be 228 mosmol l-1, 15 mosmol l-1 above the bathing solution osmolarity. In the case where the clamp current was carried entirely by K+ flowing from the oocyte into the electrode, a minimal estimate of JS of 300 X 10-14 mol s-1 was obtained. In this case O () was calculated to be 210.5 mosmol l-1, 2.5 mosmol l-1 below the bathing solution osmolarity.
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REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionAppendixReferences |
|---|
| BORGNIA M., NIELSEN, S., ENGEL, A. & AGREE, P. (1999). Cellular and molecular biology of the aquaporin water channels. Annual Review of Biochemistry 68, 425-458 | [Abstract/Full Text] |
| CARPI-MEDINA P., LEÓN, V., ESPIDEL, J. & WHITTEMBURY, G. (1988). Diffusive water permeability in isolated kidney proximal tubular cells: Nature of the cellular water pathways. Journal of Membrane Biology 104, 35-43 | [Medline] |
| DAINTY J. & HOUSE, C. R. (1966). Unstirred layers in frog skin. Journal of Physiology 182, 66-78 | [Medline] |
| DEMPSTER J. A., VAN HOEK , A. N., DEJONG, M. D. & VAN OS , C. H. (1991). Glucose transporters do not serve as water channels in renal and intestinal epithelia. Pflügers Archiv 419, 249-255 | [Medline] |
| GONZÁLES E., CARPI-MEDINA, P., LINARES, H. & WHITTEMBURY, G. (1984). Osmotic water permeability of the apical membrane of proximal straight tubular (PST) cells. Pflügers Archiv 402, 337-339 | [Medline] |
| GREEN R. & GIEBISCH, G. (1984). Luminal hypotonicity: a driving force for fluid absorption from the proximal tubule. American Journal of Physiology 246, F167-174 | [Medline] |
| HEDIGER M. A., TURK, E. & WRIGHT, E. M. (1989). Homology of the human intestinal Na+/glucose and Escherichia coli Na+/proline cotransporters. Proceedings of the National Academy of Sciences of the USA 86, 5748-5752 | [Medline] |
| HEESWIJK M. P. E. & VAN OS, C. H. (1986). Osmotic water permeabilities of brush border and basolateral membrane vesicles from rat renal cortex and small intestine. Journal of Membrane Biology 92, 183-193 | [Medline] |
| LOIKE J. D., HICKMAN, S., KUANG, K., XU, M., CAO, L., VERA, J. C., SILVERSTEIN, S. C. & FISCHBARG, J. (1996). Sodium-glucose cotransporters display sodium- and phlorizin-dependent water permeability. American Journal of Physiology 271, C1774-1779 | [Medline] |
| LOO D. D. F., HAZAMA, A., SUPPLISSON, S., TURK, E. & WRIGHT, E. M. (1993). Relaxation kinetics of the Na+/glucose cotransporter. Proceedings of the National Academy of Sciences of the USA 90, 5767-5771 | [Abstract] |
| LOO D. D. F., ZEUTHEN, T., CHANDY, G. & WRIGHT, E. M. (1996). Cotransport of water by the Na+/glucose cotransporter. Proceedings of the National Academy of Sciences of the USA 93, 13367-13370 | [Abstract/Full Text] |
| LOO D. F., HIRAYAMA, B. A., MEINILD, A.-K., CHANDY, G., ZEUTHEN, T. & WRIGHT, E. (1999). Passive water and ion transport by cotransporters. Journal of Physiology 518, 195-202 | [Abstract/Full Text] |
| MACAULAY N., GETHER, U., KLAERKE, D. A. & ZEUTHEN, T. (2000). Water transport by the Na+-coupled glutamate cotransporter. Journal of Physiology 530, 367-378. | [Abstract/Full Text] |
| MACKENZIE B., LOO, D. D. F. & WRIGHT, E. M. (1998). Relationship between Na+/glucose cotransporter (SGLT1) currents and fluxes. Journal of Membrane Biology 162, 101-106 | [Medline] |
| MEINILD A.-K., KLAERKE, D. A., LOO, D. D. F., WRIGHT, E. M. & ZEUTHEN, T. (1998a). The human Na+-glucose cotransporter is a molecular water pump. Journal of Physiology 508, 15-21 | [Abstract/Full Text] |
| MEINILD A.-K., KLAERKE, D. A. & ZEUTHEN, T. (1998b). Bidirectional water fluxes and specificity for small hydrophilic molecules in aquaporins 0 to 5. Journal of Biological Chemistry 273, 32446-32451 | [Abstract/Full Text] |
| MEINILD A.-K., LOO, D. D. F., HIRAYAMA, B. A., ZEUTHEN, T. & WRIGHT, E. M. (2000). Water transport by the Na+/dicarboxylate cotransporter. American Journal of Physiology 278, F777-783 | [Abstract/Full Text] |
| PAPPENHEIMER J. R. & REIS, K. Z. (1987). Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat. Journal of Membrane Biology 100, 123-136 | [Medline] |
| PARENT L., SUPPLISSON, S., LOO, D. D. F. & WRIGHT, E. M. (1992). Electrogenic properties of the cloned Na+/glucose coransporter: I. Voltage-clamp studies. Journal of Membrane Biology 125, 49-62 | [Medline] |
| PRATZ J., RIPOCHE, P. & CORMAN, B. (1986). Osmotic water permeability and solute reflection coefficients of rat kidney brush-border membrane vesicles. Biochimica et Biophysica Acta 861, 395-397 | [Medline] |
| SCHNERMANN J., CHOU, C.-L., MA, T., TRAYNOR, T., KNEPPER, M. A. & VERKMAN, A. S. (1998). Defective proximal tubular fluid reabsorbtion in transgenic aquaporin-1 null mice. Proceedings of the National Academy of Sciences of the USA 95, 9660-9664 | [Abstract/Full Text] |
| SOREQ H. & SEIDMAN, S. (1992). Xenopus oocyte microinjection: from gene to protein. Methods in Enzymology 207, 225-265 | [Medline] |
| VALLON V., VERKMAN, A. S. & SCHNERMANN, J. (2000). Luminal hypotonicity in proximal tubules of aquaporin-1-knockout mice. American Journal of Physiology 278, F1030-1033 | [Abstract/Full Text] |
| VERKMANN A. S. (1999). Lessons on renal physiology from transgenic mice lacking aquaporin water channels. Journal of the American Society of Nephrology 10, 1126-1135 | [Abstract/Full Text] |
| VERKMAN A. S. & IVES, H. E. (1986). Water permeability and fluidity of renal basolateral membranes. American Journal of Physiology 250, F633-643 | [Medline] |
| WORMAN H. J. & FIELD, M. (1985). Osmotic water permeability of small intestinal brush-border membranes. Journal of Membrane Biology 87, 233-239 | [Medline] |
| WRIGHT E. M., LOO, D. D. F., PANAYOTOVA-HEIERMANN, M., HIRAYAMA, B. A., TURK, E., ESKANDARI, S. & LAM, J. (1998). Structure and function of the Na+/glucose cotransporter. Acta Physiologica Scandinavica 16, 1217-1224 | |
| ZAMPIGHI G. A., KREMAN, M., BOORER, K. J., LOO, D. D. F., BEZANILLA, F., CHANDY, G., HALL, J. E. & WRIGHT, E. M. (1995). A method for determining the unitary functional capacity of cloned channels and transporters expressed in Xenopus laevis oocytes. Journal of Membrane Biology 148, 65-78 | [Medline] |
| ZEUTHEN T. (1991). Secondary active transport of water across ventricular cell membrane of choroid plexus epithelium of Necturus maculosus. Journal of Physiology 444, 153-173 | [Abstract] |
| ZEUTHEN T. (1994). Cotransport of K+, Cl- and H2O by membrane proteins from choroid plexus epithelium of Necturus maculosus. Journal of Physiology 478, 203-219 | [Abstract] |
| ZEUTHEN T. (1996). Molecular Mechanisms of Water Transport. R. G. Landes Company, Texas and Springer, Berlin | |
| ZEUTHEN T. (2000). Molecular water pumps. Reviews of Physiology, Biochemistry and Pharmacology 141, 97-151 | [Medline] |
| ZEUTHEN T., HAMANN, S. & LA COUR, M. (1996). Cotransport of H+, lactate and H2O by membrane proteins in retinal pigment epithelium of bullfrog. Journal of Physiology 497, 3-17 | [Abstract] |
| ZEUTHEN T., MEINILD, A.-K., KLAERKE, D. A., LOO, D. D. F., WRIGHT, E. M., BELHAGE, B. & LITMAN, T. (1997). Water transport by the Na+/glucose cotransporter under isotonic conditions. Biology of the Cell 89, 307-312 | [Medline] |
Acknowledgements
The Danish Research Council, the Novo-Nordisk Foundation, and US Fonds DK 19567 are thanked for financial support. The technical assistance of Tove Soland, Birthe Lynderup and Svend Christoffersen is gratefully acknowledged.
Corresponding author
T. Zeuthen: The Panum Institute, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark.
Email: zeuthen@mfi.ku.dk
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