Sulfate-sodium cotransport by brush-border membrane vesicles isolated from rat ileum

GASTROENTEROLOGY

1981;80:22-30

Sulfate-Sodium Cotransport by BrushBorder Membrane Vesicles Isolated from Rat Ileum HEINRICH and HEINI

LUCKE, MURER

GERTRAUD

STANCE,

Max-Planck-Institut fur Biophysik, Frankfurt am Main, Germany; and Division of Gastroenterology and Metabolism, Department of Medicine, University of Gottingen, Gtittingen, Germany

Uptake of inorganic sulfate into brush-border membrane vesicles isolated by a calcium precipitation method from rat small intestine was investigated using a rapid filtration technique and %rIfur acid as tracer. Sulfate uptake by membrane vesicles was osmotically sensitive, suggesting transport into an intravesicular space rather than binding to or incorporation into the membrane. Transport of sulfate into brush-border vesicles isolated from rat ileum was only stimulated by sodium ions as compared with other monovalent cations. A typical “overshoot” phenomenon was observed in the presence of an inwardly directed NaCl gradient. Tracer sulfate exchange was faster in the presence of sodium than in the presence of potassium. Addition of the ionophores for monovalent cations, monactin, or gramicidin D, decreased the sodium gradient-driven sulfate uptake. Sulfate uptake showed a saturation phenomenon only in the presence of sodium. Transstimulation of sodium-dependent sulfate transport was shown with Mo04’--, but not with PO,*- and WO,+. Changing the electrical potential difference across the membrane vesicles by establishing different diffusion potentials (anion replacement; potassium gradient & valinomycin) did not alter sodiumdependent sulfate uptake. Stimulation of sulfate transport by sodium was greater in membrane vesicles from ileal segments than from duodenum or jeReceived August 30, 1979. Accepted July 11, 1980. Address requests for reprints to: Dr. Heinrich Lttcke, Division of Gastroenterology and Metabolism, Department of Medicine, University of Gijttingen, Robert Koch Strasse 40, 3400 Giittingen, Germany. Dr. Murer’s present address is: Institut fur Physiologische Chemie, Universitat Freiburg, Freiburg, Switzerland. This study was supported by Deutsche Forschungsgemeinschaft (Lu 263/l). We are grateful to Professor Dr. K. J. Ullrich for valuable discussion during the preparation of the manuscript. 0 1981 by the American Gastroenterological Association OOlS-5085/81/010622-09$02.50

junum. It is concluded that isolated brush-border membranes of rat ileum contain an electroneutral sodium-sulfate cotransport system. From studies carried out in vivo or in vitro it is known that inorganic sulfate is absorbed in the small intestine (l-4).Using in vitro techniques [everted sacs (2-4),excised rings (3)], several groups were able to provide evidence for a mucosal-toserosal transport of inorganic sulfate against its concentration gradient in ileal segments from rat, rabbit, hamster, and guinea pig (2-4).Furthermore, the study of Anast et al. (4)suggested that sodium plays a major role in the transepithelial transport of iqorganic sulfate in the ileum. In micropuncture experiments on the reabsorption of inorganic sulfate in the rat and hamster proximal tubule it was recently demonstrated that transepithelial transport of inorganic sulfate across the renal proximal tubular epitheiium is entirely dependent on the presence of sodium and can be inhibited by ouabain, the inhibitor of the (Na’ + K+)-stimulated adenosine triphosphatase (5). Thus, sulfate transport in the ileum seems to involve mechanisms similar to those present in the renal proximal tubule. Subsequent studies with brush-border membrane vesicles isolated from rat kidney cortex have documented that these membranes contain a sodium-sulfate cotransport system (6).These findings suggested secondary active transepithelial renal proximal tubular transport of sulfate coupled to the primary active transport of sodium via a flux coupling mechanism between sodium and sulfate flux located in ’ the brush-border membrane. Secondary active sodium-dependent transport systems in renal proximal tubular as well as in small intestinal epithelia have thus far been described for many other substrates including inorganic phos-

January 1981

phate, taurocholate, sugars, and amino acids (7,8). The results of the present study demonstrate that brush-border membrane vesicles isolated from rat ileum contain a sodium-sulfate cotransport system, similar to that described for rat proximal tubular brush-border membranes (6).

Materials and Methods

SULFATE-SODIUM COTRANSPORT IN RAT ILEUM

Kinne (15). All enzyme assays were performed with a LKB reaction rate analyzer (model 8600) at 37’C. The maltase activity of the brush-border membrane vesicles isolated from the ileal segment of’rat small intestine was enriched 26.3 f 8.3-fold (mean + SD of 34 determinations) compared with the starting homogenate, whereas (Na++K+)stimulated adenosine triphosphatase was not enriched (0.6 f 0.3; mean f SD of 34 determinations). All experiments were performed in duplicate and were repeated at least four times with similar results.

Methods Membrane preparation. Brush-border membranes of rat duodenal, jejunal, and distal ileal segments were prepared from male Wistar rats of 180-220 g body wt by a modification of the method of Schmitz et al. (9) as described by Litcke et al. (10). Mucosal scrapings were used instead of a suspension of epithelial cells. Briefly, after homogenization of the mucosal scrapings in a hypoosmotic medium and addition of CaCl, (final concentration 10 mM) the brush-border membranes were purified by differential centrifugation at o-4%. The brush-border membranes thus obtained were suspended in 100 mM mannitol, 20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) adjusted to pH 7.4 with Tris base unless indicated otherwise. Transport studies. Uptake of labeled compounds by isolated brush-border membrane vesicles was measured by a rapid filtration technique as described previously (11). In essence, flux measurements were started by adding a portion of 20 pl of membrane suspension to 100 pl of incubation medium (kept at 25%). The exact compositions of the incubation media are given in the figures, legends, and tables. Uptake was stopped by adding 29~1 aliquots of the incubation mixture to 1 ml of ice-cold stopping solution. The resultant suspension was rapidly filtered through a cellulose acetate filter (0.6 pm pore size, Sartorius, Gottingen, Germany) and washed immediately with 5 ml of ice-cold stopping solution. The stopping solution contained 100 mM mannitol, 20 mM HEPES-Tris, pH 7.4, 100 mM choline chloride, and 25 mM MgSO,. In the experiments on the osmotic sensitivity of sulfate uptake, the stopping solution contained the same concentration of cellobiose as the incubation media. Radioactivity retained by the filters was counted in a liquid scintillation counter using Rotizint 33 as scintillation cocktail (Roth, Karlsruhe, Germany). The uptake values were expressed as picomoles (calculated from the specific activity of inorganic sulfate in the medium) per milligram of membrane protein pipetted onto the filter. All the values were corrected for the radioactivity retained on the filters in an experiment with an incubation medium using only buffer instead of membrane protein (blank value). Protein and enzyme determination. Protein was determined as described by Lowry et al. (12) with bovine serum albumin (Behring-Werke, Marburg, Germany) as standard. Maltase (EC 3.2.1.20) a marker of the brush-border membrane, was assayed as described by Evers et al. (13). Activity of (Na++K+)-stimulated adenosine triphosphatase (EC 3.6.1.3), a marker of the basolateral plasma membrane (14) was measured as reported by Berner and

23

Materials 35Sulfuric acid (sp act 43 Ci/mg, theoretical maximum) and D-[l-3H(N)]glucose (sp act 15-30 Ci/mmol) were purchased from New England Nuclear Corp., (Boston, Mass.). N-2-hydroxyethylpiperazine-IV-2-ethane sulfonic acid and valinomycin were obtained from Serva (Heidelberg, Germany). Enzymes and substrates needed for maltase and (Na++K+)-stimulated ATPase assays as well as gramicidin D were obtained from Boehringer (Mannheim, Germany). Monactin was a gift from Ciba-Geigy AG (Basel, Switzerland).

Results In Figure 1, the uptake of inorganic %ulfate (a), and of D-[“HIglucose (b) by brush-border membrane vesicles isolated from rat duodenum, jejunum, or distal ileum is compared. In the presence of a sodium gradient directed from outside to inside of the membrane vesicles, the ileal brush-border membranes show the highest initial sulfate uptake rate as compared with the presence of a potassium gradient. Stimulation of sulfate uptake by sodium was 1%fold in ileum, fivefold in duodenum, and fourfold in jejunum. In contrast, sodium stimulation of glucose uptake was most pronounced in the vesicles isolated from duodenum followed by jejunum and ileum. Since our main interest was to study a sulfate-sodium cotransport system, only the ileal membranes were investigated in more detail in further experiments. The question whether the uptake of sulfate by brush-border membranes represents binding to the membrane surface and/or transport into an intravesicular space was examined by analyzing the effect of the osmolarity of the incubation medium on uptake of sulfate after prolonged incubation (equilibrium). As shown in Figure 2, the amount of sulfate taken up by the vesicles after 60 min of incubation was inversely related to the osmolarity in the medium varied by different concentrations of impermeant cellobiose. Only a little uptake was obtained by extrapolating to infinite osmolarity (zero intravesicular

space).

These

results

indicate

predominantly,

transport into an intravesicular space rather than binding to, or incorporation into, the membrane.

24

LiiCKE

ET AL.

GASTROENTEROLOGY

o-@

Na Cl grad.

-4

KCI

duodenum

grad.

w-e

Na Cl grad.

-

K Cl

unum

grad.

e

Na Cl grad.

&A

K Cl

grad.

1

Vol. 80, No. 1

ileum

b

a

Incubation

time

(min)

Figure 1. Comparison

between sulfate (a) and D-glucose (b) uptake by brush-border membrane vesicles prepared from duodenum, jejunum, and distal ileum. The experiments were carried out in an incubation medium containing 100 mM mannitol, 20 mM HEPES/Tris (pH 7.4), 100 mM NaCl or KC1 as appropriate and 0.075 mM %04-* (a) or 0.1 mM D-[3H]glucose (b). Equilibrium values for sulfate (a) uptake in pmoles/milligram of protein were: Duodenum: 57 f 33 (n = 4) in the presence of NaCl (u); 58 f 39 (n = 4) in the presence of KC1 (0- -0). lejunum: 57 * 30 (n = 13) in the presence of NaCl @ I); 57 f 36 (n = 13) in the presence of KC1 (U-Cl). Ileum: 60 f 32 (n = 25) in the presence of NaCl (A-A); 61 f 37 (n = 25) in the presence of KC1 (A- -A). Equilibrium values for a-D-glucose(b) uptake in pmoles/milligram of protein were: Duodenum: 32 f 30 (n = 5) in the presence of NaCl (U); 40 + 24 (n = 5) in the presence of KC1 (0- -0). Jejunum: 59 f 38 (n = 7) in the presence of NaCl @- r); 49 t- 7 (n = 7) in the presence of KC1 m. Ileum: 68 f 50 (n = 19) in the presence of NaCl (A-A); 69 f 36 (n = 6) in the presence of KC1 (A- -A). Results are means f SD. Incubation temperature was 25%

Effect of Sodium on Sulfate

Uptake

When ileal brush-border membranes were prepared in a sodium-chlbride free medium and incubated in a sodium-chloride containing buffer, the uptake of sulfate was very rapid and transiently exceeded the values reached after 1, 2, and 60 min (equilibrium) of incubation (Figure 3, upper curve). This “overshoot” suggests an intravesicular accumulation of sulfate above the equilibrium concentration which can occur because of the persistence of a sodium gradient when the intravesicular sulfate has already reached the concentration in the incubation medium (7,16). In the presence of a potassium-chloride gradient (Figure 3, lower curve), the initial uptake of sulfate was one-fifteenth that with sodiumchloride, and the “overshoot” did not occur. In the absence of salt and chemical sulfate gradients, i.e., under tracer exchange conditions, the uptake of labeled sulfate proceeded more rapidly (2.5fold) in the presence of sodium (Figure 4, upper

curve) than in the presence of potassium (Figure 4, lower curve). Since there was no gradient as driving force, an “overshoot” uptake of sulfate cannot occur under these experimental conditions. Addition of monactin or gramicidin D, ionophores for monovalent cations (17), reduced the sodium gradient driven sulfate uptake and prevented the “overshoot” (Table 1) without changing the equilibrium value for sulfate. If the sulfate movement across the membrane would be primarily stimulated by a vesicle inside positive diffusion potential exerted by the sodium gradient, the ionophores, enhancing the membrane conductance for sodium, should cause an increased initial uptake rate of sulfate, due to indiffusion creased positivity of the 1 ansmembrane potential. The decreased uptake rate for sulfate in the presence of the ionophores, as shown in Table 1, can therefore be taken as a further evidence for a direct coupling between sodium flux and sulfate flux. In such sodium cotransport systems the enhanced breakdown of the sodium gradient in the presence of

January

SULFATE-SODIUM

1983

COTRANSPORT

IN RAT

Incubation

Figure

L

1

1

2

2. Influence isolated

I

I

I

I

4 5 6 3 Osmolarity (osrnol~’1

of osmolarity brush-border

of medium membrane

Figure

1

I

I3

on sulfate uptake by vesicles: The uptake

of 0.075 mM Na,%O., was determined in the presence of 100 mM mannitol, 20 mM HEPES/Tris (pH 7.4), 10 mM NaCl and sufficient cellobiose to give the indicated osmolarity. The values given represent equilibrium values obtained after 60 min of incubation at 25°C.

sodium ionophores must lead to a reduction of the sodium gradient driven substrate transport (uncoupling effect). Cation Specificity and Sodium Sensitivity the Transport System

of

Table 2 shows the effect of different cation gradients on sulfate transport. In the presence of a sodium-chloride gradient an “overshoot” phenome-

I

g250 % 6200

*NaCI-gradient 0 K Cl -gradient

ILEUM

25

tlme(mln)

4. Effect of Na+ on the sulfate tracer exchange by brushborder membrane vesicles: Membrane vesicles prepared in 100 mM mannitol, 20 mM HEPES/Tris (pH 7.4), 0.075 mM Na,S04 (unlabeled) and 100 mM NaCl (A) or 0.075 mM K,S04 (unlabeled) and 100 mM KC1 (V) were incubated at 25% in the same medium containing tracer ‘%-sulfate. Equilibrium values were 47 -+ 24 pmol/mg of protein in the presence of NaCl and 54 f 23 pmol/mg of protein in the presence of KCl. Number of experiments was 15 with NaCl and 10 with KCl. Results were means f SD.

non for sulfate uptake was observed. Gradients of other chloride salts did not stimulate sulfate transport as compared with the uptake in the presence of choline-chloride. Sodium increased the transport rate of sulfate about 13 times. Also the addition of valinomycin did not provoke stimulation of sulfate uptake in the presence of a potassium-chloride gradient. This lack of a valinomycin effect suggests that diffusion potential driven uptake of the sulfate anion into the vesicles is very low. In Figure 5 the initial sulfate uptake in the presence of 0.075 mM sodium sulfate is shown as a function of increasing sodium concentrations in the medium. Sodium was present under preequilibrated conditions where the sodium concentration outside is equal to that inside the vesicles: thus, increasing sodium concentrations will not alter the driving force conditions for a sodium-solute cotransport sys-

Table

2.

Eflect

of Monactin

and Gramicidin

D on Sulfate

Transport Sulfate (pmol/mg Conditions in medium (100 mM NaCl) Figure

3 Effect of Na+ and K+ gradients on sulfate uptake by brush-border membrane vesicles: Membrane vesicles were prepared in 100 mM mannitol, 20 mM HEPES/ Tris (pH 7.4). and incubated at 25°C in the same medium containing also 0.075 mM Naz3%04 and 100 mM NaCl (0) or 0.075 mM Kz3%0, and 100 mM KC1 (0). Equilibrium values were 60 f 32 pmol/mg of protein in the presence of NaCl and 61 f 37 pmol/mg of protein in the presence of KCl. Results are means f SD. Number of experiments was 25.

NaCl gradient NaCl gradient monactin NaCl gradient gramicidin

uptake, of protein)

0.33 min

1 min

60 min equil.

plus

132 34

77 35

52 51

plus

40

46

51

D

The experiments were carried out in an incubation medium as described in Figure 1. Monactin (10 mg/ml in ethanol), when present, was 12 pg/mg of protein. Gramicidin D (20 mg/ml in ethanol), when present, was 24 pg/mg of protein.

26

LiiCKE ET AL.

Table 2.

GASTROENTEROLOGY

Effect of Cation Replacement

on Sulfate Uptake into Brush-Border

Membrane

Vol. 60, No. 1

Vesicles

Sulfate uptake Percentage of equilibrium Salt in incubation medium (100 mM)

0.33 min

LiCl NaCl KC1 RbCl CsCl Choline chloride KC1 + valinomycin

1 min

13 f4

167 f 14 f 16 f 11 f 9f6 18 f

30f7

45 5 7 7

163 f 25 f 30 f 29 f 18 f 32 f

3

The experiments were carried out in an incubation tein. Results are means f SD.

of the Transport

System with

The “initial” sulfate uptake rates (20-s values) as a function of different sulfate concentrations in the medium in the presence of a sodium-chloride gradient were compared with the uptake of sulfate in the presence of a potassium-chloride gradient (Figure 6). Uptake of sulfate showed only a saturation phenomenon in the presence of a sodium gradient (Figure 6, upper curve). The smaller and not saturable uptake of sulfate in the presence of a potassium gradient might indicate uptake by simple diffusion (Figure 6, lower straight line). Therefore it

2 min 46f14

61f16

5

55 f 54 f 40 f 50 f 45 f 59 f

5 5 5 5 3 3

127 * 39 f 35 f 43 f 28 f 53 f

medium as described

tern. Half maximal saturation of the stimulatory effect of sodium can be observed between 5 and 10 mM sodium. Interaction Anions

14 4 14 15 11 7

19 9 21 23 13 11

in Figure 1. Valinomycin,

0

$5

T

1. 0

when present, was 20 &mg

mnus KCI-gradmt

Sulfate

25

.a

50 Sodium

15 chloride

100 (mM)

Figure 5. Influence of Na+ concentration on sulfate uptake into Na+-preequilibrated vesicles: Membranes were prepared in 100 mM mannitol, 20 mM HEPES/Tris (PH 7.4). and preincubated for 1 h in the same medium containing also 0.075 mM Na,%04 and different NaCl concentrations as given in the figure (l-100 mM); KC1 was added at various concentrations (99-O mM) to give a constant final salt concentration (100 mM). Number of experiments was 4. Results are means f SD.

of pro-

NaCt-gradnnt

0 K a -gradient . NaU -gradlent

2

16 16 19 13 10 11

can be assumed that the carrier-mediated sodiumdependent sulfate transport is represented by the difference between the uptake in the presence of sodium and the uptake in the presence of potassium (Figure 6, lower curve). The specificity of the transport system for anions was investigated by trans-stimulation experiments. Substrates present at the transmembrane side and exerting a stimulatory effect on the transmembrane flux of a second substrate (trans-stimulation) most probably share common transport pathways. Table 4 demonstrates that sodium-dependent tracer sulfate uptake occurred faster into vesicles preloaded with unlabeled SO,‘- and MOO,“- than into nonpreloaded

0

z

No. of experiments

pmol/mg of protein (60-min equil.)

ImMl

Figure 6. Sulfate uptake in relation to the sulfate concentration in the incubation medium: Membrane vesicles were prepared in 100 mM mannitol, 20 mM HEPES/Tris (pH 7.4) and incubated in a medium containing 100 mM mannitol, 20 mM HEPES/Tris (pH 7.4) in the presence of 100 mM NaCl (0) or in the presence of 100 mM KC1 (0). The specific Na+-dependent sulfate uptake (A) is obtained by uptake in the presence of a NaCl gradient (0) minus uptake in the presence of a KC1 gradient (0). The amount taken up by the vesicles at 25°C after 20 s is given. Number of experiments is 4. Results are means f SD.

January

Table

SULFATE-SODIUM

1981

3.

Trans-stimulation

of Na+-Dependent

Sulfate

COTRANSPORT

IN RAT

27

ILEUM

Uptake by SO,-‘, MOO,-*, WO,-‘, and PO,-’ Sulfate

uptake

(pmol/mgofprotein) No. of experiments Conditions

inside

No further

addition

0.33 min

vesicles

plus unlabeled SO,-’ No further addition plus unlabeled MOO,-’ No further addition plus unlabeled WO,-’ No further addition plus unlabeled PO,-’

17 f5 31f8'= 12 + 5 26&l'

(0.6 mM)

(0.6 mM)

14 f 3 (0.6 mM)

17 +-2

(0.6 mM)

23 f 5 20 f 1

1 min

25 f4 33 fP 25*4 31+2d 21f6 25 f3 26f4 25 lt5

60-min

equil.

41f 10 41f 6 46+-l 45 +-2 40+8 42 f3 38f2 38f2

4 4 4 4 4 4 4 4

The amount of SO,-z taken up was measured during the first 209, at 1 min, and at equilibrium after 60 min. Membrane vesicles loaded 20 mM HEPES/Tris (pH 7.4) (control) and in addition with the listed unlabeled substances were incubated at with 100 mM mannitol, 25°C in a medium containing 100mM mannitol, 20 mM HEPES/Tris (pH 7.4), and 100 mM NaCl. In the incubation media sulfate concentration was 0.06 mM Naz3%0,. The concentration in the incubation media of the different anions tested as substrates to induce transstimulation was also 0.06 mM (also in the individual controls). o 2 p < 0.025; b g p < 0.02; c 2 p < 0.005; d 2 p < 0.05. Results are means zk SD

This observation indicates that SO,“- and MOO,*- are transported via the same cotransport system. This trans-stimulation was not observed with PO,‘- and WO,“- (Table 3). The transport rates are low in this experiment, because the experiment was performed with sodium equilibrated vesicles. To examine for the existence of a chloride-sulfate exchange system brush-border membrane vesicles were preloaded with different sodium salts using anions such as chloride, nitrate, cyclamate, gluconate, and thiocyanate, and incubated in a medium containing the same salt at the same concentration and labeled sulfate in addition. Under these conditions there was no significant difference between transport of sulfate in the presence of the different sodium salts (except inhibition in the presence of thiocyanate, data not shown). This finding suggests that chloride does not interact with the sodium-dependent sulfate transport system. Dipyridamol, H,DIDS (4,4’-diisothiocyanodihydrostilbene-2,2’-disulfonic acid), phloretin, and furosemide, known inhibitors of anion transport in Ehrlich ascites tumor cells and red blood cells, were without significant effects on sodium-dependent transport of sulfate in brush-border membrane vesicles isolated from rat ileum (data not shown). controls.

Influence Uptake

of Membrane

Potential

on Sulfate

The membrane potential of the brush-border membrane vesicles can be manipulated by imposing artificial diffusion potentials (18). Since diffusion potentials depend on the relative mobility of cations and anions, they can be modified by means of anion

replacement (lipophilic anions vs. hydrophilic anions) or by the use of ionophores. As shown in Table 4, replacement of chloride by the lipophilic anion nitrate reduces the initial uptake of sulfate to a small, but not significant extent. An inhibition is seen with thiocyanate-another lipophilic anion-which seems to inhibit sulfate transport. Since the inhibition of sulfate uptake in the presence of the thiocyanate anion was also observed under thiocyanate preequilibrated conditions (data not shown), this “anion effect” must reflect inhibition of sulfate uptake via diffusion potential independent effects. The more hydrophilic anions, cyclamate and gluconate, did not alter sodium-dependent sulfate uptake significantly as compared with chloride. Also valinomycin, an ionophore which mediates potential-sensitive movements of potassium across lipid bilayers (17), was used in membrane vesicles preloaded with potassium-gluconate (potassium inside vesicles > potassium outside vesicles) to vary the transmembrane diffusion potential (increased inside negativity). Valinomycin did not influence sodium gradient-dependent uptake of sulfate under these conditions (Table 5a); D-ghCOSe, known to be transported in a potential-sensitive manner in rat small intestine (18), was stimulated in the same experiments about threefold in the presence of valinomycin compared with its uptake in the absence of valinomycin (Table 5b). Thus, alterations of the electrical diffusion potentials across the brush-border membrane are not able to alter sodium-dependent transport of sulfate. This insensitivity of sodium-dependent sulfate uptake towards transmembrane diffusion potential suggests that sodium-dependent sulfate uptake is an electro-

28

LijCKE ET AL.

Table

4.

Effect of Anion

GASTROENTEROLOGY Vol. 80, No. 1

Replacement

on Sulfate

Uptake into Brush-Border

Membrane

Vesicles

Sulfate uptake Percentage of equilibrium Salt in incubation

medium (100 mM) NaNO, NaSCN

NaCl Na-cyclamate Naigluconate

0.33 min

1 min

pmol/mg of protein 60 min equil.

No. of experiments

150 f 53 113 f17 179 f 25

134 f 19 111 f 12 155 f 18

44 f 15 32 f 13 59 f 15

3 3 3

14Of22 183 f 41

130 f 18 145 f 36

69 f 16 62 f 12

3 3

The experiments were carried out in an incubation medium as described in Figure 1.Results are means f SD.

neutral process. An alternate explanation for the observed potential insensitivity is unlikely, i.e., that an increased inside negativity of the transmembrane electrical diffusion potential difference would inhibit the free diffusion of the anion sulfate to the same extent as increased inside negativity stimulates sodium coupled sulfate movement carrying a positive charge. As can be seen from Figure 6 (sulfate uptake in the presence of potassium), free diffusion seems to represent only a minor portion of total transmembrane sulfate flux and is not significantly altered by valinomycin/potassium-induced diffusion potentials (Table 2).

mucosa of rabbit ileum that sulfate influx from mucosal medium into the epithelium is sodium-coupled and electrically silent. The properties of the sulfatesodium cotransport system in rat ileal brush-border membranes are strikingly similar with respect to sodium stimulation, substrate specificity, and electroneutrality to those observed in brush-border membrane vesicles of rat kidney proximal tubule (6). However, ileal and proximal tubular brush-border membranes show different transport characteristics of sulfate transport as compared with other plasma membranes such as erythrocyte membranes or Ehrlich ascites tumor cell membranes where sulfate transfer seems to proceed via an anion exchange system (20-22). An advantage of studies with isolated membrane vesicles is that the driving forces of the transport across the membrane can be studied in the absence of any undefined “cellular” energy source such as ATP or unknown intracellular concentrations of substances interfering with the transport system. Based on our study, at least two different driving forces have to be considered for the coupled transport of sodium and sulfate: the concentration differences of (a) sulfate and (b) sodium across the vesicular membrane. That a sodium concentration difference provides directly a driving force for trans-

Discussion The results presented in this paper indicate that the transfer of inorganic sulfate across the brush-border membrane of rat ileum occurs via a sodium-dependent transport system. The experimental evidence for a direct effect of sodium rather than a diffusion potential mediated effect are (a) specific stimulation by sodium, (b) stimulation by sodium in the absence of a sodium gradient, and (c) saturation phenomenon only in the presence of sodium. In agreement with our results, Smith et al. (19) demonstrated by flux measurements across short-circuited

Table

5.

Effect of Valinomycin

on Sulfate

Transport

in Potassium-Preloaded

Membranes

Sulfate uptake (pmol/mg of protein) No. of Conditions in incubation medium Sodium-gluconate gradient Sodium-gluconate gradient plus valinomycin

60 min equil.

1 min

0.33 min a

b

a

123 f 34 107 f 14

97 * 12 283 f 23

116 f 20 lOOf

b 86f7 16Of55

experiments

a

b

a

104f18 99 f 19

82 f 31 81 f 30

4 4

b 4 4

The membranes were prepared in buffer containing 100 mM mannitol, 20 mM HEPES/Trts (pH 7.4) and, in addition, 50 mM potassiumgluconate. Sulfate uptake was initiated by adding 1 vol of K+-preloaded membranes to 11 vol of incubation medium with sodium-gluconate (100 mM), mannitol (100 mM), HEPES/Tris (20 mM, pH 7.4) and %ulfate (0.075 mM) (a) or [3H]n-glucose (0.1 mM) (b) as substrate. Valinomycin, when present, was 19 pg/mg of protein. Results are means f SD.

January

1981

membrane sulfate movement via a flux coupling mechanism is demonstrated by the experiments using the ionophores monactin or gramicidin D (Table 1). On the other hand the driving force is related to the sulfate concentration difference documented in the trans-stimulation experiments (Table 3). In the brush-border membrane vesicles, sodium gradient-induced substrate transport against its concentration gradient can only be transient in contrast to the intact cell where sodium-coupled transport systems are part of secondary active transepithelial transport mechanisms. Via flux coupling of sulfate and sodium, sulfate can be transported across the luminal membrane against its concentration gradient driven by the concentration difference for sodium. Thereby, a high intracellular sulfate concentration will build up within the epithelial cells and provide the driving force for the efflux from the cell into the interstitium. This exit mechanism is thus far unknown and should be a subject of further investigations. It might be possible that the exit of sulfate proceeds via an anion exchange mechanism known to be present in other plasma membranes of eucariotic cells (20-22). Evidence for such an exit mechanism was recently provided by Smith et al. (19) who showed in short-circuited mucosa of rabbit ileum that serosal addition of &acetamido-4’-isothiocyanostilbine-2,2’-disulfonic acid reduced mucosa to serosa flux of sulfate by 95% and serosa to mucosa flux by 75% thus abolishing net flux. They further demonstrated a decreased mucosa-to-serosa flux and an increased serosa-to-mucosa flux by replacing Cl with gluconate, nearly abolishing net flux. The concentration difference for sodium across the luminal membrane necessary for the coupled sodiumsulfate influx is maintained by a sodium pump located at the contraluminal cell side (14,23). Furthermore, our results suggest that sulfate transport in rat ileum occurs via a mechanism separate from those involved in the absorption of other inorganic anions such as chloride or phosphate. Our investigations also documented that sulfate present in the intestinal lumen at concentrations
SULFATE-SODIUM

COTRANSPORT

constant for chloride transfer membrane is above 200 mM.

IN RAT

ILEUM

29

in the brush-border

References 1. Dziewiatkowski DD. On the utilisation of exogenous sulfate sulfur by the rat in the formation of ethereal sulfates as indicated by the use of sodium sulfate labeled with radioactive sulfur. J Biol Chem 1949;178:389-93. of radiosulfate by the ileum of 2. Deyrup IJ. In vitro transport the rat. Fed Proc 1963;22:332. JWL. Species differences in the intestinal response 3. Robinson to sulfate ions. Febs Lett 1969;5:157-60. 4. Anast C, Kennedy R, Volk G, Adamson L. In vitro studies of sulfate transport by the small intestine of the rat, rabbit and hamster. J Lab Clin Med 1965;65:963-11. reabsorption in the 5. Ullrich KJ, Rumrich G, Kloss S. Sulphate proximal convolution of the rat kidney. Pfltigers Arch Suppl 1979;379:R17. co-transport by 6. Lticke H, Stange G, Murer H. Sulfate-sodium brush-border membrane vesicles isolated from rat kidney cortex. Biochem J 1979;182:223-29. and coupling of transport proc7. Murer H, Kinne R. Sidedness esses in small intestinal and renal epithelia. In: Semenza G, Carafoli E, eds. Biochemistry of membrane transport. Berlin: Springer, 1977;292-304. 6. Liicke H, Stange G, Kinne R, Murer H. Taurocholate-sodium co-transport by brush-border membrane vesicles isolated from rat ileum. Biochem J 1978;174:951-58. D. et al. Purification of the 9. Schmitz J, Preiser H, Maestracci human intestinal brush border membrane. Biochim Biophys Acta 1973;323:98-112. 10. Lucke H, Berner W, Menge H, Murer H. Sugar transport by brush border membrane vesicles isolated from human small intestine. Pfliigers Arch 1978;373:243-48. 11. Berner W, Kinne R, Murer H. Phosphate transport into brushborder membrane vesicles isolated from rat small intestine. Biochem J 1976;166:467-74. 12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75. 13. Evers C, Haase W, Murer H, Kinne R. Properties of brush border vesicles isolated from rat kidney cortex by calcium precipitation. Membrane Biochem 1978;1:203-19. 14. Fujita M, Matsui H, Natano K, Nakao M. Differential isolation of microvillus and basolateral membranes from intestinal mucosa. Biochim Biophys Acta 1972;274:336-47. of p-aminohippuric acid by 15. Berner W, Kinne R. Transport plasma membrane vesicles isolated from rat kidney cortex. Pfliigers Arch 1976;361:269-77. uptake in iso16. Evers J, Murer H, Kinne R. Phenylalanine lated renal brush border vesicles. Biochim Biophys Acta 1976;426:598-615. 17. Henderson PJF, McGivan JB, Cappell JB. The action of certain antibiotics on mitochondrial, erythrocyte and artificial phospholipid membranes. Biochem J 1969;111:521-35. of electrogenic Na+-de16. Murer H, Hopfer U. Demonstration pendent o-glucose transport in intestinal brush border membranes. Proc Nat1 Acad Sci USA 1974;71:484-88. S, Field M. Sulfate transport in rabbit 19. Smith PL, Orellana ileum: evidence for separate carrier-mediated steps at brushborder and basolateral membranes. Fed Proc 1986;39:285. and sulfate transport in Ehrlich ascites 20. Levinson C. Chloride

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LUCKE ET AL.

tumour cells: evidence for a common mechanism. J Cell Physiol 1978;95:23-32. 21. Gunn RB. Considerations of a titratable carrier model for sulfate transport in human red blood cells. In: Hoffman JF, ed. Membrane transport processes, Vol. 1. New York: Raven Press, 197881-77. 22. Lepke S, Fasold H, Pring M, Passow H. A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4’-diisothiocyanostil-

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bene-2,2’-disulfonic acid (DIDS) and its dihydro derivative (H,DIDS). J Membr Biol1976;29:147-77. 23. Hopfer U, Sigrist-Nelson K, Murer H. Intestinal sugar transport: studies with isolated plasma membranes. Ann NY Acad Sci 1975;284z414-27. 24. Liedke CM, Hopfer U. Chloride sodium symport versus chloride/hydroxide antiport or chloride uniport as mechanisms for chloride transport across rat intestinal brush-border membrane. Fed Proc 1980;39:734.