Camp.Biochm. Physiol. Vol. IOIA,No. 4, Printed in Great Britain

pp. 759-767,

0300-9629/92

1992

$5.00 + 0.00

0 1992Pergamon Press plc

CHARACTERIZATION OF THE TRANSPORT OF TRI- AND DICARBOXYLATES BY PIG INTESTINAL BRUSH-BORDER MEMBRANE VESICLES SIEGFRIEDWOL~AM,* CHRISTIANHAGEMANN,BEAT GRENACHERand ERWIN SCHARRER Institute of Veterinary Physiology, Winterthurerstras~ 260, CH-8057 Zurich, Switzerland. Tefephone: (01) 36.5-1347; Fax: (01) 365-1323 (Received 17 July 1991) Abstract-l. Transport of citrate and fumarate across the pig intestinal brush-border membrane (BBM) was investigated using isolated BBM vesicles. 2. Citrate and fumarate uptake was stimulated by an inwardly directed Na+ gradient consistent with Na+/citrate (fumarate) co-transport. Cis-inhibition and (runs-stimulation experiments strongly suggest the existence of a common transport site for tri- and dicarboxylates. 3. The protonated forms of citrate (citrate-‘, citratem2) seem to be much better transported than

eitratem3,indicated by the strong stimulation of citrate uptake at an extravesicular pH of 5.6 compared to pH 7.8. 4. Uptake of tri- and dicarboxylates seems to be potential-sensitive since citrate and in particular fumarate transport was enhanced by an inside negative potential difference. 5. Kinetics of succinate transport revealed a single carrier-mediated component with apparent kinetic constants of 0.43 nmol/mg protein-3 s (I’,,,) and 0.14 mmol/l (K,).

The tricarboxylic acid

citric acid has been shown to occur in the milk of various species (e.g. cow, goat, sheep, rabbit, pig, man) at concentrations of 2-lOmmol/l (Faulkner and Peaker, 1982; Konar et oil., 1971; Linzell et al., 1976; Peaker et al., 1981). Organic acids including citrate and fumarate are also present in plants (Prior et al., 1973; Russel and Van Soest, 1984) and are therefore ingested in particular by herbivores and omnivores. Furthermore, citrate is normally secreted with the gastric and pancreatic juice (Boustiere et al., 1985; Kuroshima et al., 1988; Lohse et al., 1981; Piper et al., 1967). Another potential source of dietary citric acid and also of the dicarboxylic acid fumaric acid is the use of those substances as feed additives in animal nutrition. Several investigators have reported improvement of growth performance and digestibility of protein and dry matter in piglets fed diets with citric or fumaric acid (Falkowski and Aherne, 1984; Henry ef af., 1985; Kirchgessner and Roth, 1982). Moreover, citrate has been shown to stimulate sodium and water absorption in human jejunum (Rolston et al., 1986) as well as in rabbit ileum (Newsome et al., 1983). These experiments substantiate the value of including citrate in oral rehydration solutions for the therapy of acute diarrhoea (Newsome er al., 1983; Rolston et al., 19861, aIthough the mechanisms of the effects of citrate remain to be clarified. The experiments of Newsome, Burgess and Holman (Newsome et al., 1983) suggest stimulation of a chloride-dependent epithelial uptake of sodium ions in the presence of citrate. *To whom correspondence

should be addressed.

In spite of the presence of citrate, fumarate, and other tri- and dicarboxylic acids in food and their growth-promoting effects, knowledge about intestinal transport of tri- and dicarboxylic acids is incomplete. Evidence for active Na+-dependent absorption of citrate has been presented in rabbits (Newsome et al., 1983) and hamsters (Browne et al., 1978). Additionaily, citrate was found to be rapidly absorbed from the gastrointestinai tract of dogs and a dosedependent increase in citrate con~ntration in the blood following intragastric administration has been reported (Verine et al., 1982). The most comprehensive study using various preparations of intact intestinal mucosa was carried out by Browne et al. (1978). They found citrate to be absorbed by a saturable Nat-dependent mechanism by sacs of everted intestine of hamsters. Furthermore, some evidence was presented for the transfer of intact citrate across the intestinal wall against a con~ntration gradient. Browne et nl. (1978) concluded from the inhibitory effect of di- and tricarboxylates on citrate transport that a specific, common transport site exists for the transfer of citric acid cycle intermediates. Furthermore, they suggested from their results that an additional saturable transport system might be involved in succinate absorption. Evidence for active Natdependent transport of dicarboxylates across the intestinal brush-order membrane (BBM) was presented by Moe et al. (1988) and for di- and tricarboxyiates in a more detailed study by Wolffram et al. (1990) using isolated brush-border membrane vesicles (BBMV). These studies lend further support to the findings of Na+-gradient-driven co-transport of triand dicarboxylates across the intestinal BBM. The results suggest the existence of a similar mechanism as found in the proximal tubule of the kidney. 759

SIEGFRIEDWOLFFRAM et al.

760

However, some differences appear to exist between the respective intestinal and renal transport mechanisms. For example transport across the intestinal BBM seems to occur by a potential-insensitive process (Moe et al., 1988; !&hell et al., 1983; Wolffram et al., 1990), whereas transport across the luminal membrane of proximal tubular cells was shown to be clearly influenced by a trans-membrane electrical potential difference (Grass1 et al., 1983; Hirayama and Wright, 1986; Jorgensen et af., 1983; Schell and Wright, 1985; Wright et al., 1983, 1982a,c). We now have investigated the mechanism(s) of citrate and fumarate transport across the intestinal brush border membrane using BBMV isolated from pig proximal jejunum. The influence of a transmembrane Na+ gradient, of various pH conditions, and of a transmembrane electrical potential difference on the uptake of citrate and fumarate by the vesicles was investigate. To gain insight into the substrate specificity of the transporter(s) for citrate and fumarate, some c&-inhibition as we11 as trans-stimulation experiments were also performed. Furthermore, the transport kinetics of succinate were determined. The findings obtained extend the observations by Moe et al. (1988) in pig intestinal BBMV with regard to substrate affinity, pH dependence and potential sensitivity of the carrier for tri- and dicarboxylates of the intestinal BBM. In conjunction with our previous study performed with calf BBMV, the results further indicate that certain species differences appear to exist as to the pH dependence of the respective transport mechanism for dicarboxylates. In addition, a potential sensitivity of fumarate transport across the intestinal BBM in calves was found in the present study. MATERIAL AND METHODS Preparation

of BBMV

and uptake experiments

The proximal jejunum of pigs and calves was removed from freshly killed animals at the local slaughter house. The intestine was immediately opened along the mesenterical border, cleaned with chilled saline and transported in icecold saline to the laboratory within 15-20 min. All subsequent isolation steps were performed at 4°C. BBMV were prepared from mucosal scrapings according to the method of Kessler et al. (1978a). The vesicles were preloaded with buffers as indicated in the legends of the Figures and Tables. Aliquots of 400~1 were stored under liquid nitrogen until use (Stevens et al., 1982). Purification of BBM from pig small intestine was routinely checked by determination of the BBM marker enzyme alkaline phosphatase (AP, EC 3.1.3.1) (test kit, Boehringer Mannheim GmbH, Germany). The specific AP activity (mUnits/mg protein: 633.5 f 110.4 and 8592.7 + 1678 in the homogenate and BBM suspension, respectively; mean + SEM from four preparations) was several-fold enriched in the final BBM fraction (13.6 & 2.4). Crosscontamination of these BBM preparations with basolateral membranes (BLM) was neghgibie since the specific activity of the Na+, K+-ATPase, a marker enzyme for basolateral membranes (EC 3.6.1.3) (Biber et al., 1981) was reduced in the final vesicle fraction compared with the activity of the mucosa homogenate (mUnits/mg protein: 124.7 f 25.3 and 59.9 + 36 in the homogenate and BBM suspension, respectively; mean j, SEM from four preparations). Protein was determined by using the Bio-Rad protein assay kit with bovine albumin as standard (Bio-Rad Lab., Gla~b~~, Switzerland).

The utility of the porcine BBMV was demonstrated by means of cumulative o-glucose transport (D-gtucose concentration: 0.1 mmol/l). Uptake of [3H]o-ghrcose (NEN, Boston, MA) was strongly Na+-dependent and showed a peak overshoot (at 30 set incubation)/equilibrium uptake (at 60 min incubation) ratio of 24.9 f 8.7 (mean &-SEM; duplicate determinations from four different vesicle preparations) in the presence of an inwardly directed Na+gradient (initial Na+-gradient: 100 mmol/I) whereas uptake under Na+-free conditions only slowly reached equilib~um. Mean eq~lib~~ space was ~calculated to be i.02 + 0.02 and 1.32 f 0.1 I ullma protein (mean f SEM of d&&ate determinations from-four different vesicle preparations) under Na+-gradient and Na+-free conditions, respectively. The enzymatic as well as functional characterization of BBMV prepared from the small intestine of calves has been reported previously (Wolffram et al., 1990). Uptake of ‘H-labeiled o-glucose (NEN, Boston, MA) or 14C-labelledcitric, fumaric, or succinic acid (Amersham Int., Amersham, England) was determined by a rapid filtration technique, Short-term incubations (incubation time < 10 set) were performed using a semi-automatic incubation device (Innovativ AG. Wallisellen. Switzerland) described first by Kessler et al. (1978b). Unless otherwise stated uptake was started by adding a 10 JII of membrane suspension to 20 or 40~1 of the incubation medium and quenched by adding 3 ml of ice-cold stop solution of the same composition as the final reaction mixture without the addition of substrate. The diluted mixture was immediately sucked through a nitro-cellulose filter (0.45 pm pore size, Schleicher and Schiill, Feldbach, S~tzerland) and rinsed twice with the stop solution. The composition of the reaction media is indicated in the legends of the Figures. Blanks without the addition of vesicle suspension were performed for correction of unspecific binding of radioactivity to the filters. Substrate uptake was measured at room temperature. Where indicated, valinomycin was added from an ethanolit stock solution to the thawed vesicle suspension I hr prior to the experiment to achieve concentrations of 20&ml vahnomycin and 0.1% ethanoi in the final reaction mixture. During this hour vesicles were kept on ice. The radioactivity remaining on the filters was determined by liquid scintillation counting in a Beta-counter (Betamatic I, Kontron AG, Zurich, Switzerland). Statistical evaluations Values are presented as means with the standard error of the mean (SEM). The differences between two means were statistically evaluated using the Student’s t-test (Sachs, 1984). If differences between more than two means were statistically evaluated a one way analysis of variance with pair comparisons according to Helm (1979) was performed. RESULTS

The time course of citrate uptake (concentration: 0.05 mmol/l) in the presence of an inwardly directed gradient (Na& or initial Na+ or choline+ choline,: = choline;, = 100 mmol/l, Nat: or 0 mmol/l) is shown in Fig. 1. In these experiments the pH of the intra- and extravesicular medium was varied to achieve the subsequent pH conditions @H,,,/pH,): 7.8/7.8, 5.6/5.6 and S/7.8. The possible influence of a transmembrane electrical potential difference on citrate uptake was ruled out by clamping the potential difference at zero (addition of the K+-ionophore valinomycin in the presence of a K+equilibrium of 100 mmol/l). Uptake under Na”-gradient conditions clearly exceeded uptake under Na+-free conditions (choline+ gradient) independently of the pH of the incubation media (Fig. 1).

Intestinal transport of tri- and dicarboxylates

761

15 set to 3 min, four determinations from two different vesicle preparations, each determined in dupli7.w7.0 5.6/5.6 5.60.6 cate). tW A n . Citrate uptake in the presence of an initial Na+ChliM’ •I A 0 gradient exhibited a clear pH dependence with uptake 1.o-5 values being highest in the presence of an inwardly s directed H+ gradient and lowest in the presence of the g 0.8. H+ equilibrium at pH 7.8. Citrate uptake at an extraand intravesicular pH of 5.6 was intermediate. In the P absence of Na+ citrate uptake was not substantially 2 0.6influenced by pH (Fig. 1). c In further experiments, the Na+- and pH-dependence of the uptake of the dicarboxylic acid fumaric * 0.4 acid across the intestinal BBM was also investigated. 5 4 Analogous to citrate uptake, transport of fumarate was also significantly stimulated by an inwardly s 0.2 m -A----+ ” directed Na+-gradient compared with Na+-free conb 4 ditions (Fig. 2). However, lowering the extravesicular ” I4 0.0 pH from 7.8 to 5.6 caused a significant reduction of 180 I 2 3 0 fumarate uptake in the presence of an inwardly time min directed Na+-gradient whereas uptake under Na+Fig. 1. Na+- and pH-dependence of citrate uptake. Values free conditions was slightly stimulated (Fig. 2). are presented as means f SEM of six determinations from In additional experiments the influence of a three different vesicle preparations, each determined in transmembrane electrical potential difference (PD) duplicate. Where absent, SEM was smaller than symbol. on citrate and fumarate uptake by the BBMV Vesicles were prepared in (mmol/l): 300 D-maIInitOl, 100 Kwas investigated under Na+-gradient as well as under gluconate, 35 N-2-hydroxyethylpiperaxine-N’-2-ethane Na+-free (choline gradient) conditions. Uptake of sulfonic acid (HEPES) adjusted with Tris(hydroxymethyl)aminomethane (Tris) to pH 7.8 (A, A, 0, 0) or HOOD- citrate and fumarate was measured either under

‘z

mannitol, 100 K-gluconate, 42 2-morpholinoethanesulfonic acid (MES), 15 HEPES, and 13 Tris, pH 5.6 (m, 0) and

incubated in a transport buffer resulting in final concentrations of (mmol/l): 100 n-mannitol, 100NaCl (A, n , 0) or choline-Cl (A, 0, O), 100K-gluconate, 0.05 ‘“C-lahelled citric acid and either 35 HEPES adjusted with Tris to pH 7.8 ((A, A) or 42 MES, 15 HEPES, 13 Tris, pH 5.6 (m, 0, 0, 0). Vesicles were zero-voltage clamped by the addition of valinomycin (see text for further details). Significant differences (r-test, P < 0.01) were obtained between uptake in the presence of a Na+ gradient and the respective uptake under choline+ gradient conditions at all pH conditions with the exception of uptake at 180min incubation time at pH 5.6/7.8. Differences between the uptake values in the presence of a Na+ gradient at different pH conditions were statistically significant [one way analysis of variance with pair comparisons according to Holm (lo), P < 0.051 between pH conditions 7.8/7.8 and 5.6/5.6 as well as between 7.8/7.8 and 5.6/7.8 at all incubation times and between 5.6/5.6 and 5.6/7.8 up to an incubation time of 1 min.

Furthermore, citrate was accumulated inside the vesicles under Na+-gradient conditions about 2-5-fold (3 min incubation) over citrate equilibrium (3 hr incubation). Thus, citrate accumulation inside the intestinal BBMV appears to be energized by the transmembrane Na+-gradient. The differences between uptake values after an incubation period of 180 min (Fig. 1) might be explained by an extremely slow equilibration of citrate across the BBM (Grass1 et al., 1983; Moe et al., 1988). Similar to experiments with renal BBMV (&hell and Wright, 1985; Wright, 1985; Wright and Wunz, 1987), uptake of citrate by pig intestinal BBMV in the presence of an inwardly directed Na+ gradient was strongly inhibited in the presence of Li+ in the incubation medium (Li+ concentration: 10 mmol/l; about 70% inhibition of citrate uptake at a concentration of 0.05 mmol/l at incubation periods from

c

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0.4

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5.617.8

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.

cholim*

A

D

0

1

0

t-l-

1

2

3

180

time, min

Fig. 2. Na+- and pH-dependence of fumarate uptake. Values are presented as means f SEM of six determinations from three different vesicle preparations, each determined in duplicate. Where absent, SEM was smaller than symbol. Vesicles were prepared and incubated as described in the legend of Fig. 1 with the exception of O.O5mmol/l 14Clabelled fumaric acid as transport substrate. Vesicles were zero-voltage clamped by the addition of valiomycin (for further details see text). Significant differences (r-test, P c 0.01) were obtained between uptake under Na+- and choline+-gradient conditions at all pH conditions up to 3 min incubation. Differences between the uptake values in the presence of a Na+ gradient at different pH conditions were statistically significant [one way analysis of variance with pair comparisons according to Holm (lo), P < 0.051 between pH conditions 7.8/7.8 and 5.6/5.6 as well as between 7.8/7.8 and 5.6/7.8 at incubation times of 15 set, 30 set, 1 min and 180 min.

SmFiuED Wow

et

al.

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I

2

3

180

0

1

2

3

180

tine, rin

time, min Fig. 3. Influence of a transmembrane electrical potential difference on citrate uptake. Values are presented as means f SEM of six (pH 5.6/5.6) or seven @H 7.8/5.6) different membrane preparations, each determined in duplicate. Where absent, SEM was smaller than symbol. Open symbols represent uptake under zero-voltage clamped conditions whereas filled symbols represent respective uptake in the presence of an inside negative membrane potential. Vesicles were prepared in (mmolfl): 300 o-mannitol, lOOK-gluconate, 35 HEPES adjusted with Tris to pH 7.8 or in 300 D-mannitol, lOOK-gluconate, 42 2-mo~holinoe~an~~fonic acid (MES), 15 HEPES, and 13 Tris, pH 5.6 and incubated in a transport b&r resulting in final con~nt~tions of (~ol/l): 260 D-mannitol, 100 NaCl, 20 K-gluconate, 42 MES, 15 HEPES, 13 Tris, pH 5.6 and 0.05 %Xabelled citric acid (@, inside negative K+ diffusion potential) or in 100 D-mannitol, 100 NaCl, WOK-gluconate, 42 MES, 15 HEPES, 13 Tris, pH 5.6 and 0.05 “Cc-labelied citric acid (0, zero-voltage clamped). Valinomycin was added as described in Materials and Methods. See text for further information.

zero-voltage clamped conditions or in the presence of an inside negative valinomycin-induced K+-diffusion potential. For control, glucose uptake under the same conditions was also determined. Nat/glucose cotransport (concentration of D-&$UCOSe: 0.1 mmoljl) was stimulated 5.2-fold at an incubation time of 15 set in the presence of an inside negative membrane potential compared to zero-voltage clamped conditions, proving successful manipulation of the membrane potential under the experimental conditions described above. As shown in Fig. 3, uptake of citrate under two different pH conditions ~H~“~/p~i~ = 5.6j5.6 or 5.6~7.8) in the presence of an inwardly directed Na+-gradieut appeared to be moderately stimulated by a transmembrane PD compared with zero-voltage clamped conditions. The effect occurred in all vesicle preparations tested and thus was statistically significant with the paired r-test (P --z0.05) at 15 set and 30 set @H 5.6/X6) or at 15 set @H 5.6/7.8). Furthermore, uptake of fumarate under Na+-gradient conditions was signifi~ntly enhanced by an inside negative K+-diffusion potential (Fig. 4). Uptake of both substrates under Na+-free conditions was not altered by manipulating the electrical PD (results not shown). Thus, Na+-dependent fumarate uptake across the intestinal BBM of the pig appears to be mediated at least partially by a potential-sensitive mechanism. Although to a lesser extent, citrate uptake by BBMV from pig small intestine seems also to be influenced by a transmembrane elect&a1 PD. Since in our previous study on citrate and fumarate transport across the intestinal BBM of calves

(Wolffram et al., 1990), the influence of a transmembrane PD was only investigated using citrate as transport substrate, some additional experiments with calf BBMV were performed to evaluate the potential ~nsitivity of fumarate transport. In contrast to citrate uptake (Wotffram et al., 1990), fumarate uptake (concentration: 0.1 mmol/l) by calf BBMV under Na+-gradient conditions, but not in the absence of Na+, was clearly stimulated by an inside negative K+ diffusion potential compared with zerovoltage clamped conditions (Fig. 5). To find out whether tri- and dicarboxylates share a common transport site, some cam-inhibition as well as bran-stim~ation experiments were performed. In the presence of an inwardly directed Na+-gradient and a H+ equilibrium at pH 5.6 uptake of citrate (concentration: 0.1 mmol/l) after 10 set incubation was strongly inhibited by 10 mmol/l of unlabelled citrate, fumarate or succinate, respectively (Fig. 6). citrate (concentration Additionally, uptake 0.2 mmol/l) in the presence of a Na+ equilibrium was clearly enhanced by preloading the vesicles with 10 mmoI/l succinate {Fig. 7). For the evaluation of the kinetics of succinate transport across the intestinal BBM, initial uptake of succinate (concentration 0.5 mmol/l) was determined in some preliminary experiments under Na+ as well as choline+ gradient conditions. Substrate uptake in the presence of the Nat-gradient was reasonably linear up to an incubation time of 5 sec. Uptake under Na+-free conditions (choline+ gradient) was only 10% or less of the respective uptake under Na+-gradient conditions (Fig. 8). Therefore,

763

Intestinal transport of tri- and dicarboxylates

o.oow

h

180

time, ain Fig. 4. Influence of a transmembrane electrical potential difference on fumarate uptake. Values are presented as means + SEM of nine determinations from three different membrane preparations (each determined in triplicate). Where absent, SEM was smaller than symbol. Open symbols represent uptake under zero-voltage clamped conditions whereas filled symbols represent respective uptake in the presence of an inside negative membrane potential. Vesicles were prepared in (mmol/l): 300 o-mannitol, 100 Kgluconate, 35 HEPES adjusted with Tris to pH 7.8 and incubated in a transport buffer resulting in final concentrations of (mmol/l): 260 D-mannitol, 100 NaCl, 20 K-gluconate, 42 MES, 15 HEPES, 13 Tris, pH 5.6 and 0.05 “‘C-labelled fumaric acid (0, inside negative K+ diffusion potential) or in 100 o-mannitol, 100 NaCl, 100 K-gluconate, 42 MES, 15 HEPES, 13 Tris, pH 5.6 and 0.05 “‘C-1abelled fumaric acid (0, zero-voltage clamped). Valinomycin was added as described in Materials and Methods. Significant differences (t-test, P < 0.05) between uptake under zerovoltage clamped conditions and in the presence of an inside negative potential are indicated by an asterisk.

succinate uptake as a function of the extravesicular substrate concentration was determined at an incubation time of 3 set in the presence of an inwardly directed Na+ gradient. Succinate uptake as a function of the extravesicular substrate concentration, ranging from 0.05 to 0.6mmol/l clearly showed saturation at increasing substrate concentrations (Fig. 9). Nonlinear fitting of the data to a Michaelis-Menten type equation indicates a single saturable mechanism with apparent kinetic parameters P’,,,,Xand Km of 0.43 nmol/mg protein .3 s and 0.14 mmol/l, respectively (Fig. 9). DISCUSSION

Despite the fact that diets of man and animals can contain considerable amounts of tri- and dicarboxylic acids, knowledge about the intestinal transport of these substances is incomplete. Some evidence for active Na+-dependent absorption of citrate has been obtained in rabbits (Newsome et al., 1983) as well as in hamsters (Browne et al., 1978) using intact intestinal preparations. However, interpretation of the results from transport studies using intact intestinal preparations is hampered by the fact that Krebs cycle

intermediates are extensively metabolized by the intestinal wall (Browne et al., 1978; Moe et al., 1988). Thus, the underlying mechanisms of the transfer of tri- and dicarboxylic acids across the cell envelope of intestinal epithelial cells, e.g. the brush-border as well as the basolateral membranes, can be best investigated using isolated membrane vesicles. Moe et al. (1988) and Wolffram et al. (1990) have presented evidence for Na+-dependent uptake of di- (Moe et al., 1988; Wolffram et al., 1990) and tricarboxylates (Wolffram et al., 1990) using brush border membrane vesicles isolated from pig (Moe et al., 1988) and calf (Wolffram et al., 1990) small intestine, respectively. In the present study we have tried to characterize the transport of tri- and dicarboxylic acids across the brush border membrane of pig small intestine with special reference to Na+, pH, and electrical potential dependence of citrate and fumarate transport. The results clearly demonstrate that citrate as well as fumarate crosses the BBM by a Na+-gradientdependent mechanism. Furthermore, results from &-inhibition (80% inhibition of citrate uptake by Ns*

0.6

1

choline*

zero-voltlps clupd

0

A

K* diffusion ptntial

0

A

8 :

O.Orn”

180

time, min

Fig. 5. Influence of a transmembrane electrical potential difference on fumarate uptake by calf BBMV. Values are presented as means k SEM of five different vesicle preparations, each determined in duplicate. Where absent, SEM was smaller than symbol. Open symbols represent uptake under zero-voltage clamped conditions whereas filled symbols represent respective uptake in the presence of an inside negative membrane potential. Vesicles were prepared in (mmol/l): 300 o-mannitol, 100 K-gluconate, 42 (MES), 15 HEPES, and 13 Tris, pH 5.6 and incubated in a transport buffer resulting in final concentrations of (mmol/l): 260 D-mannitol, lOONaC1 (0) or Choline-Cl (A), 20 K-gluconate, 42 MES, 15 HEPES, 13 Tris, pH 5.6 and 0.1 ‘%-labelled fumaric acid (0, A, inside negative K+ diffusion potential) or in 100 o-mannitol, IOONaCl (0) or choline-Cl (A), 100 K-gluconate, 42 MES, 15 HEPES, 13 Tris, pH 5.6 and 0.1 Y-labelled fumaric acid (0, A, zero-voltage clamped). Valinomycin was added as described in Materials and Methods. Significant differences (t-test, P < 0.05) between uptake under zero-voltage clamped conditions and in the presence of an inside negative potential are indicated by an asterisk.

764

to be only moderately, but si~fi~n~y influenced by a transmembrane electrical potential difference, transport capacity of the respective BBM preparation might be of crucial importance for demonstration of potential sensitivity of the uptake of dicarboxylates. The smaller effect of a transmembrane electrical potential difference on citrate transport by pig BBMV compared with the effect on fumarate uptake found in this study might be explained by a certain a&&y of the trivalent form of citrate to the respective carrier, thus partially masking the potential dependence of the transport of dicarboxylates (and the divalent form of tricarboxylates) across the intestinal BBM. This may also explain the absence of a significant effect of an electrical potential difference on citrate uptake by calf intestinal BBMV (Wolffram et al., 1990). Uptake of Krebs cycle intermediates, including citrate, across the renal BBM has been shown to occur by a potential-~nsitive process (Grass1 et al., 1983; Hirayama and Wright, 1986; Jorgensen et al., 1983; Schell and Wright, 1985; Wright et af., 1983, 1982a; Wright and Wunz, 1987) due to co-transport of three Nat ions with each divalent citrate (citrate*-). It should be mentioned, that uptake of triand dicarboxylates by renal BBMV exceeds uptake of these substrates by intestinal BBMV by about %-fold (Stevens et al., 1982). In renal BBMV an inside negative membrane potential resulted in a decrease of the apparent affinity constant (A’,) of the Na+/succinate co-transport mechanism (Wright et al., 1983).

Fig. 6. Cis-inhibition of citrate uptake under Na+-gradient conditions by fumarate and succinate. Values are presented as means f SEM of nine determinations from three different membrane preparations each determined in triplicate. Vesicles were prepared in (mmol/l): 300 D-mannitol, 100 K&con&e, 42 MES, 15 HEPES and 13 Tris, pH 5.6 and incubated in a transport buffer resulting in &al concentrations of (mmolll): 100 r+mannitol. 100 NaCl, 100 K-aluconate, 42 ties, I’S’HEPES, 13 Tris, PH 5.6,O. 1 “C-labeiled citric acid and 10 of the indicated substance. The incubation period was 10 sec. Citrate uptake was significantly reduced by all inhibitors investigated [one way analysis of variance with pair comparisons according to Holm (IO), P < 0.051. fumarate and succinate) and rrans-stimulation experiments (enhanced citrate uptake by BBMV preloaded

with succinate in the absence of a Na+-gradient) strongly indicates the existence of a common transport site for tri- and dicarboxylates in the intestinal BBM of the pig. Since an inside negative valinomycin-induced K+-diffusion potential clearly enhanced fumarate uptake and also moderately stimulated citrate uptake compared to zero-voltage clamped conditions, transport of tri- and dicarboxylates across the intestinal BBM seems to occur by a ~tential-~nsit~ve process. With respect to the influence of a transmembrane electrical potential difference our results are not in keeping with those of Moe et al. (1988) who found a Na+-dependent, electroneutral transport of succinate across the brush border of pig small intestine. Furthermore, in a study on citrate transport across the intestinal BBM of calves no evidence for an influence of a transmembrane potential difference was obtained ~olffmm et al., 1990). However, additional experiments with calf intestinal BBMV suggest that fumarate transport is also clearly potential-sensitive in this species (Fig. 5). The difference between the results of Moe et al. (1988) and the present study might be due to differences in the transport capacity of the vesicles. Uptake of succinate by intestinal BBMV from pigs prepared by Moe et al. (1988) was considerably lower compared with the fumarate uptake in the present study. Since the uptake of fumarate across the intestinal BBM seems

tin+, min Fig. 7. Trans-stimulation of citrate uptake by succinate. Values are presented as means of two different vesicle preparations each determined in duplicate. Vesicles were prepared in (mmol/l): lOONaC1, 50 K-gluconate, 100 Dmannitol, 42 MES, 15 HEPES, 13 Tris, pH 5.6 with (0) or without (0) the addition of 10 succinic acid; osmolarity was kept constant by the addition of D-mannitol. A 10 ~1 vesicle suspension was added to 490 ~1 of transport buffer resulting in final concentrations of (mmol/l): 100 NaCl, 50 K-gluconate, 120 mmannito10.2 succinate, 42 MES, 15 HEPES, 13 Tris, pH 5.6 and 0.2 l%-labelled citric acid. Vesicles were zero-voltage chunped by the addition of v~i~ycin (for further details see text).

165

Intestinal transport of tri- and dicarboxylates 1.0

2

0.6

f 2 4

0.4

,.

0.0

0.2 concentration,

0

0

2

0

0

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4

6

8

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10

time, 6 Fig. 8. Initial time course of succinate uptake. Values are presented as means of two different vesicle preparations each determined in duplicate. Vesicles were prepared in (mmol/l): 300 n-mannitol, 100 K-gluconate, 35 HEPES adjusted ‘v&h Tris to pH 7.8 and incubated in a transport buffer resulting in final concentrations of 100 NaCl (0) or choline-Cl (c), 100 n-mannitol, 100 K-gluconate,’ 35 HEPES adjusted with Tris to pH 7.8 and 0.5 “C-1abelled succinic acid. Vesicles were zero-voltage clamped by the addition of valinomycin. In the presence of an inwardly directed transmembrane Na+-gradient citrate uptake was strongly stimulated by lowering the extravesicular pH. The

stimulatory effect of a more acidic pH (5.6) on citrate transport is best explained by the assumption that the protonated forms of citrate (citrate-’ and citrate-‘) are much better transported than the trivalent species (citratee3) which is the predominant form at pH 7.8. Unlike in intestinal BBMV of calves (Wolffram et al., 1990), an inwardly directed H+ gradient appears to further stimulate Na+-gradient driven citrate uptake by pig intestinal BBMV. Similar findings were reported by Grass1 et al. (1983) who investigated citrate transport across the brush-border membrane of the proximal tubule of calf kidney. Unlike citrate, fumarate uptake was somewhat reduced at low extravesicular pH. Since the dicarboxylic acid fumaric acid is almost completely dissociated at pH 5.6 as well as 7.8, different dissociation of fumaric acid cannot explain this finding. A possible explanation for reduced fumarate uptake at low extravesicular pH might be a direct influence of pH on the carrier or an unspecific alteration of the membrane environment of the carrier which in turn influences the transport characteristics. The reduction of fumarate uptake at a low pH is not in discrepancy with the stimulation of citrate uptake under the same conditions since a reduction of citrate uptake which might occur at low pH is overruled by a strong stimulation of citrate uptake due to the shift of

0.4

0.6

mmol/l

Fig. 9. Succinate uptake as a function of extravesicular succinate concentration. Values are means from three different vesicle preparations each determined in duplicate. Vesicles were prepared and incubated as described in the legend to Fig. 8. The initial extra-vesicular concentration of succinate was varied between 0.05 and 0.6 mmol/l as indicated on the abscissa; osmolarity was kept constant by the addition of D-mannitol. The curve was calculated with nonlinear regression analysis based on a Michaelis-Menten type equation with one saturable component. The apparent kinetic parameters are shown in the inset (V,,, = maximal transport velocity, K,,, = affinity constant, r* = nonlinear regression coefficient).

citratee3 to the preferentially transported protonated forms of citrate at a low pH. It should be mentioned that fumarate uptake by calf intestinal BBMV was not influenced by lowering the pH from 7.8 to 5.6 (Wolffram et al., 1990), suggesting that species differences in membrane composition (Gruber and Deuticke, 1973) might be involved in this discrepancy. Uptake of succinate as a function of the extravesicular substrate concentration, ranging from 0.05 to 0.6 mmol/l, indicates saturable uptake which is mediated by a single carrier-mechanism with apparent transport constants of 0.441 nmol/mg protein. 3 s (I’,,,,,) and 0.14 mmol/l (K,,,). Taken together our results indicate Na+-dependent saturable transport of tri- and dicarboxylates across the intestinal BBM of the pig consistent with secondary active Na+/di(tri)carboxylate co-transport. Uptake seems to be mediated by a single saturable component and only small diffusional uptake. The present study shows for the first time that the transfer of dicarboxylates and to a lesser extent of tricarboxylates across the intestinal BBM can be influenced by a transmembrane electrical potential difference. Thus uptake of tri- and dicarboxylates as described in the present study shows similar characteristics as the transport mechanism for Krebs cycle intermediates identified in the BBM of the proximal tubule of the kidney (Barac-Nieto, 1984; Grass1 et al., 1983; Jorgensen et al., 1983; Wright, 1985; Wright et al., 1983, 1980, 1982a,b,c; Wright and Wunz, 1987). With respect to the pH dependence of fumarate and citrate

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transport, results of this study using pig BBMV differs partially from previous findings obtained with calf BBMV (Wolffram er al., 1990) since, unlike in the previous study, fumarate uptake was decreased by lowering extravesicular pH and citrate transport was additionally stimulated by an inwardly directed H+ gradient. Acknowledgement-This

work was supported by the Schweizerische Nationalfonds (Grant No. 3.808-0.87).

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Characterization of the transport of tri- and dicarboxylates by pig intestinal brush-border membrane vesicles.

1. Transport of citrate and fumarate across the pig intestinal brush-border membrane (BBM) was investigated using isolated BBM vesicles. 2. Citrate an...
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