Taurocholate transport by human ileal brush border membrane vesicles

GASTROENTEROLOGY

1987:93:925-33

Taurocholate Transport by Human Ileal Brush Border Membrane Vesicles J. A. BARNARD

and F. K. GHISHAN

Vanderbilt University Gastroenterology/Nutrition,

Medical Center, Nashville,

Department Tennessee

Human ileal brush border membrane vesicles were prepared from intestines obtained from cadaveric renal allograft donors. The energetics and kinetics of taurocholate transport were studied. Fifty-five percent of equilibrium uptake (picomoles per mg protein) resulted from binding to the vesicle surface or incorporation into an osmotically insensitive compartment. The initial rate of transport was stimulated fourfold by an inwardly directed Na+ gradient when compared with a K+ gradient, and cation gradient-dependent differences persisted throughout the initial 5 min of incubation [p < 0.05). Taurocholate uptake was half-maximally stimulated by a Na+ concentration of 23 t 4 mM. A Hi11 transformation of this plot gave a slope [n) of 0.97, indicating a 1:~ [moI/moI) Nat -taurochoIate coupling ratio. Generation ofa negative inside diffusion potential by anion substitution or valinomycin-induced K+ diffusion potential failed to alter bile salt uptake, suggesting an electroneutral transport mechanism. When Na+dependent uptake velocity (10 s) was examined over a range of taurocholate concentration (0.036-0.9 mM), the plot described a rectangular hyperbola. The mean apparent Michaelis constant was 0.037 ? 0.007 mM and maximum velocity was 1093 + 329 pmol taurocholate per milligram protein per 10 s. These data confirm and extend animal studies of ilea bile salt transport. Taurocholate uptake by the human ileal brush border occurs by a Na+-dependent, carrier-mediated electroneutral mechanism. According to this model, a single Na ion is coupled with a single taurocholate anion and transported

Received May 7, 1986. Accepted May 4, 1987. Address requests for reprints to: John A. Barnard, M.D., Vanderbilt University Medical Center, Department of Pediatrics, Division of Pediatric Gastroenterology and Nutrition, Nashville, Tennessee 37232. This work was supported in part by training grants AM 07083-09 and R01 AM 33209-01 from the National Institutes of Health. 0 1987 by the American Gastroenterological Association 0016-5085/87/$3.50

of Pediatrics,

Division

of Pediatric

across the brush border by a carrier mechanism is driven by a transmembrane Na+ gradient.

that

The detergentlike properties of bile salts are necessary for hepatic excretion of cholesterol and other lipophilic compounds, as well as the intestinal absorption of dietary lipid. Maintenance of a sufficient concentration of bile salt molecules in the enterohepatic circulation depends upon high affinity transport systems in the hepatic sinusoidal membrane and the intestinal brush border membrane. Intestinal transport of bile salts has been extensively characterized by a variety of techniques in several mammalian and avian species (l-5). These studies demonstrate that intestinal transport of conjugated bile salts occurs predominantly as a Na+ gradient-dependent, carrier-mediated process located in the ileal brush border membrane. The electrochemical potential difference for Na+ across the brush border membrane is maintained by the activity of sodium-potassium-stimulated adenosine triphosphatase located in the basolateral membrane; thus, bile salt transport is termed “secondarily active.” The relative contributions of intestinal absorption by ionic and nonionic diffusion to the enterohepatic bile salt pool is not yet fully defined (6). Investigations of human intestinal bile salt transport have been limited to perfusion studies in volunteer subjects and, in general, have corroborated results from animal experiments (2). In this report we examine human ileal brush border vesicle transport of taurocholate. Membrane vesicle transport techniques permit examination of the driving forces and kinetics of solute transport in an experimental setting that distinguishes carrier-mediated from nonmediated uptake, minimizes unstirred water layer effects, and permits a precise definition of electrochemical conditions on both sides of the membrane.

926

GASTROENTEROLOGY

BARNARD AND GHISHAN

Materials

and Methods

Table

1.

Vol. 93, No. 5

Enzyme Specific Activities for Human Brush Border Membrane Prenaration

Ileal

Methods Preparation of human brush border membrane vesicles. Three human small intestines were obtained at the time of renal allograft harvest. There was no history of intestinal disease. The small intestine was removed from the ligament of Treitz to the cecum, flushed with ice-cold normal saline, and kept at 0”-4°C during the 2 h in transit. After division into 1%15cm lengths, the intestine was opened longitudinally and the mucosa was removed by scraping with a metal spatula. The 8-12 distal aliquots were considered ileal mucosa and were kept in separate tubes at -70°C. This method was adapted from Rajendran et al. (7; personal communication). All experiments were done within 90 days of tissue storage. One-third to one-half of a frozen aliquot was quick thawed at 37°C. Ileal brush border membrane vesicles were isolated by a modification of Kessler’s divalent cation precipitation technique (8), which we have described in detail elsewhere (9). The final pellet was resuspended in preincubation media, the composition of which is noted in each figure legend. The final protein concentration was 3-10 mg/ml and was determined according to the method of Lowry et al. (10) using bovine serum albumin as the standard. Purity of the vesicle preparation. Purity of the brush border membrane preparation was determined by marker enzyme activity. Leucine aminopeptidase activity was measured using a kit from Sigma Chemical Co. (St. Louis, MO.). Sodium-potassium-stimulated adenosine triphosphatase, cytochrome c oxidase, and nicotinamide adenine dinucleotide phosphate cytochrome c reductase activities were determined by published methods (11,12). Morphologic purity was assessed by transmission electron microscopy. Transport measurements. Uptake of radiolabeled taurocholate was measured by a rapid filtration technique. Incubations were initiated by the addition of 20 ~1 of vesicle suspension to 60 ~1 of incubation solution. Experiments were conducted at 37°C and a pH of 7.4. After the desired incubation time interval, transport was terminated [IOO by abrupt dilution in 1 ml of ice-cold “stop solution” mM mannitol, 20 mM HEPESiTris (pH 7.41, 20 mM MgSO+ 100 mM choline chloride, and 0.1 mM taurocholate]. The reaction mixture was then immediately pipetted onto a prewetted filter (cellulose nitrate, 0.45-pm pore size; Sartorius Filters, Inc., Hayward, Calif.) and kept under suction. The filter was rinsed with 5 ml of ice-cold stop solution and prepared for scintillation counting. Binding of the radiolabel to the filter was determined by filtration of incubation media without vesicle protein and was considered in the calculation of results. Results are expressed as picomoles of taurocholate uptake per milligram of vesicle protein. Most experiments were performed on two to four occasions using freshly prepared vesicles and repeated at least once when the second human intestine became available; only uptake velocity versus concentration experiments were done on the third intestine. The number of experiments and number of individual incubations that contribute to each data

Enzyme

495 2 62 (81

Leucine

Brush border

Crude homogenate 4765

+ 374 (8)

Enrichment 9.6

aminopeptidase” Cytochrome c

17.4 + 9.2 (4)

6.0 + 1.0 (4)

0.34

oxidase” NADH cytochrome

30.0 2 7.0 (4)

10.0 ? 6.7 (4)

0.33

c reductase” Nat, K+-ATPase’

0.97

0.76 -t 0.12 (7)

0.78

+ 0.20 (7)

NADH, nicotinamide adenine dinucleotide phosphate; Na-. K’ATPase, sodium-potassium-stimulated adenosine triphosphatase. Values are mean 5 SEM; values in parentheses are number of preparations. ” Measured in micromoles of /?-NPHA per milligram of protein per hour. ‘I Measured in micromoles per milligram of protein per minute. Measured in micromoles of Pi per milligram of protein per minute.

point are noted in the figure legends. Statistical significance was analyzed by the unpaired t-test or, where indicated, by analysis of variance. Estimation of K,, and V ,1,%Xwere determined by a weighted least-squares computer fit of the sigmoidal curve according to the method of Vaughn et al. (13).

Materials [“H]Taurocholic acid (6.6 Ciimmol) and [“HIglucose (33.1 Ciimmol) were purchased from New England Nuclear [Boston, Mass.]. Taurocholic acid was purchased from Sigma Chemical. All other chemicals were reagent grade.

Results Marker

Enzyme

Studies

Marker enzyme activities were determined for each intestine (Table 1).Leucine aminopeptidase, an enzyme localized primarily in the brush border membrane, was lo-fold enriched when the initial mucosal homogenate was compared with the final pellet. Sodium-potassium-stimulated adenosine triphosphatase, an enzymic marker for the basolateral membrane; cytochrome c oxidase, a marker for the mitochondrial membrane; and nicotinamide adenine dinucleotide phosphate cytochrome c reductase, a marker for the endoplasmic reticulum, all demonstrated no increase in specific activity.

Electron

Microscopy

Two weeks after storage of the mucosa, a freshly prepared, 2% glutaraldehyde-fixed vesicle preparation was examined by electron microscopy. A homogenous vesicle population was minimally contaminated with cellular debris and membrane sheets (Figure 1).

November

1987

TAUROCHOLATE

Figure

Glucose

1. Electron

micrograph

of human

brush

Experiments

Nat gradient-stimulated o-glucose uptake was used as a functional measure of the capacity for “uphill” (concentrative) transport in the human ileal brush border membrane vesicle preparation. Glucose transport was examined within 1 wk of storage of the mucosa (intestine Nos. 1 and 31, and after 1 and 3 mo of storage (intestine No. 2). In each instance, a 100 mM inwardly directed Na+ gradient effected an initial transient accumulation of glucose above equilibrium uptake. As shown in Figure 2A, after 3 mo of was noted after 20 s of freezing an “overshoot” incubation and it was eightfold greater than the uptake value at equilibrium. The glucose uptake value at equilibrium can be used to derive an estimation of intravesicular volume. The intravesicular volume of the human ileal brush border membrane preparation was 1.1 j_d/mg protein, a value similar to previously reported volumes for intestinal brush border vesicles (14). Brush border membrane vesicles are osmotically sensitive compartments (15). Figure 2B demonstrates that ileal brush border membrane vesicle uptake of glucose varied as an inverse linear func-

border

membrane

vesicles.

TRANSPORT

Magnification,

BY ILEAL VESICLES

927

x 26,600.

tion of incubation solution osmolarity. These osmotic sensitivity data indicate that glucose uptake primarily results from transport into the intravesicular space. Extrapolation of the relationship to infinite osmolarity (zero intravesicular volume) shows that only 16% of equilibrium uptake at physiologic tonicity (300 mosmol) is due to binding or transport into an osmotically insensitive compartment.

Osmolarity When taurocholate uptake was examined as a function of osmolarity, a linear relationship was found (Figure 3). Fifty-five percent of bile salt uptake at equilibrium resulted from binding or transport into an osmotically sensitive compartment. In a single additional experiment, 53% of uptake after 2 min of incubation resulted from binding (data not shown).

Sodium

Dependence

Transport of taurocholate was determined function of time and cationic gradient. Vesicles prepared in Na+- and K+-free buffer solutions incubated in a NaS - or K+-containing buffer

as a were and such

928

BARNARD

GASTROENTEROI,OGY

AND GHISHAN

VALIDATION A. Glucose

STUDIES

WITH

Vol. 93. No. 5

GLUCOSE B. Glucose

uptoke

uptake

versus

osmolorrty

TIME (mmutes) Glucose transport by human ileal brush border membranes. Pnnel A: glucose uptake versus time. Membranes were preloaded with 280 mM mannitol and 20 mM HEPES/Tris (pH 7.4). Incubation was done at 37°C for 30 min (equilibrium] in a medium containing 0.1 mM [3H]glucose, 100 mM NaCl (top curve) or 100 mM KC1 (bottom curve), 20 mM HEPESiTris (pH 7.4). and 100 mM mannitol. Each data point represents four incubations performed on a single vesicle preparation. Panel 13: effect of osmolarity on glucose uptake. Membranes were preloaded with 280 mM mannitol and 20 mM HEPESiTris (pH 7.4). Incubation was done at 37°C for 30 min in a medium containing (final concentration) 0.1 mM j”H]glucose, 40 mM NaCl. 20 mM HEPESiTris (pH 7.41, and mannitol in sufficient concentration to give the indicated osmolarity. Each data point represents four incubations performed on a single vesicle preparation.

that a 100 mM cationic the extravesicular to the presence of an inwardly was a rapid accumulation first 2 min of incubation a slight efflux of bile

Figure

gradient was directed from intravesicular space. In the directed Na+ gradient there of taurocholate during the and, after 5 min, there was salt until equilibrium was

3. Effect of osmolarity on taurocholate uptake. Membranes were preloaded with 280 mM mannitol and 20 mM HEPESiTris (pH 7.4). Incubation was done at 37°C for 30 min (equilibrium) in a medium containing (final concentration) 0.375 mM [“Hltaurocholate, 40 mM NaCl, 20 mM HEPESiTris (pH 7.4), and mannitol in sufficient concentration to give the indicated osmolarity. Data were obtained from three separate vesicle preparations and two human ilea. Each point represents the mean 5 SEM for 5-14 determinations.

achieved at 30 min (Figure 4). Taurocholate uptake in the presence of an inwardly directed K+ gradient proceeded at a fourfold slower initial rate and was significantly less than Nat-stimulated uptake at 20 s and 1. 2, and 5 min of incubation (p < 0.05, analysis of variance). The inset in Figure 4 shows the initial rate of Na+-stimulated taurocholate uptake. Linearity is maintained for at least 20 s (r = 0.99). Taurocholate uptake by jejunal brush border membrane vesicles was examined under conditions identical to those described above. Uptake after 10 and 20 s and 2 and 5 min of incubation was not significantly stimulated by an inwardly directed Nat gradient. These observations corroborate the regional specificity of intestinal taurocholate transport (2,3) (data not shown). Corcelli and Storelli (16) recently observed an increased rate of glutamate uptake by renal brush border membrane vesicles in the presence of an outwardly directed 100 mM K+ gradient. In a single additional experiment, an outwardly directed 100 mM K’ gradient did not influence the initial (10 s) rate of taurocholate uptake. Uptake of taurocholate as a function of sodium concentration is shown in Figure 5. The rate of uptake increased as a hyperbolic function of Na ’ concentration from 5 to 125 mM. Taurocholate uptake was half-maximally stimulated at a Na+ concentration of 23 ? 4 mM. A Hill transformation (log (V/V,,,;,,-V) versus log [Na]) of this data yields a slope = n, which

November

1987

TAUROCHOLATE

TRANSPORT

BY ILEAL VESICLES

929

TIME (minutes) Figure

4. Effect of inwardly directed cation gradient and time on taurocholate uptake. Brush border vesicles were preloaded with 280 mM mannitol and 20 mM HEPESiTris (pH 7.4). Incubations were conducted at 37°C in a medium containing 100 mM NaCl or Data were obtained from four 100 mM KCl, 100 mM mannitol, 20 mM HEPES/Tris (pH 7.4), and 0.375 mM [3H]taurocholate. separate vesicle preparations and two human ilea. Each time point represents the mean -t SEM for 3-10 incubations. The initial taurocholate uptake rate is shown in the inset. The plot is linear from 5 to 30 s of incubation (r = 0.99). These data were calculated from two membrane preparations and two human ilea with incubations performed three to six times. Open circles, Na’ gradient: closed circles, K+ gradient.

represents the apparent number of binding sites for Naf. As shown in the inset, the slope is 0.97 indieating a 1: 1 sodium/taurocholate coupling ratio. Effect of Electrical

Potential

The influence of an imposed electrochemical membrane potential was studied using two methods,

anion substitution and valinomycin-induced K+ diffusion potentials. In the former experiment, anions with a range of lipid permeabilities were selected. Initial incubation with relatively lipid permeable anion would result in a relativel) negative intravesicular potential and thereby affect an electrogenic component of taurocholate transport. As shown in Table 2, the initial rate (10 s) of taurocholate uptake

1800-

T

160014001200-

Y=o.Wx-,.X

J

mM NaCl Figure

5.

Effect of extravesicular sodium concentration on 10-s taurocholate uptake. Brush border membrane vesicles were preloaded with 280 mM mannitol and 20 mM HEPESiTris (pH 7.4). Incubation media contained a final concentration of 0.375 mM [3H]taurocholate, 20 mM HEPES/Tris (pH 7.41, the indicated concentration of NaCl, and sufficient mannitol to maintain isotonicity (300 mosmol). Uptake at 0 mM NaCl (passive uptake) was subtracted from each data point. The inset shows a Hill transformation of the data. The slope = n or the number of Na+ binding sites. These data were obtained from three fresh vesicle preparations and two human ilea, and each point represents the mean t SEM for 12 determinations

930

BARNARD

Table

2.

GASTROENTEROLOGY

AND GHISHAN

Effect

of Anion

Table

on Taurocholute

Replacement

3.

Uptake

2134 2296 2297

1086 -t- 64 1286 ? 67 1353 t 56

NaSCN NaCl Na,SO,

Potential

Ten seconds (pmol TCimg protein)

Equilibrium (pm01 ‘I’Cimg protein 30 min)

Ten seconds (pm01 TClm:: protein)

Sodium salt

Effect of Electrical Transport

Inside diffusion Voltage clamped Significance

i- 227 f 273 f 284

potential

1006 t 59 1041 r 40 p 3’ 0.05

Vol. 93, No. 5

on Tuurocholute

Thirty minutes (pm01 1’Cimg protein] 1971 !I 136 2375 t 200 p 3- 0.05

TC, taurocholate. Membrane vesicles were preloaded with 280 mM mannitol and 20 mM HEPESiTris (pH 7.4). Incubations were carried out in (final concentration] 100 mM NaSCN. 100 mM NaCl, or 50 mM Na,SO,, 20 mM HEPESiTris (pH 7.4). and sufficient mannitol to maintain physiologic tonicity. Ten-second uptakes are mean + SEM of nine incubations performed 011 two fresh vesicle preparations. Equilibrium uptakes are mean + SEM of 13 incubations on four fresh vesicle preparations.

TC. taurocholate. Brush border membrane vesicles were preloaded with 100 mM KCl. 100 mM mannitol, and 20 mM HEPES:Tris (pH 7.4). Incubations were initiated in a medium containing 100 mM KCI. 50 mM NaCl. 20 mM HEPES!Tris (pH 7.4). 0.375 mM [“Hltaurocholate. and 10 ~g valinomycin/mg protein (lwltage clamped or K’ equilibrated): or 50 mM NaCI. 200 mM mannitol. 20 mM HEPESiTris (pH 7.4). 0.375 mM I,‘H]taurocholate, and 10 pg \ralinornycin per milligram protein (diffusion potential). The 10-s incubations were done on three separate vesicle preparations and each value represents the mean t SEM for 11 determinations. The equilibrium (30 min) \ralues were derived from two vesicle

is similar for the thiocyanate [high lipid permeability), chloride (intermediate lipid permeability), or sulfate (low lipid permeability) salts of sodium. When initial uptake rates are expressed as a function of equilibration uptake, no differences are appreciated. These results suggest an electroneutral transport process. A second experimental approach to the electrogenie nature of taurocholate transport was used. Intestinal brush border membrane vesicles were preloaded with 100 mM KCl. Incubations were initiated in a buffer containing 50 mM NaCl and the K+-ionophore valinomycin. Controls were voltage clamped by incubation in 50 mM NaCl, valinomytin, and 100 mM KC1 (K+ in = Kf out). Rapid Kt efflux in the experimental incubation generates a transmembrane electrochemical potential with the intravesicular compartment relatively negative. Results are shown in Table 3. Taurocholate uptake at 10 s and equilibrium was the same for the imposed potential and voltage-clamped incubations. These results also suggest an electroneutral transport process.

preparations

and 7 incubations.

linear, whereas under K+ gradient conditions velocity was a linear function of concentration from 0.036 to 0.9 mM. The Na’ gradient-dependent, carriermediated taurocholate transport was determined by the difference between uptake in the presence of a Na+ gradient and in the absence of Naf (Kt gradient). Saturability experiments were performed only on the three most distal segments (35-45 cm) of ileum. The apparent K,, values for each were not significantly different, but V,,,,, was significantly greater in the first intestine than in the second or third (Table 4). Figure 6 shows representative kinetic data from a single intestine.

Discussion

The relationship between uptake velocity and increasing taurocholate concentration was examined under initial flux (10 s), zero-trans conditions. Under Na+ gradient conditions uptake velocity was curvi-

Intestinal transport studies in human subjects have been limited in scope because of inherent technical and mechanical difficulties as well an inability to fully manipulate the transport system pharmacologically. The report herein describes taurocholate transport by intestinal brush border membrane vesicles prepared from human intestinal tissue obtained from renal allograph donors. The electron microscopic appearance and enzyme enrichment studies suggest that the predominant com-

Table

Membrane

Saturability

4.

Kinetic Parameters

for Human

Intestine Apparent

0.052

1

+ 0.015

Ileal Brush

Border Intestine

0.032

2

2 0.013

Taurocholate

Transport

Intestine 0.028

3

* 0.014

Mean 0.037

t SEM 2 0.007

K,, (mM)

V,,,,X

1751

Ifr 115

TC, taurocholate. V,,.,, values are picomoles Figure 6. Each experiment was done using preparations were used from each intestine

770 ? 50

757 2 63

lUY2 z!z 329

of taurocholate per milligram of protein per 10 s. Experimental conditions are given in vesicles prepared from the distal 35-45 cm of ileum (terminal ileum). Two fresh vesicle (n = 3-6 incubations at each bile salt concentration).

November

1987

TAUROCHOLATE

‘Ooo; 0.2

Figure

a4 0.6 Taurocho late ( m M )

0.0

1.0

6. Taurocholate uptake velocity versus concentration. Membrane vesicles were preloaded with 280 mM mannitol and 20 mM HEPESiTris (pH 7.41. Incubations were initiated in a medium containing a final concen20 tration of 100 mM NaCl or KCI, 100 mM mannitol, mM HEPESiTris (pH 7.4),and [3H]taurocholate ranging from 0.03 to 0.9 mM. The difference between Na’gradient and K+-gradient uptake is the Na+-dependent component of transport from which kinetic parameters are calculated. The data were obtained from two separate terminal ileal vesicle preparations on a single intestine. Each time point represents the mean t SEM for three determinations.

ponent of the final membrane pellet was a population of sealed brush border membrane vesicles. The functional integrity of the human ileal brush border membrane vesicle preparation was tested by examination of uphill glucose transport. A characteristic glucose overshoot eightfold above equilibrium was demonstrated after 20 s of incubation, and osmolarity experiments indicated that uptake occurred into an osmotically sensitive compartment. These observations persisted for as long as 3 mo after storing mucosal aliquots at -70°C. Previous reports have also documented the use of frozen intestinal mucosa in the preparation of intestinal membrane vesicles (14). A method for preparation of human intestinal brush border membrane vesicles from mucosa obtained at surgical margins has been described (17,18); however, this method is limited by the quantity of tissue available for vesicle preparation as well as the underlying disease process that has necessitated surgical intervention. Recently, human intestinal mucosa obtained from cadaveric renal allograft donors was used in the study of dipeptide transport (7). The present study further suggests that cadaveric renal allograft donor intestine is a potentially valuable source of tissue for the preparation of isolated brush border membrane vesicles, but the authors caution that marker enzyme recoveries were not quantified and more comprehensive studies are required before the described method is considered fully validated for transport studies.

TRANSPORT

BY ILEAL VESICLES

931

We studied taurocholate transport by human ileal brush border membrane vesicles. Osmotic sensitivity analysis suggests that a substantial portion of total taurocholate uptake results from binding to the internal and external vesicle surface or transport into an osmotically insensitive compartment. This observation is not surprising in light of the amphiphilic nature of the conjugated trihydroxy bile salt molecule and data from nonvesiculated rat intestinal brush border membranes that suggest binding is determined in part by partitioning between the aqueous phase and membrane lipid (19). Binding also appears to be a significant component of taurocholate uptake into rat liver plasma membrane vesicles (20). The Na+-dependent nature of taurocholate transport is evidenced by marked stimulation of uptake in the presence of a Na+ gradient directed from the extravesicular to the intravesicular space. Although uptake after 5 min of incubation significantly exceeded that equilibrium, the blunted nature of the curve may represent volume changes during the initial incubation interval rather than an actual “overshoot” phenomenon (21). Alternatively, a brisk “overshoot” may be obscured by the high degree of nonspecific binding and uptake found with the amphiphilic taurocholate molecule. When taurocholate uptake was examined as a function of Na+ concentration, a rectangular hyperbolic curve was found. Half-maximal bile salt uptake occurred at a Na+ concentration of 23 -+ 4 mM. Further analysis of this data by a Hill (linear) transformation yields a slope of n or apparent number of binding sites for Nat. When the taurocholate concentration was 0.373 mM, the Hill plot was linear (r = 0.98) with a slope of 0.97. This suggests, but does not prove, a 1 :l taurocholate/Na+ coupling ratio, i.e., a single monovalent anionic bile salt molecule accompanied by a single Na ion resulting in a electroneutral translocation from the extravesicular to the intravesicular space. A more complete description of Na+ dependence would include additional experiments performed under voltage-clamped conditions. The effect of an electrical potential on intestinal bile salt transport is controversial. Rouse and Lack (22), using anion substitution techniques, concluded that taurocholate transport across guinea pig ileal brush border vesicles was electroneutral. In our laboratory, valinomycin-induced K+ diffusion potentials did not influence taurocholate transport in developing rats (9). The Na+-dependent taurocholate uptake by renal brush border vesicles and liver plasma membrane vesicles is considered electroneutral (20,23). By contrast, both anion substitution and valinomycin-induced diffusion potential data reported by Lucke et al. (24) suggested an electrogenic

932

BARNARD

GASTROENTEROLOGY

AND GHISHAN

transport process in the rat ileum. Wilson and Treanor (25) found that glycodeoxycholate transport in the rat was electrogenic. In the present study, we used both anion substitution and valinomycininduced diffusion potentials to evaluate taurocholate transport by human ileal vesicles. Both techniques demonstrated an electroneutral transport process; however, before these studies can be considered definitive, they should be corroborated by “control” experiments using a substrate such as glucose, which is transported by an electrogenic mechanism. Initial-flux, zero-trans kinetic experiments were performed in brush border vesicles prepared from the terminal ileum. The Na+-dependent uptake velocity varied as a hyperbolic function of taurocholate concentration. The mean apparent K, value was 0.037+ 0.007mM. Previous studies have derived an apparent K, value for the perfused human ileum of 0.6 mM (2) and for in vitro transport in the rat, 0.23 mM (5). Inasmuch as solute uptake across the intestinal mucosa is influenced by penetration of the unstirred water layer as well as the intestinal brush border membrane, factors that diminish the thickness of the unstirred water layer will affect calculation of K,. For example, K, for taurocholate in everted intestinal sacs is twofold greater in “stirred” versus “unstirred” incubations (26). Although controversial, brush border membrane vesicle transport techniques presumably minimize unstirred water layer effects on carrier-mediated taurocholate transport. Thus, estimation of the K, from the present data more likely approximates the true K, for the ileal brush border membrane bile salt carrier. The present study demonstrates that the taurocholate transport process in the human intestinal brush border membrane is similar to that described by in vivo and in vitro human and animal experiments. That is, the carrier-mediated process is localized to the brush border membrane of the ileum (2, 3). The requirement for Na+ in the cotransport process is similar to that described for the rat, hamster, and guinea pig (1,4,24). A more detailed and complete analysis of the importance of a membrane potential in human Na+-taurocholate cotransport will require determination of stoichiometry by direct dualisotope fluxes, the use of potential-sensitive dyes, and control experiments with a substrate known to be transported by an electrogenic mechanism, e.g., glucose. The preliminary data presented here suggest that human ileal brush border membrane transport of taurocholate results from the coupling of a single Na ion with a single taurocholate anion and translocation across the brush border by a carrier mechanism that is driven by a transmembrane Na+ gradient.

Vol. 93, No. 5

References 1. Bee&y RC, Faust KG. Sodium ion-coupled taurocholate by brush border membrane vesicles.

uptake of Biochem J

1979;178:299-303. 2. Krag E, Phillips SF. Active and passive bile acid absorption in man: perfusion studies of the ileum and jejunum. J. Clin Invest 1974;53:1686-2694. 3. Lack L, Weiner IM. Intestinal absorption of bile acids and some biological implications. Gastroenterology 1963;22: 1334-8. 4. Rouse DJ, Lack L. Ion requirements for taurocholate transport by ileal brush border membrane vesicles. Life Sci 1979;25:45JL.

5. Schiff ER, Small NC, Dietschy JM. Characterization of the kinetics of the passive and active transport mechanism for bile acid absorption in the passive and active colon of the rat. J Clin Invest 1972;51:1351-62. 6. McClintock C, Yih-tu S. Jejunum is more important than terminal ileum for taurocholate absorption in rats. Am J Physiol 1983;244:G507-14, 7. Rajendran VM, Ansari SA, Harig JM, Adams MB, Khan AH, Ramaswamy K. Transport of glycyl+proline by human intestinal brush border membrane vesicles. Gastroenterology 1985;89:1298-304, 8. Kessler M, Acute 0, Storelli C, Murer H, Muller M, Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from the small intestinal brush border membrane. Biochim Biophys Acta 1978;506:136-54. 9. Barnard JA, Ghishan FK, Wilson FA. Ontogenesis of taurocholate transport by rat ileal brush border membrane vesicles. J Clin Invest 1985;75:869-73. 10. Lowry DH, Rosenbrough JN, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75. 11. Hodges TK, Leonard RT. Purification of a membrane bound adenosine triphosphatase from plant roots. Methods Enzymol 1974;32:392-406. 12. Scharchmidt BF, Keefe EB, Blankenship NM, Ockner RK. Validation of a recording spectrophotometric method for measurement of membrane associated Mg- and Na-K-ATPase activity. J Lab Clin Med 1979;93:790-9. 13. Vaughn WK, Neal RA, Anderson AJ. Computer estimation of the parameters of sigmoid kinetic model. Comput Biol Med 1976;6:1-7. 14. Kessler M, Toggenburger G. A laboratory manual on transport and bioenergetics. New York: Springer-Verlag 1979:1-24. 15. Hopfer UK, Nelson K, Perrotto J, Isselbacher KJ. Glucose transport in isolated brush border membranes from rat intestine. J Biol Chem 1973;248:25-32. 16. Corcelli A, Storelli C. The role of potassium and chloride ions on the Na+iacidic amino acid cotransport system in rat intestinal brush border membrane vesicles. Biochim Biophys Acta 1983;732:24-31. 17. Bluett M, Abumrad N, Arab N, Ghishan FK. Transport of n-glucose by human jejunal and ileal brush border membrane vesicles. Biochem J 1986;237:229-34. 18. Lucke H, Berner W, Menge H, Murer H. Sugar transport in brush border membrane vesicles isolated from human small intestine. Pflugers Arch 1978;168:529-32. 19. Wilson FA, Treavor L. Characterization of bile acid binding to the intestinal brush border membranes. J Membr Biol 1977:33:213-30. 20. Duffy MC, Blitzer BL, Boyer JL. Direct determination of the driving forces for taurocholate uptake into rat liver plasma membrane vesicles. J Clin Invest 1983;72:1470-81. 21. Gunther RD, Wright EM. Na’, Li+, and Cll transport by brush

November

border

TAUROCHOLATE

1987

membranes

from

rabbit

jejunum.

J Membr

Biol

1983;74:85-94. 22. Rouse DJ, Lack L. Short-term studies of taurocholate uptake by ileal brush border membrane vesicles. Anion effects. Biochim Biophys Acta 1980;599:324-9. 23. Wilson FA, Buchardt G, Murer H, Rumrich G, Ulrich KJ. Sodium-coupled taurocholate transport in proximal convolution of the rat kidney in vivo and in vitro. J. Clin Invest 1981;67:1141-50.

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24. Lucke H, Stange G, Kinne R, Murer H. Taurocholate-sodium cotransport by brush border membrane vesicles isolated from rat ileum. Biochem J 1978;174:951-8. 25. Wilson FA, Treanor LL. Glycodeoxycholate transport in brush border membrane vesicles isolated from rat jejunum and ileum. Biochim Biophys Acta 1979;554:430-40. 26. Wilson FA, Dietschy JM. The intestinal unstirred layer: its surface area and effect on active transport kinetics. Biochim Biophys Acta 1974;353:112-26.